WorldWideScience

Sample records for western blot results

  1. Western blot analysis.

    Science.gov (United States)

    Hirano, Seishiro

    2012-01-01

    Electrophoresis and the following western blot analysis are indispensable to investigate biochemical changes in cells and tissues exposed to nanoparticles or nanomaterials. Proteins should be extracted from the cells and tissues using a proper method, especially when phosphorylated proteins are to be detected. It is important to select a good blocking agent and an appropriate pair of primary and peroxidase-tagged secondary antibodies to obtain good results in western blot analysis. One thing that may be specific to nanomaterials, and that you should keep in mind, is that some proteins may be adsorbed on the surface of particulate nanomaterials. In this chapter the whole process of western blot analysis, from sample preparation to quantitative measurement of target proteins, is described.

  2. The western blot

    Science.gov (United States)

    Western blotting is a technique that involves the separation of proteins by gel electrophoresis, their blotting or transfer to a membrane, and selective immunodetection of an immobilized antigen. This is an important and routine method for protein analysis that depends on the specificity of antibod...

  3. Western Blot Techniques.

    Science.gov (United States)

    Kim, Brianna

    2017-01-01

    The Western blot is an important laboratory technique that allows for specific identification and characterization of proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins are electophoretically transferred to a polyvinylidene fluoride (PVDF) membrane which is then incubated with specific antibodies, then developed to show the protein of interest. Here, we describe the transfer and detection of Outer surface protein A (OspA), a protein only found on the surface of Borrelia burgdorferi, the bacteria responsible for Lyme disease.

  4. Lectin-probed western blot analysis.

    Science.gov (United States)

    Sato, Takeshi

    2014-01-01

    Lectin-probed western blot analysis, the so-called lectin blot analysis, is a useful method to yield basic information on the glycan structures of glycoproteins, based on the carbohydrate-binding specificities of lectins. By lectin blot analysis, researchers can directly analyze the glycan structures without releasing the glycans from glycoproteins. Here, the author describes protocols for standard analysis, and applies analysis in combination with glycosidase digestion of blot.

  5. Western blot: technique, theory, and trouble shooting.

    Science.gov (United States)

    Mahmood, Tahrin; Yang, Ping-Chang

    2012-09-01

    Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot.

  6. Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results.

    Science.gov (United States)

    Kuramitsu, Madoka; Sekizuka, Tsuyoshi; Yamochi, Tadanori; Firouzi, Sanaz; Sato, Tomoo; Umeki, Kazumi; Sasaki, Daisuke; Hasegawa, Hiroo; Kubota, Ryuji; Sobata, Rieko; Matsumoto, Chieko; Kaneko, Noriaki; Momose, Haruka; Araki, Kumiko; Saito, Masumichi; Nosaka, Kisato; Utsunomiya, Atae; Koh, Ki-Ryang; Ogata, Masao; Uchimaru, Kaoru; Iwanaga, Masako; Sagara, Yasuko; Yamano, Yoshihisa; Okayama, Akihiko; Miura, Kiyonori; Satake, Masahiro; Saito, Shigeru; Itabashi, Kazuo; Yamaguchi, Kazunari; Kuroda, Makoto; Watanabe, Toshiki; Okuma, Kazu; Hamaguchi, Isao

    2017-09-01

    Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens. Copyright © 2017 American Society for Microbiology.

  7. Single cell-resolution western blotting.

    Science.gov (United States)

    Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E

    2016-08-01

    This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4-6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine.

  8. Interpretation criteria in Western blot diagnosis of Lyme borreliosis.

    Science.gov (United States)

    Mavin, S; McDonagh, S; Evans, R; Milner, R M; Chatterton, J M W; Ho-Yen, D O

    2011-01-01

    This study reviews the Lyme borreliosis Western blot interpretation process, including what bands are classed as specific, the number of bands needed for a positive result, the role of band intensity and the use of clinical information. In 2008, 3688 patients (4223 serum samples) were tested by enzyme immunoassay (EIA), with 832 patients tested by confirmatory in-house IgG Western blot: 272 patients were Western blot-positive, 170 were weak positive, 156 were equivocal and 234 were negative. These results were assessed, and a review of interpretation criteria from both the USA and Europe was carried out. New interpretation criteria and a testing algorithm were developed. The revised criteria changed the results in 109/3688 (3%) patients and produced significantly more Western blot-positive and weak-positive patients than with the current criteria (485 vs. 442, P blot interpretation and improved the sensitivity and robustness of their Western blot method. Using a protocol tailored to patients that incorporates clinical characteristics means that the entire process will be easier and will aid the management of patients.

  9. SDS -PAGE and Western Blotting Techniques.

    Science.gov (United States)

    Blancher, C; Jones, A

    2001-01-01

    The goal of Western blotting, or more correctly, immunoblotting, is to identify with a specific antibody a particular antigen within a complex mixture of proteins that has been fractionated in a polyacrylamide gel and immobilized onto a membrane. Immunoblotting can be used to determine a number of important characteristics of protein antigens-the presence and quantity of an antigen, the relative molecular weight of the polypeptide chain, and the efficiency of extraction of the antigen.Immunoblotting occurs in six stages: (1) extraction and quantification of protein samples; (2) resolution of the protein sample in sodium dodecyl sulfatepolyacrylamide denaturing gel electrophoresis (SDS-PAGE); (3) transfer of the separated polypeptides to a membrane support; (4) blocking nonspecific binding sites on the membrane; (5) addition of antibodies; and (6) detection.Sample preparation is important for obtaining accurate separation of the proteins on the basis of molecular weight. Depending on whether an antigen is primarily extracellular, cytoplasmic, or membrane-associated different procedures might be required to prepare the sample initially. Although there are exceptions, many soluble nuclear and cytoplasmic proteins can be solubilized by lysis buffers that contain the nonionic detergent Nonidet P-40 (NP-40) and either no salt at all or relatively high concentrations of salt (e.g., 0.5 M NaCl). However, the efficiency of extraction is often greatly affected by pH of the buffer and the presence or absence of chelating agents such EDTA.

  10. Use of rapid HIV assays as supplemental tests in specimens with repeatedly reactive screening immunoassay results not confirmed by HIV-1 Western blot.

    Science.gov (United States)

    Wesolowski, Laura G; Delaney, Kevin P; Meyer, William A; Blatt, Amy J; Bennett, Berry; Chavez, Pollyanna; Granade, Timothy C; Owen, Michele

    2013-09-01

    An alternate HIV testing algorithm has been proposed which includes a fourth-generation immunoassay followed by an HIV-1/HIV-2 antibody differentiation supplemental test for reactive specimens and a nucleic acid test (NAT) for specimens with discordant results. To evaluate the performance of five rapid tests (Alere Clearview, Bio-Rad Multispot, OraSure OraQuick, MedMira Reveal, and Trinity Biotech Unigold) as the supplemental antibody assay in the algorithm. A total of 3273 serum and plasma specimens that were third-generation immunoassay repeatedly reactive and Western blot (WB) negative or indeterminate were tested with rapid tests and NAT. Specimens were classified by NAT: (1) HIV-1 infected (NAT-reactive; n=184, 5.6%), (2) HIV-status unknown (NAT nonreactive; n=3078, 94.2%) or by Multispot, (3) HIV-2 positive (n=5), and (4) HIV-1 and HIV-2 positive (n=6). Excluding HIV-2 positive specimens, we calculated the proportion of reactive rapid tests among specimens with reactive and nonreactive NAT. The proportion of infected specimens with reactive rapid test results and negative or indeterminate WB ranged from 30.4% (56) to 47.8% (88) depending on the rapid test. From 1% to 2% of NAT-negative specimens had reactive rapid test results. In these diagnostically challenging specimens, all rapid tests identified infections that were missed by the Western blot, but only Multispot could differentiate HIV-1 from HIV-2. Regardless of which rapid test is used as a supplemental test in the alternative algorithm, false-positive algorithm results (i.e., reactive screening and rapid test in uninfected person) may occur, which will need to be resolved during the baseline medical evaluation. Published by Elsevier B.V.

  11. Selected Reaction Monitoring (SRM) frente a Western Blot

    OpenAIRE

    Heredia Ponce, Zahira Maria; Urbano Gámez, Jose Alberto

    2016-01-01

    We propose the SRM technology as a complementary method to the Western Blot for the detection and quantification of proteins in a sample. The technique Western Blot has its own limitations: i) only a protein-of-choice is detected, ignoring any non-relevant proteins, ii) the sensitivity of the technique depends on the specificity of the antibody and iii) Western Blot is expensive and time-consuming. The advantages of SRM with respect Western Blot are remarkable: i) you can detect up to h...

  12. Western Blot of Stained Proteins from Dried Polyacrylamide Gels

    Science.gov (United States)

    Gruber, Claudia; Stan-Lotter, Helga

    1996-01-01

    Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

  13. Dealing with large sample sizes: comparison of a new one spot dot blot method to western blot.

    Science.gov (United States)

    Putra, Sulistyo Emantoko Dwi; Tsuprykov, Oleg; Von Websky, Karoline; Ritter, Teresa; Reichetzeder, Christoph; Hocher, Berthold

    2014-01-01

    Western blot is the gold standard method to determine individual protein expression levels. However, western blot is technically difficult to perform in large sample sizes because it is a time consuming and labor intensive process. Dot blot is often used instead when dealing with large sample sizes, but the main disadvantage of the existing dot blot techniques, is the absence of signal normalization to a housekeeping protein. In this study we established a one dot two development signals (ODTDS) dot blot method employing two different signal development systems. The first signal from the protein of interest was detected by horseradish peroxidase (HRP). The second signal, detecting the housekeeping protein, was obtained by using alkaline phosphatase (AP). Inter-assay results variations within ODTDS dot blot and western blot and intra-assay variations between both methods were low (1.04-5.71%) as assessed by coefficient of variation. ODTDS dot blot technique can be used instead of western blot when dealing with large sample sizes without a reduction in results accuracy.

  14. Studying protein-protein interactions via blot overlay/far western blot.

    Science.gov (United States)

    Hall, Randy A

    2015-01-01

    Blot overlay is a useful method for studying protein-protein interactions. This technique involves fractionating proteins on SDS-PAGE, blotting to nitrocellulose or PVDF membrane, and then incubating with a probe of interest. The probe is typically a protein that is radiolabeled, biotinylated, or simply visualized with a specific antibody. When the probe is visualized via antibody detection, this technique is often referred to as "Far Western blot." Many different kinds of protein-protein interactions can be studied via blot overlay, and the method is applicable to screens for unknown protein-protein interactions as well as to the detailed characterization of known interactions.

  15. Validation of western blot for Histoplasma capsulatum antibody detection assay.

    Science.gov (United States)

    Almeida, Marcos de Abreu; Pizzini, Cláudia Vera; Damasceno, Lisandra Serra; Muniz, Mauro de Medeiros; Almeida-Paes, Rodrigo; Peralta, Regina Helena Saramago; Peralta, José Mauro; Oliveira, Raquel de Vasconcelos Carvalhaes; Vizzoni, Alexandre Gomes; de Andrade, Carla Lourenço Tavares; Zancopé-Oliveira, Rosely Maria

    2016-02-24

    Histoplasmosis is worldwide systemic mycoses caused by the dimorphic fungus Histoplasma capsulatum. The isolation and identification of H. capsulatum in culture is the reference test for histoplasmosis diagnosis confirmation. However, in the absence of it, serology has been used as a presumptive diagnosis through antibody and antigen detection. The purpose of the present study was to validate an immunoassay method (western blot) for antibodies detection in the diagnosis of histoplasmosis. To validate the western blot (WB) a study was conducted using 118 serum samples from patients with histoplasmosis and 118 serum controls collected from January 2000 to December 2013 in residents of the Rio de Janeiro State, Brazil. Diagnostic validation parameters were calculated based on the categorization of results obtained in a 2 × 2 table and subjected to statistical analysis. In addition, the viability of deglycosylated histoplasmin antigen (ptHMIN) onto nitrocellulose membranes previously sensitized was evaluated during the same period. The WB test showed sensitivity of 94.9 %, specificity of 94.1 %, positive predictive value of 94.1 %, negative predictive value of 94.9 %, accuracy of 94.5 %, and almost perfect precision. Besides, the strips have proved to be viable for using at least 5 years after ptHMIN antigen sensitization. Western blot test using ptHMIN provides sensitive, specific, and faster results. Therefore, could be considered a useful tool in the diagnosis of histoplasmosis being used by public health system, even in situations where laboratory facilities are relatively limited.

  16. The fastest Western in town: a contemporary twist on the classic Western blot analysis.

    Science.gov (United States)

    Silva, Jillian M; McMahon, Martin

    2014-02-05

    The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

  17. HIV-1/2 indeterminate Western blot results: follow-up of asymptomatic blood donors in Belo Horizonte, Minas Gerais, Brazil

    Directory of Open Access Journals (Sweden)

    CARNEIRO-PROIETTI A.B.F.

    1999-01-01

    Full Text Available The clinical and public health importance of indeterminate results in HIV-1/2 testing is still difficult to evaluate in volunteer blood donors. At Fundação Hemominas, HIV-1/2 ELISA is used as the screening test and, if reactive, is followed by Western blot (WB. We have evaluated 84 blood donors who had repeatedly reactive ELISA tests for HIV-1/2, but indeterminate WB results. Sixteen of the 84 donors (19.0% had history of sexually transmitted diseases; 18/84 (21.4% informed receiving or paying for sex; 3/84 (3.6% had homosexual contact; 2/26 women (7.6% had past history of multiple illegal abortions and 3/84 (3.6% had been previously transfused. Four out of 62 donors (6.5% had positive anti-nuclear factor (Hep2, with titles up to 1:640. Parasitological examination of the stool revealed eggs of S. mansoni in 4/62 (6.4% donors and other parasites in 8/62 (12.9%. Five (5.9% of the subjects presented overt seroconversion for HIV-1/2, 43/84 (51.2% had negative results on the last visit, while 36/84 (42.9% remained WB indeterminate. Although some conditions could be found associated with the HIV-1/2 indeterminate WB results and many donors had past of risky behavior, the significance of the majority of the results remains to be determined.

  18. A defined methodology for reliable quantification of Western blot data.

    Science.gov (United States)

    Taylor, Sean C; Berkelman, Thomas; Yadav, Geetha; Hammond, Matt

    2013-11-01

    Chemiluminescent western blotting has been in common practice for over three decades, but its use as a quantitative method for measuring the relative expression of the target proteins is still debatable. This is mainly due to the various steps, techniques, reagents, and detection methods that are used to obtain the associated data. In order to have confidence in densitometric data from western blots, researchers should be able to demonstrate statistically significant fold differences in protein expression. This entails a necessary evolution of the procedures, controls, and the analysis methods. We describe a methodology to obtain reliable quantitative data from chemiluminescent western blots using standardization procedures coupled with the updated reagents and detection methods.

  19. The Design of a Quantitative Western Blot Experiment

    Directory of Open Access Journals (Sweden)

    Sean C. Taylor

    2014-01-01

    Full Text Available Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013 and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

  20. The design of a quantitative western blot experiment.

    Science.gov (United States)

    Taylor, Sean C; Posch, Anton

    2014-01-01

    Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

  1. Evaluating strategies to normalise biological replicates of Western blot data.

    Science.gov (United States)

    Degasperi, Andrea; Birtwistle, Marc R; Volinsky, Natalia; Rauch, Jens; Kolch, Walter; Kholodenko, Boris N

    2014-01-01

    Western blot data are widely used in quantitative applications such as statistical testing and mathematical modelling. To ensure accurate quantitation and comparability between experiments, Western blot replicates must be normalised, but it is unclear how the available methods affect statistical properties of the data. Here we evaluate three commonly used normalisation strategies: (i) by fixed normalisation point or control; (ii) by sum of all data points in a replicate; and (iii) by optimal alignment of the replicates. We consider how these different strategies affect the coefficient of variation (CV) and the results of hypothesis testing with the normalised data. Normalisation by fixed point tends to increase the mean CV of normalised data in a manner that naturally depends on the choice of the normalisation point. Thus, in the context of hypothesis testing, normalisation by fixed point reduces false positives and increases false negatives. Analysis of published experimental data shows that choosing normalisation points with low quantified intensities results in a high normalised data CV and should thus be avoided. Normalisation by sum or by optimal alignment redistributes the raw data uncertainty in a mean-dependent manner, reducing the CV of high intensity points and increasing the CV of low intensity points. This causes the effect of normalisations by sum or optimal alignment on hypothesis testing to depend on the mean of the data tested; for high intensity points, false positives are increased and false negatives are decreased, while for low intensity points, false positives are decreased and false negatives are increased. These results will aid users of Western blotting to choose a suitable normalisation strategy and also understand the implications of this normalisation for subsequent hypothesis testing.

  2. Determination of Diagnostic Antigens in Cattle Amphistomiasis Using Western Blotting

    Directory of Open Access Journals (Sweden)

    A Halajian

    2009-05-01

    Full Text Available "nBackground: Mixed infection with amphistomes seems common in native cattle of Iran. The aim of this study was to determine diagnostic antigens in cattle mixed amphistomiasis."nMethods: Specific antigens of Cotylophoron cotylophorum, Gastrothylax crumenifer and Paramphisto­mum cervi (mixed infection, the most common species, were collected from cattle was deter­mined. Adult trematodes were collected from the rumen of naturally infected cattle at meat inspec­tion. After their homogenization and centrifugation, somatic antigens were prepared and ana­lyzed by SDS-PAGE. Specific antigens were determinated by western blot with homologous and heterolo­gous sera. SDS-PAGE of whole worms extract was performed at different concentrations and subse­quent gels staining. Immunoblotting analysis using sera from cattle naturally infected with am­phistomes, Dicrocoelium dendriticum, Fasciola spp. and hydatid cyst was performed."nResults: Electrophorese analysis of somatic antigens revealed the presence of 10 and 21 protein bands at 4 µgr/ml and 8 µgr/ml with molecular weights ranging from 25-120 and 25-150 kDa, respectively. The best result was taken at 8 mg/ml concentration. Although western blot of these proteins demon­strate 5 major antigenic polypeptides ranging from 50 to 100 kDa which were recognized by serum of cat­tle naturally infected with mixed amphistomes.

  3. Clinical, immunohistochemical, Western blot, and genetic analysis in dystrophinopathy.

    Science.gov (United States)

    Na, Sang-Jun; Kim, Won-Joo; Kim, Seung Min; Lee, Kee Ook; Yoon, Bora; Choi, Young-Chul

    2013-08-01

    Dystrophin-deficient muscular dystrophies (dystrophinopathies) are the most common form of muscular dystrophy, with variable clinical phenotypes ranging from the severe Duchenne (DMD) to the milder Becker (BMD) forms. In this study, we investigated the relationship between clinical characteristics, findings at immunohistochemistry (IHC) and Western blot, and the pattern of exon deletions in 24 male patients with dystrophinopathies. We retrospectively reviewed findings from clinical and laboratory examinations, IHC for dystrophin of muscle biopsy tissue, Western blot analysis, and multiplex polymerase chain reaction (PCR) examination of genomic DNA. All tests were performed in every patient. PCR examination revealed exon deletions in 13 patients (54.2%). At Western blot analysis, 15 patients (62.5%) were negative at all three dystrophin domains. Most of these patients had a clinical presentation consistent with the DMD phenotype. Nine (37.5%) others were weakly positive at one or more domains. Most of these patients presented clinically as BMD phenotype. One patient whose clinical presentation was consistent with BMD phenotype had normal findings at IHC and was weakly positive at all three domains on Western blot analysis; however, with the exception of this patient, the findings at IHC and Western blot were consistent for individual patients. Based on these findings, we conclude that Western blot analysis appears useful for confirmation of dystrophinopathy in BMD patients with normal staining on IHC. Exon deletion analysis by multiplex PCR using peripheral blood is also a simple and useful test for the diagnosis of dystrophinopathy, although it has limited sensitivity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Western blot analysis of adhesive interactions under fluid shear conditions: the blot rolling assay.

    Science.gov (United States)

    Sackstein, Robert; Fuhlbrigge, Robert

    2015-01-01

    Western blotting has proven to be an important technique in the analysis of receptor-ligand interactions (i.e., by ligand blotting) and for identifying molecules mediating cell attachment (i.e., by cell blotting). Conventional ligand blotting and cell blotting methods employ non-dynamic (static) incubation conditions, whereby molecules or cells of interest are placed in suspension and overlaid on membranes. However, many cell-cell and cell-matrix adhesive interactions occur under fluid shear conditions, and shear stress itself mediates and/or facilitates the engagement of these physiologically appropriate receptors and ligands. Notably, shear forces critically influence the adhesion of circulating cells and platelets to vessel walls in physiologic cell migration and hemostasis, as well as in inflammatory and thrombotic disorders, cancer metastasis, and atherosclerosis. Use of non-dynamic blotting conditions to analyze such interactions can introduce bias, overtly missing relevant effectors and/or exaggerating the relative role(s) of non-physiologic adhesion molecules. To address this shortfall, we have developed a new technique for identifying binding interactions under fluid shear conditions, the "blot rolling assay." Using this method, molecules in a complex mixture are resolved by gel electrophoresis, transferred to a membrane that is rendered semitransparent, and the membrane is then incorporated into a parallel-plate flow chamber apparatus. Under controlled flow conditions, cells or particles bearing adhesion proteins of interest are then introduced into the chamber and interactions with individual immobilized molecules (bands) can be visualized in real time. The substrate molecule(s) supporting adhesion under fluid shear can then be identified by staining with specific antibodies or by excising the relevant band(s) and performing mass spectrometry or microsequencing of the isolated material. This method thus allows for the identification, within a complex

  5. Identification of immunodiagnostic antigens for cerebrospinal filariasis in horses by western blot analysis.

    Science.gov (United States)

    Takesue, Masataka; Osaka, Yuki; Muranaka, Masanori; Katayama, Yoshinari; Ikadai, Hiromi

    2016-01-01

    In the present study, the serum and cerebrospinal fluid of horses diagnosed with Setaria digitata cerebrospinal filariasis were analyzed by western blot. The results revealed S. digitata protein bands measuring 65, 34, 22, and 18 kDa in molecular weight. In particular, the 18 kDa band is a possible candidate for clinical immunodiagnosis on the basis of western blot findings.

  6. The line blot assay: problems with titrating first and second antibodies for Western blot and immunohistochemistry assays?

    Science.gov (United States)

    Rojas-Espinosa, O; Silva-Miranda, M; Wek-Rodriguez, K; Arce-Paredes, P

    2006-01-01

    We describe a technique designed to assess the optimal dilution of primary and secondary antibodies, to be used in Western blot, dot blot, the multi-antigen print immunoassay (MAPIA) and immunohistochemistry assays. The method that we call "line blot" is not an alternative but a practical, complementary tool for the above techniques that assures definitive results are obtained from single assays, so there is no need to repeat the assay. As with most immunoenzymatic assays, the line blot assay is very sensitive, allowing the detection of absolute amounts of antigen as low as 2.5 ng in the 0.5 cm-long segment line (see Results), depending on the strength of the secondary, enzyme-labelled antibody.

  7. Routine Western blot to check autophagic flux : Cautions and recommendations

    NARCIS (Netherlands)

    Gomez-Sanchez, Ruben; Pizarro-Estrella, Elisa; Yakhine-Diop, Sokhna M. S.; Rodriguez-Arribas, Mario; Bravo-San Pedro, Jose M.; Fuentes, Jose M.; Gonzalez-Polo, Rosa A.

    2015-01-01

    At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible

  8. Application of Intermittent Microwave Irradiation to Western Blot Analysis.

    Science.gov (United States)

    Liu, Yu-Ting; Toyokuni, Shinya

    2015-01-01

    We established a shortened protocol for Western blot analysis using intermittent microwave irradiation. With this method, the procedure is completed within 1 h after applying the primary antibody, and thus greatly saves time. This procedure appears to be applicable to any antibody based on our experience of several years.

  9. Routine Western blot to check autophagic flux : Cautions and recommendations

    NARCIS (Netherlands)

    Gomez-Sanchez, Ruben; Pizarro-Estrella, Elisa; Yakhine-Diop, Sokhna M. S.; Rodriguez-Arribas, Mario; Bravo-San Pedro, Jose M.; Fuentes, Jose M.; Gonzalez-Polo, Rosa A.

    2015-01-01

    At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible disadvanta

  10. Luminex xMAP combined with Western blot improves HIV diagnostic sensitivity.

    Science.gov (United States)

    Kong, Weiwei; Li, Yan; Cheng, Shaohui; Yan, Chen; An, Shiping; Dong, Zheng; Yan, Lina; Yuan, Yuhua

    2016-01-01

    Currently, Western blot is used to confirm the initial serodiagnosis of HIV infection by antibody detection. However, a major deficiency of the Western blot relates to a lack of sufficient sensitivity in detecting HIV antibodies. This report describes a simple, sensitive and inexpensive bead-based assay for detection of early HIV infection. A panel of 138 positive specimens including 105 blood donors and 33 MSM with known Western blot results were evaluated using Luminex xMAP at Tianjin Centers for Disease Control and Prevention (CDC). We demonstrate a superior sensitivity of Luminex xMAP compared with Western blot. Of the 87 confirmed HIV positive blood donors, Western blot only confirmed 65 cases with 74.7% (65/87) sensitivity while Luminex xMAP identified 72 cases with 82.8% (72/87) sensitivity (pWestern blot and Luminex xMAP verified 13 and 19 of 33 MSM specimens, respectively. The sensitivity was 39.4% (13/33) for Western blot and 57.6% (19/33) for Luminex xMAP (pWestern blot improves the diagnostic sensitivity of HIV infection at an early stage, and reduces the chances of missed diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Characterization of Nora Virus Structural Proteins via Western Blot Analysis

    Directory of Open Access Journals (Sweden)

    Brad L. Ericson

    2016-01-01

    Full Text Available Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs. The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles and ~1.33 g/mL (complete virions. Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.

  12. A duplex approach for immunochemical staining and typing of protein in western blots

    NARCIS (Netherlands)

    Kuczius, T.; Brandstädter, L.; Karch, H.; Langeveld, J.P.M.

    2011-01-01

    The qualitative and semiquantitative Western blotting technique enables the detection of separate proteins and the determination of subtypes and fragments by specific immunological reactions. Protein typing on immunoblots is restricted to antibody-specific determination, with the result of a

  13. Cy5 total protein normalization in Western blot analysis.

    Science.gov (United States)

    Hagner-McWhirter, Åsa; Laurin, Ylva; Larsson, Anita; Bjerneld, Erik J; Rönn, Ola

    2015-10-01

    Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Antibody performance in western blot applications is context-dependent.

    Science.gov (United States)

    Algenäs, Cajsa; Agaton, Charlotta; Fagerberg, Linn; Asplund, Anna; Björling, Lisa; Björling, Erik; Kampf, Caroline; Lundberg, Emma; Nilsson, Peter; Persson, Anja; Wester, Kenneth; Pontén, Fredrik; Wernérus, Henrik; Uhlén, Mathias; Ottosson Takanen, Jenny; Hober, Sophia

    2014-03-01

    An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well-designed conditions. Here, we have analyzed 13 000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application-specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full-length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full-length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context- and application-dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross-reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Evaluation of an indigenous western blot kit for human immunodeficiency virus

    Directory of Open Access Journals (Sweden)

    Lakshmi V

    2002-01-01

    Full Text Available PURPOSE: The Western Blot test is considered a gold standard test for the confirmation of an ELISA and/or rapid assay screened reactive sample in the diagnosis of HIV infection, especially in the low risk population. In this study, an indigenously developed HIV W. Blot kit (J.Mitra & Co., New Delhi, India was compared for its performance characteristics with a widely used Western Blot kit, HIV Blot 2.2 (Genelabs, Singapore. Antigens of both HIV-1 and the indicator antigen gp36 of HIV-2 are included in the strips. METHODS: A panel of 150 clinical serum samples was used in the evaluation. All the sera were tested simultaneously by both the kits. RESULTS: The HIV W. Blot kit had high performance characteristics (100% sensitivity and 100% specificity, like the HIV Blot 2.2. The test procedure was easy to perform. There was clear delineation of the bands. CONCLUSIONS: The interpretation of the results on the HIV W. Blot was less prone to subjective errors. The test gave positive bands at even very high serum dilutions in the test kit. This fact indicates that HIV W. Blot probably has a potential application in early phases of infection, when the antibody concentrations are still very low.

  16. Quantum dot-based western blot for sensitive detection of pig serum antibody to actinobacillus pleuropneumoniae

    Science.gov (United States)

    Cişmileanu, Ana; Sima, Cornelia; Grigoriu, Constantin

    2007-08-01

    A quantum dot - immunoglobulin conjugate specific for pig IgG, was obtained by carbodiimide chemistry. We used a Western blot technique for detecting specific antibodies against Actinobacillus pleuropneumoniae (A. pp), which cause porcine pleuropneumonia. The antigen used in this technique was Apx haemolysin which is an important virulence factor of A. pp and it induces protective immunity in vaccined pigs. The detection on Western blot membrane was possible at 1/50 dilution of quantum dot conjugate at a dilution of pig serum till 1/6400. The results for pig serum demonstrated a higher sensitivity of QD-based Western blot technique for the presence of antibodies specific for Apx haemolysin in comparison with similar classical techniques (with coloured substrate for enzyme present in secondary antibody conjugate).

  17. A Streamlined Western Blot Exercise: An Efficient and Greener Approach in the Laboratory Classroom

    Science.gov (United States)

    Ness, Traci L.; Robinson, Rebekah L.; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H.

    2015-01-01

    SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes…

  18. A Streamlined Western Blot Exercise: An Efficient and Greener Approach in the Laboratory Classroom

    Science.gov (United States)

    Ness, Traci L.; Robinson, Rebekah L.; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H.

    2015-01-01

    SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes…

  19. More specific bands in the IgG western blot in sera from Scottish patients with suspected Lyme borreliosis.

    Science.gov (United States)

    Evans, Roger; Mavin, Sally; McDonagh, Susan; Chatterton, Jean M W; Milner, Rachel; Ho-Yen, Darrel O

    2010-08-01

    To identify further Western blot bands that may be specific in the diagnosis of Lyme borreliosis. The Borrelia burgdorferi antibody profiles of 270 western blot positive patients and 241 western blot negative patients from 2008 were examined. 27 different non-specific bands were detected in both groups. Six of 27 (22%) of the non-specific bands were detected significantly more in the western blot positive patients compared to the western blot negative patients (20 kDa, p<0.0001; 28 kDa, p<0.002; 36 kDa, p<0.002; 37 kDa, p<0.007; 48 kDa, p<0.023; 56 kDa, p<0.028; two-tailed F test). Results suggest that the 20, 28 and 48 kDa bands should be regarded as specific.

  20. Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting

    Science.gov (United States)

    Omotola, Oluwabukola B.; Heda, Rajiv P.; Avery, Jamie

    2016-01-01

    SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin’s transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting. PMID:27582639

  1. Blame it on Southern, but it's a western blot.

    Science.gov (United States)

    Klionsky, Daniel J

    2017-01-02

    Edwin M. Southern is a professor emeritus at the University of Oxford. He is perhaps best known for development of the "Southern blot" (Dr. Southern was at the University of Edinburgh when he wrote his landmark paper). The Southern blot provided a scientific breakthrough by allowing scientists to detect a particular DNA sequence without first purifying it from the rest of the genome; the basic method involves the transfer of the DNA to a membrane, followed by detection with a specific probe. Although few people perform Southern blots as originally carried out by Southern, due in part to the more recent technique of the polymerase chain reaction, the basic concept continues to play an important role in molecular biology.

  2. Total protein or high-abundance protein: Which offers the best loading control for Western blotting?

    Science.gov (United States)

    Thacker, Jonathan S; Yeung, Derrick H; Staines, W Richard; Mielke, John G

    2016-03-01

    Western blotting routinely involves a control for variability in the amount of protein across immunoblot lanes. Normalizing a target signal to one found for an abundantly expressed protein is widely regarded as a reliable loading control; however, this approach is being increasingly questioned. As a result, we compared blotting for two high-abundance proteins (actin and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) and two total protein membrane staining methods (Ponceau and Coomassie Brilliant Blue) to determine the best control for loading variability. We found that Ponceau staining optimally balanced accuracy and precision, and we suggest that this approach be considered as an alternative to normalizing with a high-abundance protein.

  3. HIV‑2 antibody detection after indeterminate or negative HIV‑1 Western blot in Cuba, 2005-2008.

    Science.gov (United States)

    Díaz, Dervel F; Ortiz, Eva; Martín, Dayamí; Nibot, Carmen; Rizo, Adis; Silva, Eladio

    2012-01-01

    INTRODUCTION Differentiating between HIV-1 and HIV-2 infection is the first step to understanding HIV transmission, epidemiology and pathogenesis in geographical areas where both viruses circulate. In Cuba, positive results in mixed HIV-1/2 screening assays are confirmed by HIV-1 Western blot. Indeterminate results constitute the main limitation of this test and HIV-2 infection is among their possible causes; hence the importance of second-stage screening and confirmatory tests for HIV-2 infection. OBJECTIVE Investigate the contribution of HIV-2 antibodies to negative or indeterminate HIV-1 Western blot results in serum samples from 2005 through 2008 in Cuba. METHODS HIV-2 reactivity was studied using the ELISA DAVIH-VIH-2 diagnostic kit (Cuba) in 1723 serum samples with negative or indeterminate results for HIV-1 Western blot from January 2005 through December 2008. Duplicate sera reactive by ELISA were confirmed by HIV-2 Western blot, results interpreted according to WHO criteria. The epidemiological interview established by Cuba's National Program for Prevention and Control Sexually-Transmitted Diseases and HIV/AIDS was applied to HIV-2 Western blot-positive patients. RESULTS Among all sera studied, HIV-2 ELISA identified 12 reactive serum samples (0.70%) and 1711 non-reactive (99.30%). Western blot analysis of the 12 ELISA-reactive samples confirmed two positive samples (16.67%), 4 negative (33.33%) and 6 indeterminate (50%). Positive samples reacted against the p16, p26, gp36, p53, p56, p68 and gp105 proteins. All 12 ELISA-reactive samples belonged to the HIV-1 Western blot indeterminate group. The two HIV-2-positive samples showed well defined reactivity to gp160, p53, p55 and p34 of HIV-1. HIV-1 seroconversion was observed in all 10 remaining samples during serological followup. CONCLUSIONS Two new HIV-2 seropositive cases were diagnosed using DAVIH-VIH-2 and HIV-2 Western blot in indeterminate HIV-1 Western blot samples. Results support the recommendation

  4. When less is more: a simple Western blotting amendment allowing data acquisition on human single fibers

    DEFF Research Database (Denmark)

    Jensen, Thomas Elbenhardt; Richter, Erik A

    2011-01-01

    This editorial discusses a simple western blotting-amendment allowing rapid data-acquisition on single fibers obtained from freeze-dried human skeletal muscle biopsies.......This editorial discusses a simple western blotting-amendment allowing rapid data-acquisition on single fibers obtained from freeze-dried human skeletal muscle biopsies....

  5. A Laboratory Exercise Illustrating the Sensitivity and Specificity of Western Blot Analysis

    Science.gov (United States)

    Chang, Ming-Mei; Lovett, Janice

    2011-01-01

    Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the…

  6. Positive IgG Western Blot for Borrelia burgdorferi in Colombia

    Directory of Open Access Journals (Sweden)

    Palacios Ricardo

    1999-01-01

    Full Text Available In order to evaluate the presence of specific IgG antibodies to Borrelia burgdorferi in patients with clinical manifestations associated with Lyme borreliosis in Cali, Colombia, 20 serum samples from patients with dermatologic signs, one cerebrospinal fluid (CSF sample from a patient with chronic neurologic and arthritic manifestations, and twelve serum samples from individuals without clinical signs associated with Lyme borreliosis were analyzed by IgG Western blot. The results were interpreted following the recommendations of the Centers for Diseases Control and Prevention (CDC for IgG Western blots. Four samples fulfilled the CDC criteria: two serum specimens from patients with morphea (localized scleroderma, the CSF from the patient with neurologic and arthritic manifestations, and one of the controls. Interpretation of positive serology for Lyme disease in non-endemic countries must be cautious. However these results suggest that the putative "Lyme-like" disease may correlate with positivity on Western blots, thus raising the possibility that a spirochete genospecies distinct from B. burgdorferi sensu stricto, or a Borrelia species other than B. burgdorferi sensu lato is the causative agent. Future work will focus on a survey of the local tick and rodent population for evidence of spirochete species that could be incriminated as the etiologic agent.

  7. Use of a Western blot technique for the serodiagnosis of glanders

    Directory of Open Access Journals (Sweden)

    de Souza Marcilia MA

    2011-01-01

    Full Text Available Abstract Background The in vivo diagnosis of glanders relies on the highly sensitive complement fixation test (CFT. Frequently observed false positive results are troublesome for veterinary authorities and cause financial losses to animal owners. Consequently, there is an urgent need to develop a test with high specificity. Hence, a Western blot assay making use of a partly purified lipopolysaccaride (LPS containing antigen of three Burkholderia mallei strains was developed. The test was validated investigating a comprehensive set of positive and negative sera obtained from horses and mules from endemic and non endemic areas. Results The developed Western blot assay showed a markedly higher diagnostic specificity when compared to the prescribed CFT and therefore can be used as a confirmatory test. However, the CFT remains the test of choice for routine testing of glanders due to its high sensitivity, its feasibility using standard laboratory equipment and its worldwide distribution in diagnostic laboratories. Conclusions The CFT should be amended by the newly validated Western blot to increase the positive likelihood ratio of glanders serodiagnosis in non endemic areas or areas with low glanders prevalence. Its use for international trade of horses and mules should be implemented by the OIE.

  8. Automated capillary Western dot blot method for the identity of a 15-valent pneumococcal conjugate vaccine.

    Science.gov (United States)

    Hamm, Melissa; Ha, Sha; Rustandi, Richard R

    2015-06-01

    Simple Western is a new technology that allows for the separation, blotting, and detection of proteins similar to a traditional Western except in a capillary format. Traditionally, identity assays for biological products are performed using either an enzyme-linked immunosorbent assay (ELISA) or a manual dot blot Western. Both techniques are usually very tedious, labor-intensive, and complicated for multivalent vaccines, and they can be difficult to transfer to other laboratories. An advantage this capillary Western technique has over the traditional manual dot blot Western method is the speed and the automation of electrophoresis separation, blotting, and detection steps performed in 96 capillaries. This article describes details of the development of an automated identity assay for a 15-valent pneumococcal conjugate vaccine, PCV15-CRM197, using capillary Western technology. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Banding pattern indicative of echinococcosis in a commercial cysticercosis western blot

    Directory of Open Access Journals (Sweden)

    Tappe D

    2009-09-01

    Full Text Available Abstract Objective A commercial cysticercosis Western blot was evaluated for serological cross-reactivity of sera from patients with alveolar (AE and cystic echinococcosis (CE. Methods A total of 161 sera were examined, including 31 sera from AE-patients, 11 sera from CE-patients, 9 sera from patients with other parasitic diseases and 109 sera from patients with unrelated medical conditions. All AE-and CE-sera were also examined by the echinococcosis Western blot. Results More sera from patients with AE than with CE showed cross-reactivity in the form of ladder-like patterns ("Mikado aspect" and untypical bands at 6-8 kDa (71% and 77.4% versus 27.3% and 45.5%, respectively. In contrast, triplets of bands in the area above 50 kDa and between 24 and 39-42 kDa were more frequent in CE than in AE sera. The fuzzy band at 50-55 kDa typical for cysticercosis was absent in all AE and CE sera. Conclusions Atypical banding patterns in the cysticercosis Western blot should raise the suspicion of a metacestode infection different from Taenia solium, i.e. Echinococcus multilocularis or E. granulosus, especially when the Mikado aspect and an altered 6-8 kDa band is visible in the absence of a fuzzy 50-55 kDa band.

  10. The diagnostic value of Western blot method in patients with cystic echinococcosis.

    Science.gov (United States)

    Aslan, Mustafa; Yüksel, Pelin; Polat, Erdal; Cakan, Huseyin; Ergin, Sevgi; Öner, Y Ali; Zengin, Kagan; Arıkan, Soykan; Saribas, Suat; Torun, Muzeyyen Mamal; Kocazeybek, Bekir

    2011-04-01

    Cystic echinococcosis (CE) is the larval cystic stage (called echinococcal cysts) of a small taeniid-type tapeworm (Echinococcus granulosus). Carnivores such as dogs are usually definitive hosts. Intermediate hosts are typically herbivores such as sheep and cattle. CE can be detected using various imaging techniques such as ultrasonography or radiology. Moreover the primary diagnosis has to be confirmed by serological tests since the clinical signs of the disease are non-specific. This study examined the antigenic band patterns useful for serologic diagnosis of hydatidosis. We also report on the post-operative evolution of patients treated for this disease and also determined the diagnostic performance of Western blot IgG kit. Twenty-five (16 females and 9 males) non-operated patients with hydatid cysts (NOP) and 33 (21 females and 12 males) operated patients with hydatid cysts (OP) were included as study group and 22 healthy individuals (14 females and 8 males) with no known chronic diseases were included as a control group. The ages of the patients and control group individuals were between 16-83 years. Patient and control groups were matched for age and sex. Cyst hydatid IgG antibodies were detected in the sera from all patient groups but no antibodies were found in the sera from the control group using ELISA IgG method. Twenty-three (92%) non-operated patients and 18 (54.5%) operated patients exhibited positive results when Western blot IgG kit was used. The P7 band pattern was detected in the sera from all operated and non-operated patients. Twenty-seven of these positive cases had p7 and (p7+p16/18), (p7+p24/26) or (p7+p16/18+p24/26). No antibodies against p7, p16/18 ve p24/26 band patterns were seen in sera from the control group A statistically significant difference was detected between operated and nonoperated patients for Western blot positivity.(pWestern blot kit for 25 cases with CE and 22 healthy controls were calculated as 92%, 100%, 100% and 91

  11. Comparisons of ELISA and Western blot assays for detection of autophagy flux.

    Science.gov (United States)

    Oh, Sung-Hee; Choi, Yong-Bok; Kim, June-Hyun; Weihl, Conrad C; Ju, Jeong-Sun

    2017-08-01

    We analyzed autophagy/mitophagy flux in vitro (C2C12 myotubes) and in vivo (mouse skeletal muscle) following the treatments of autophagy inducers (starvation, rapamycin) and a mitophagy inducer (carbonyl cyanide m-chlorophenylhydrazone, CCCP) using two immunodetection methods, ELISA and Western blotting, and compared their working range, accuracy, and reliability. The ELISAs showed a broader working range than that of the LC3 Western blots (Table 1). Table 2 showed that data value distribution was tighter and the average standard error from the ELISA was much smaller than those of the Western blot, directly relating to the accuracy of the assay. Test-retest reliability analysis showed good reliability for three individual ELISAs (interclass correlation, ≥ 0.7), but poor reliability for three individual Western blots (interclass correlation, ≤ 0.4) (Table 3).

  12. Immunochemische detectiemethoden na western blotting van cytochroom P-450 iso-enzymen

    NARCIS (Netherlands)

    Laan CA; Jansen EHJM

    1992-01-01

    In this report a number of staining techniques on Western blots have been compared with respect to sensitivity, background staining, practical applicability and cost aspects. After electrophoresis of a rat microsomal liver sample followed by blotting, an incubation was performed of a primary antibo

  13. Use of Nonradioactive Detection Method for North- and South-Western Blot.

    Science.gov (United States)

    Franke, Claudia; Gräfe, Daniel; Bartsch, Holger; Bachmann, Michael P

    2015-01-01

    Many proteins bind to nucleic acids. For the first characterization of novel proteins, a fast and simple technique for testing their nucleic acid binding capabilities is desirable. Here we describe the use of a North-western and South-western blot protocol for the evaluation of the DNA and RNA binding abilities of a novel putative methyl transferase HSPC133 (METTL5).

  14. Protein analysis through Western blot of cells excised individually from human brain and muscle tissue.

    Science.gov (United States)

    Koob, A O; Bruns, L; Prassler, C; Masliah, E; Klopstock, T; Bender, A

    2012-06-15

    Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Novel chemiluminescent Western blot blocking and antibody incubation solution for enhanced antibody-antigen interaction and increased specificity.

    Science.gov (United States)

    Schwartz, Kimberly; Bochkariov, Dmitry

    2017-07-13

    Western blotting is a ubiquitous tool used in protein and molecular biology research, providing information about the presence, size, relative abundance, and state of a protein in a mixture. First, the proteins in a sample are separated by size using SDS-PAGE then transferred onto a membrane for detection with a set of primary and secondary antibodies. High-quality Western data requires high signal-to-noise ratios, which depend upon reduction of nonspecific antibody interactions. Blocking is a critical step in the Western blot method as it prevents the antibodies from binding nonspecifically to the membrane and irrelevant proteins. A solution of nonfat dry milk (NFDM) in physiological buffer is commonly used for this purpose, but does not perform well with every type of antibody and is not optimal for low-abundance proteins. We present a novel blocking solution for chemiluminescent Western blots, AdvanBlock™-chemi, which outperforms NFDM in experiments with 20 unique antibodies by increasing signal-to-noise ratios and minimizing nonspecific binding. This solution enhances protein detection by Western blot and provides consistent results for detection of low abundant and modified proteins. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Analysis of sperm membrane antigens relevant to antisperm antibody using Western blot

    Institute of Scientific and Technical Information of China (English)

    WangHF

    2002-01-01

    Objective:To identify the sperm membrane antigens associated with antisperm antibody.Methods:The antisperm antibody in serum was tested by ELISA.Antisperm antibody positive sera from 18 infertile men and 15 infertile women were used.The molecular weight(MW) of sperm membrane antigens associated with the antisperm antibody was analyzed with antisperm antibody positive serum using Western blot.Results:Eight kinds of MW of sperm membrane antigens were identified.The ratio of identification on the 78 KD(60.7%),60KD(71.4%),51KD(14.9%) and 23KD(14.29%)sperm antigen was higher than other.Conclusion:sperm membrane antigens with MW of 78KD,60KD,51KD and 23KD were associated with antisperm antibody and immunological infertility.

  17. Two-dimensional gel-based protein standardization verified by western blot analysis.

    Science.gov (United States)

    Haniu, Hisao; Watanabe, Daisuke; Kawashima, Yusuke; Matsumoto, Hiroyuki

    2015-01-01

    In data presentation of biochemical investigation the amount of a target protein is shown in the y-axis against the x-axis representing time, concentrations of various agents, or other parameters. Western blot is a versatile and convenient tool in such an analysis to quantify and display the amount of proteins. In western blot, so-called housekeeping gene product(s), or "housekeeping proteins," are widely used as internal standards. The rationale of using housekeeping proteins for standardization of western blot is based on the assumption that the expression of chosen housekeeping gene is always constant, which could be false under certain physiological or pathological conditions. We have devised a two-dimensional gel-based standardization method in which the protein content of each sample is determined by scanning the total protein density of two-dimensional gels and the expression of each protein is quantified as the density ratio of each protein divided by the density of the total proteins on the two-dimensional gel. The advantage of this standardization method is that it is not based on any presumed "housekeeping proteins" that are supposed to be being expressed constantly under all physiological conditions. We will show that the total density of a two-dimensional gel can render a reliable protein standardization parameter by running western blot analysis on one of the proteins analyzed by two-dimensional gels.

  18. COMPARISONS OF ELISA AND WESTERN BLOT ASSAYS FOR DETECTION OF CRYPTOSPORIDIUM ANTIBODY

    Science.gov (United States)

    A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurre...

  19. A western blot protocol for detection of proteins heterologously expressed in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Jørgensen, Morten Egevang; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins...

  20. Ferulic acid enhances IgE binding to peanut allergens in western blots.

    Science.gov (United States)

    Phenolic compounds at high concentrations are known to form insoluble complexes with proteins. We hypothesized that this complex formation could interfere with Western blot and ELISA assays for peanut allergens. To verify this, three simple phenolic compounds (ferulic, caffeic, and chlorogenic acids...

  1. A Study of Rubisco through Western Blotting and Tissue Printing Techniques

    Science.gov (United States)

    Ma, Zhong; Cooper, Cynthia; Kim, Hyun-Joo; Janick-Buckner, Diane

    2009-01-01

    We describe a laboratory exercise developed for a cell biology course for second-year undergraduate biology majors. It was designed to introduce undergraduates to the basic molecular biology techniques of Western blotting and immunodetection coupled with the technique of tissue printing in detecting the presence, relative abundance, and…

  2. Western blotting as a method for studying cell-biomaterial interactions : The role of protein collection

    NARCIS (Netherlands)

    van Kooten, TG; Klein, CL; Kirkpatrick, CJ

    2001-01-01

    Research of cell-biomaterial interactions is building on knowledge and methods available in cell and molecular biology. Western blotting is one of the options to characterize protein expression in cell populations. Method transfer to biomaterial model systems is not trivial because of the structure

  3. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

    Directory of Open Access Journals (Sweden)

    Samantha L Eaton

    Full Text Available Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate

  4. Characterization of a biopharmaceutical protein and evaluation of its purification process using automated capillary Western blot.

    Science.gov (United States)

    Xu, Dong; Mane, Sarthak; Sosic, Zoran

    2015-01-01

    This paper describes the application of an automated size-based capillary Western blot system (Sally instrument) from ProteinSimple, Inc., for biopharmaceutical fusion-Fc protein characterization and evaluation of its purification process. The fusion-Fc protein column purification from an excess of single chain Fc polypeptide and removal of an enzyme coexpressed for protein maturation have been demonstrated using an automated capillary Western system. The clearance of a selected host cell protein (HCP) present in cell culture of fusion-Fc protein was also quantitatively monitored throughout the protein purification process. Additionally, the low levels of fusion-Fc product-related impurities detected by traditional slab gel Western blot were confirmed by the automated capillary Western system. Compared to the manual approach, the automated capillary Western blot provides the advantages of ease of operation, higher sample throughput, greater linearity range, and higher precision for protein quantitation. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Contribution of a Comparative Western Blot Method to Early Postnatal Diagnosis of Congenital Syphilis.

    Science.gov (United States)

    Marangoni, Antonella; Foschi, Claudio; Capretti, Maria Grazia; Nardini, Paola; Compri, Monica; Corvaglia, Luigi Tommaso; Faldella, Giacomo; Cevenini, Roberto

    2016-05-01

    Serology has a pivotal role in the diagnosis of congenital syphilis (CS), but problems arise because of the passive transfer of IgG antibodies across the placenta. The aim of this study was to assess the diagnostic value of a comparative Western blot (WB) method finalized to match the IgG immunological profiles of mothers and their own babies at birth in order to differentiate between passively transmitted maternal antibodies and antibodies synthesized by the infants against Treponema pallidum Thirty infants born to mothers with unknown or inadequate treatment for syphilis were entered in a retrospective study, conducted at St. Orsola-Malpighi Hospital, Bologna, Italy. All of the infants underwent clinical, instrumental, and laboratory examinations, including IgM WB testing. For the retrospective study, an IgG WB assay was performed by blotting T. pallidum antigens onto nitrocellulose sheets and incubating the strips with serum specimens from mother-child pairs. CS was diagnosed in 11 out of the 30 enrolled infants; 9/11 cases received the definitive diagnosis within the first week of life, whereas the remaining two were diagnosed later because of increasing serological test titers. The use of the comparative IgG WB testing performed with serum samples from mother-child pairs allowed a correct CS diagnosis in 10/11 cases. The CS diagnosis was improved by a strategy combining comparative IgG WB results with IgM WB results, leading to a sensitivity of 100%. The comparative IgG WB test is thus a welcome addition to the conventional laboratory methods used for CS diagnosis, allowing identification and adequate treatment of infected infants and avoiding unnecessary therapy of uninfected newborns. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. Native Electrophoresis and Western Blot Analysis (NEWeB): Methods and Applications.

    Science.gov (United States)

    Manoussopoulos, Ioannis N; Tsagris, Mina

    2015-01-01

    Native Electrophoresis and Western Blot Analysis (NEWeB) has been developed for the study of plant virus characteristics, among others, virus particle-protein interactions, electrophorotype formation, and strain separation. The method is based on the property of electrophoretic mobility of virus particles (VP) and proteins and combines the analytical capacity of electrophoresis with the specificity of western blot. One of its advantages is that it deals with entire VP that can be studied in cause and effect or in time-interval experiments. Some of the most interesting approaches include VP structural studies, VP interaction with host or viral proteins, and also the characterization of VP-protein complexes. In this protocol, NEWeB is used to demonstrate the interaction of Plum pox virus particles with the helper component, a virus encoded protein. It is expected that the method could be used in analogous studies of other viruses or large protein complexes, where similar principles apply.

  7. Western blot diagnosis of vivax malaria with multiple stage-specific antigens of the parasite

    OpenAIRE

    Son, Eui-Sun; Kim, Tong Soo; Nam, Ho-Woo

    2001-01-01

    Western blot analysis was performed to diagnose vivax malaria using stage-specific recombinant antigens. Genomic DNA from the whole blood of a malaria patient was used as templates to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1) of Plasmodium vivax. Each amplified DNA fragment was inserted into a pGEX-4T plasmid to induce ...

  8. Evaluation of the Western blotting method for the diagnosis of congenital toxoplasmosis,

    Directory of Open Access Journals (Sweden)

    Jaqueline Dario Capobiango

    Full Text Available Abstract Objective: To evaluate the Western blotting method for the detection of IgG anti-Toxoplasma gondii (T. gondii (IgG-WB in the serum of children with suspected congenital toxoplasmosis. Methods: We accompanied 47 mothers with acquired toxoplasmosis in pregnancy and their children, between June of 2011 and June of 2014. The IgG-WB was done in house and the test was considered positive if the child had antibodies that recognized at least one band on IgG blots different from the mother's or with greater intensity than the corresponding maternal band, during the first three months of life. Results: 15 children (15.1% met the criteria for congenital toxoplasmosis and 32 (32.3% had the diagnosis excluded. The symptoms were observed in 12 (80.0% children and the most frequent were cerebral calcification in 9 (60.0%, chorioretinitis in 8 (53.3%, and hydrocephalus in 4 (26.6%. IgM antibodies anti-T. gondii detected by chemiluminescence (CL were found in 6 (40.0% children and the polymerase chain reaction (PCR for detection of T. gondii DNA was positive in 5 of 7 performed (71.4%. The sensitivity of IgG-WB was of 60.0% [95% confidence interval (CI 32.3-83.7%] and specificity 43.7% (95% CI 26.7-62.3%. The sensitivity of IgG-WB increased to 76.0 and 89.1% when associated to the research of IgM anti-T. gondii or PCR, respectively. Conclusions: The IgG-WB showed greater sensitivity than the detection of IgM anti-T. gondii; therefore, it can be used for the diagnosis of congenital toxoplasmosis in association with other congenital infection markers.

  9. Imported intraocular gnathostomiasis with subretinal tracks confirmed by western blot assay.

    Science.gov (United States)

    Yang, Ji Ho; Kim, Moosang; Kim, Eung Suk; Na, Byoung-Kuk; Yu, Seung-Young; Kwak, Hyung-Woo

    2012-03-01

    We report a case of intraocular gnathostomiasis diagnosed by western blot assay in a patient with subretinal tracks. A 15-year-old male patient complained of blurred vision in the right eye, lasting for 2 weeks. Eight months earlier, he had traveled to Vietnam for 1 week and ate raw wild boar meat and lobster. His best-corrected visual acuity was 20/20 in both eyes and anterior chamber examination revealed no abnormalities. Fundus examination showed subretinal tracks in the right eye. Fluorescein angiography and indocyanine green angiography showed linear hyperfluorescence of the subretinal lesion observed on fundus in the right eye. Ultrasound examination revealed no abnormalities. Blood tests indicated mild eosinophilia (7.5%), and there was no abnormality found by systemic examinations. Two years later, the patient visited our department again for ophthalmologic evaluation. Visual acuity remained 20/20 in both eyes and the subretinal tracks in the right eye had not changed since the previous examination. Serologic examination was performed to provide a more accurate diagnosis, and the patient's serum reacted strongly to the Gnathostoma nipponicum antigen by western blot assay, which led to a diagnosis of intraocular gnathostomiasis. This is the first reported case of intraocular gnathostomiasis with subretinal tracks confirmed serologically using western blot in Korea.

  10. Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Wu, Yonghong; Wu, Min; He, Guowei; Zhang, Xiao; Li, Weiguang; Gao, Yan; Li, Zhihui; Wang, Zhaoyan; Zhang, Chenggang

    2012-04-01

    In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples.

  11. A new Western blot assay for the detection of porcine cytomegalovirus (PCMV).

    Science.gov (United States)

    Plotzki, Elena; Keller, Martina; Ivanusic, Daniel; Denner, Joachim

    2016-10-01

    Porcine cytomegalovirus (PCMV) may be harmful for human recipients if xenotransplantation using pig cell, tissue or organ will be performed transmitting the virus from donor pigs to human recipients. PCMV is widespread in pigs and closely related to human pathogenic herpesviruses, however there are no data concerning infection of humans. In contrast, recently it had been shown that transplantation of organs from pigs infected with PCMV into non-human primate recipients resulted in a significant reduction of the survival time compared with the transplantation of organs from uninfected pigs. To prevent transmission of PCMV in future pig to human xenotransplantations, sensitive and specific detection methods should be used. Here a new Western blot assay using recombinant proteins corresponding to two domains of the glycoprotein gB of PCMV is described. With this assay, the presence of PCMV-specific antibodies in different pig breeds was analysed. Antibodies were detected in a high percentage of animals, in one breed up to 85%. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. An alternative strategy to western blot as a confirmatory diagnostic test for HIV infection.

    Science.gov (United States)

    Feng, Xia; Wang, Jibao; Gao, Zhiyun; Tian, Yu; Zhang, Ling; Chen, Huichao; Zhang, Tong; Xiao, Lin; Yao, Jun; Xing, Wenge; Qiu, Maofeng; Jiang, Yan

    2017-03-01

    In China, western blot (WB) is the recommended procedure for the diagnosis of HIV infection. However, this technique is time consuming and labor intensive, and its complexity restricts wide application in resource-limited regions. The aim of this study was to evaluate the efficacy of a dry blood spots (DBS)-urine paired enzyme-linked immunosorbent assay (ELISA) test, instead of WB, for HIV antibody detection. Plasma, DBS, and urine samples were collected from 1213 subjects from different populations. Two diagnostic testing strategies were conducted in parallel. The equivalence of the paired ELISA and WB strategies was assessed. A diagnosis of HIV was determined in 250 subjects according to the paired ELISA test, and in 249 according to the WB strategy. The discordant case was judged HIV-positive during follow-up. In total, 18 subjects were diagnosed with possible HIV using the paired ELISA test, among whom, 11 subjects tested negative with WB, and one was confirmed to be HIV-positive during follow-up. For the remaining 945 subjects, both strategies indicated a negative result. The kappa test indicated good conformity (kappa=0.954) between the two diagnostic strategies. The DBS-urine paired ELISA could be applied as an alternative to WB in HIV diagnosis, which would be valuable in resource-limited regions owing to the associated affordability and ease of use. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

    Directory of Open Access Journals (Sweden)

    MORALES Olga Lucía

    2002-01-01

    Full Text Available Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB assay was performed using excretory/secretory antigens (E/S of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

  14. Herpes simplex virus type 2 antibody detection performance in Kisumu, Kenya, using the Herpeselect ELISA, Kalon ELISA, Western blot and inhibition testing.

    Science.gov (United States)

    Smith, J S; Bailey, R C; Westreich, D J; Maclean, I; Agot, K; Ndinya-Achola, J O; Hogrefe, W; Morrow, R A; Moses, S

    2009-04-01

    In certain parts of Africa, type-specific herpes simplex virus type 2 (HSV-2) ELISAs may have limited specificity. To date, no study has been conducted to validate HerpeSelect and Kalon type-specific HSV-2 ELISAs using both the Western blot and recombinant gG ELISA inhibition testing as reference standards. A total of 120 men who were HIV seronegative (aged 18-24 years) provided blood samples. HSV-2 IgG serum antibodies were detected using four different methods: HerpeSelect HSV-2 ELISA (n = 120), Kalon HSV-2 ELISA (n = 120), University of Washington Western blot (n = 101) and a recombinant inhibition test (n = 93). HSV-2 seroprevalence differed significantly by HSV-2 detection method, ranging from 24.8% with the Western blot to 69.8% with the HerpeSelect ELISA. Using the Western blot as the reference standard, the HerpesSelect had the highest sensitivity for HSV-2 antibody detection (100%) yet lowest specificity (40%). Similar results were obtained using the inhibition test as the reference standard. The sensitivity and specificity of the Kalon test versus the Western blot were 92% and 79%, respectively, and 80% and 82% versus the inhibition test. Using the inhibition test as the reference standard, the sensitivity of the Western blot appeared low (49%). In men in western Kenya who were HIV seronegative, the HerpeSelect and Kalon type-specific ELISAs had high sensitivities yet limited specificities using the Western blot as reference standard. Overall, the Kalon ELISA performed better than the HerpeSelect ELISA in these young men from Kisumu. Further understanding is needed for the interpretation of HSV-2 inhibition or ELISA test positive/ Western blot seronegative results. Before HSV-2 seropositivity may be reliably reported in selected areas of Africa, performance studies of HSV-2 serological assays in individual geographical areas are recommended.

  15. Investigation of telomerase activity and apoptosis on invasive ductal carcinoma of the breast using immunohistochemical and Western blot methods.

    Science.gov (United States)

    Simsek, B C; Turk, B A; Ozen, F; Tuzcu, M; Kanter, M

    2015-08-01

    Invasive ductal carcinoma (IDC) comprises the largest group of breast cancers. This study aimed to investigate telomerase activity and apoptosis using immunohistochemical and Western blot methods. In total, 75 cases that had been diagnosed as IDC and 20 cases that had undergone a freezing procedure were included. The histological sections were stained with Bax, Bcl-2, hTERT and BNIP3. The ages of the patients, as well as their hormonal status and tumour sizes and grades were evaluated, as well as the staining characteristics of the antibodies in question. A decrease in Bcl-2 positivity and an increase in Bax positivity were found immunohistochemically with increasing tumour grades. The data obtained by western blot method showed that Bcl-2 was highest in grade 1 tumours although these results were not statistically significant. The relationship between estrogen and progesterone receptor positivity and Bcl-2 was statistically significant, suggesting there is hormonal control through apoptosis. BNIP3 was found to be decreased with increasing tumour grades. Similarly, BNIP3 was found to be having the lowest value in grade 3 tumours by western blot method. Furthermore, hTERT was found to be increased with increasing tumour grades. In the western blot method, hTERT increased nearly four-fold compared to the control. In addition, hTERT, which was seen in very high levels in tumours, may be a helpful cancer marker. Both hTERT and BNIP3 are important markers that can provide information about prognosis. Big improvements can be achieved in tumour progression control with new treatment modalities that stop telomerase activity and hypoxic cell death.

  16. A Western Blot Protocol for Detection of Proteins Heterologously Expressed in Xenopus laevis Oocytes.

    Science.gov (United States)

    Jørgensen, Morten Egevang; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    Oocytes of the African clawed frog, Xenopus laevis, are often used for expression and biochemical characterization of transporter proteins as the oocytes are particularly suitable for uptake assays and electrophysiological recordings. Assessment of the expression level of expressed transporters at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins heterologously expressed in Xenopus oocytes.

  17. Proteínas inmunodominantes de Brucella Melitensis evaluadas por Western Blot

    Directory of Open Access Journals (Sweden)

    Elizabeth Anaya

    1997-01-01

    Full Text Available Se separaron extractos de proteínas totales de Brucella melitensis en gel 15% SDS-PAGE. Su seroreactividad fue analizada por Western Blot con resultados satisfactorios. Para éste propósito sueros controles negativos (n=03, sueros de pacientes con brucelosis (n=34, cólera (n=12, tifoidea (n=02 y tuberculosis (n=02 fueron usados. Esta prueba inmunodiagnóstica detectó bandas seroreactivas altamente específicas (100% correspondientes a 8,14,18, un complejo de 25-48 y 58kDa. La sensibilidad del test fue del 90% usando los sueros antes mencionados.

  18. Should we ignore western blots when selecting antibodies for other applications?

    DEFF Research Database (Denmark)

    Uhlén, Mathias

    2017-01-01

    applications and that this influences the epitopes exposed on the target protein, which might have profound consequences for the ability of a given antibody to bind specifically to its target. As an example, proteins that are analyzed by immunohistochemistry (IHC) are normally first cross-linked with formalin....... In conclusion, western blot and protein array analyses can indeed be useful tools when selecting specific antibodies for other applications. The use of these methods is encouraged both for antibody providers and users, and antibodies with signs of cross-reactivity in these applications should be treated...

  19. Antibody responses to Borrelia burgdorferi detected by western blot vary geographically in Canada.

    Science.gov (United States)

    Ogden, Nicholas H; Arsenault, Julie; Hatchette, Todd F; Mechai, Samir; Lindsay, L Robbin

    2017-01-01

    Lyme disease is emerging in eastern and central Canada, and most cases are diagnosed using the two-tier serological test (Enzyme Immuno Assay [EIA] followed by Western blot [WB]). Simplification of this algorithm would be advantageous unless it impacts test performance. In this study, accuracy of individual proteins of the IgG WB algorithm in predicting the overall test result in samples from Canadians was assessed. Because Borrelia burgdorferi strains vary geographically in Canada, geographic variations in serological responses were also explored. Metrics of relative sensitivity, specificity and the kappa statistic measure of concordance were used to assess the capacity of responses to individual proteins to predict the overall IgG WB result of 2524 EIA (C6)-positive samples from across Canada. Geographic and interannual variations in proportions of samples testing positive were explored by logistic regression. No one protein was highly concordant with the IgG WB result. Significant variations were found amongst years and geographic regions in the prevalence of samples testing positive using the overall IgG WB algorithm, and for individual proteins of the algorithm. In most cases the prevalence of samples testing positive were highest in Nova Scotia, and lower in samples from Manitoba westwards. These findings suggest that the current two tier test may not be simplified and continued use of the current two-tier test method and interpretation is recommended. Geographic and interannual variations in the prevalence of samples testing positive may be consistent with B. burgdorferi strain variation in Canada, and further studies are needed to explore this.

  20. Design, preparation and use of ligated phosphoproteins: a novel approach to study protein phosphatases by dot blot array, ELISA and Western blot assays.

    Science.gov (United States)

    Sun, Luo; Ghosh, Inca; Barshevsky, Tanya; Kochinyan, Samvel; Xu, Ming-Qun

    2007-07-01

    The study of substrate specificity of protein phosphatases (PPs) is very challenging since it is difficult to prepare a suitable phosphorylated substrate. Phosphoproteins, phosphorylated by a protein kinase, or chemically synthesized phosphopeptides are commonly used substrates for PPs. Both types of these substrates have their advantages and limitations. Phosphoproteins mimic more closely the physiologically relevant PP substrates, but their preparation is technically demanding. Synthetic phosphopeptides present advantages over proteins because they can be easily produced in large quantity and their amino acid sequence can be designed to contain potential determinants of substrate specificity. However, short peptides are less optimal compared to in vivo PP substrates and often display poor and variable binding to different matrices, resulting in low sensitivity in analysis of PP activity on solid support. In this work we utilize the intein-mediated protein ligation (IPL) technique to generate substrates for PPs, combining the advantages of proteins and synthetic peptides in one molecule. The ligation of a synthetic phosphopeptide to an intein-generated carrier protein (CP) with a one-to-one stoichiometry results in the formation of a ligated phosphoprotein (LPP). Three widely used assays, dot blot array, Western blot and ELISA were employed to study the PP activity on LPP substrates. Dephosphorylation was measured by detection of the remaining phosphorylation, or lack of it, with a phospho-specific antibody. The data show the advantage of LPPs over free peptides in assays on solid supports. LPPs exhibited enhanced binding to the matrices used in the study, which significantly improved sensitivity and consistency of the assays. In addition, saturation of the signal was circumvented by serial dilution of the assay samples. This report describes detailed experimental procedures for preparation of LPP substrates and their use in PP assays based on immobilization on

  1. Validation of endothelin B receptor antibodies reveals two distinct receptor-related bands on Western blot.

    Science.gov (United States)

    Barr, Travis P; Kornberg, Daniel; Montmayeur, Jean-Pierre; Long, Melinda; Reichheld, Stephen; Strichartz, Gary R

    2015-01-01

    Antibodies are important tools for the study of protein expression but are often used without full validation. In this study, we used Western blots to characterize antibodies targeted to the N or C terminal (NT or CT, respectively) and the second or third intracellular loop (IL2 or IL3, respectively) of the endothelin B receptor (ETB). The IL2-targeted antibody accurately detected endogenous ETB expression in rat brain and cultured rat astrocytes by labeling a 50-kDa band, the expected weight of full-length ETB. However, this antibody failed to detect transfected ETB in HEK293 cultures. In contrast, the NT-targeted antibody accurately detected endogenous ETB in rat astrocyte cultures and transfected ETB in HEK293 cultures by labeling a 37-kDa band but failed to detect endogenous ETB in rat brain. Bands detected by the CT- or IL3-targeted antibody were found to be unrelated to ETB. Our findings show that functional ETB can be detected at 50 or 37kDa on Western blot, with drastic differences in antibody affinity for these bands. The 37-kDa band likely reflects ETB processing, which appears to be dependent on cell type and/or culture condition. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Western blot analysis of sera from dogs with suspected food allergy.

    Science.gov (United States)

    Favrot, Claude; Linek, Monika; Fontaine, Jacques; Beco, Luc; Rostaher, Ana; Fischer, Nina; Couturier, Nicolas; Jacquenet, Sandrine; Bihain, Bernard E

    2017-04-01

    Food allergy is often suspected in dogs with clinical signs of atopic dermatitis. This diagnosis is confirmed with an elimination diet and a subsequent challenge with regular food. Laboratory tests for the diagnosis of food allergy in dogs are unreliable and/or technically difficult. Cyno-DIAL(®) is a Western blot method that might assist with the selection of an appropriate elimination diet. To evaluate the performance of Cyno-DIAL(®) for the selection of an elimination diet and diagnosis of food allergy. Thirty eight dogs with atopic dermatitis completed an elimination diet. Combining the results of the diet trials and the challenges, 14 dogs were classified as food allergic (FA), 22 as nonfood-allergic and two as ambiguous cases. Amongst all dogs and amongst dogs with a clinical diagnosis of FA, 3% and 7% (respectively) were positive to Royal Canin Anallergenic(®) , Vet-Concept Kanguru(®) or Vet-Concept Dog Sana(®) ; 8% and 7% to Hill's d/d Duck and Rice(®) ; 8% and 21% to Hill's z/d Ultra Allergen Free(®) ; 53% and 64% to Eukanuba Dermatosis FP(®) ; and 32% and 43% to a home-cooked diet of horse meat, potatoes and zucchini. The specificity and sensitivity of Cyno-DIAL(®) for diagnosing food allergy were 73% and 71%, respectively. Although Cyno-DIAL(®) was considered potentially useful for identifying appropriate foods for elimination diet trials, it cannot be recommended for the diagnosis of food allergy. The Cyno-DIAL(®) test performed better than some previously evaluated ELISA-based tests. © 2017 ESVD and ACVD.

  3. Evaluation of the Aspergillus Western blot IgG kit for diagnosis of chronic aspergillosis.

    Science.gov (United States)

    Oliva, A; Flori, P; Hennequin, C; Dubus, J-C; Reynaud-Gaubert, M; Charpin, D; Vergnon, J M; Gay, P; Colly, A; Piarroux, R; Pelloux, H; Ranque, S

    2015-01-01

    Immunoprecipitin detection (IPD) is the current reference confirmatory technique for anti-Aspergillus antibody detection; however, the lack of standardization is a critical drawback of this assay. In this study, we evaluated the performance of the Aspergillus Western blot (Asp-WB) IgG kit (LDBio Diagnostics, Lyon, France), a recently commercialized immunoblot assay for the diagnosis of various clinical presentations of chronic aspergillosis. Three hundred eight serum samples from 158 patients with aspergillosis sensu lato (s.l.) were analyzed. More specifically, 267 serum samples were derived from patients with Aspergillus disease, including 89 cases of chronic pulmonary aspergillosis, 10 of aspergilloma, and 32 of allergic bronchopulmonary aspergillosis, while 41 samples were from patients with Aspergillus colonization, including 15 cystic fibrosis (CF) and 12 non-CF patients. For blood donor controls, the Asp-WB specificity was 94%, while the kit displayed a sensitivity for the aspergillosis s.l. diagnosis of 88.6%, with a diagnostic odds ratio (DOR) of 119 (95% confidence interval [CI], 57 to 251). The DOR values were 185.22 (95% CI,78.79 to 435.45) and 43.74 (95% CI, 15.65 to 122.20) for the diagnosis of Aspergillus disease and Aspergillus colonization, respectively. Among the patients, the sensitivities of the Asp-WB in the diagnosis of Aspergillus colonization were 100% and 41.7% in CF and non-CF patients, respectively. The Asp-WB yielded fewer false-negative results than did IPD. In conclusion, the Asp-WB kit performed well for the diagnosis of various clinical presentations of aspergillosis in nonimmunocompromised patients, with an enhanced standardization and a higher sensitivity than with IPD, which is the current reference method.

  4. β-actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients.

    Science.gov (United States)

    Zhang, Rufang; Yang, Deyu; Zhou, Chanjuan; Cheng, Ke; Liu, Zhao; Chen, Liang; Fang, Liang; Xie, Peng

    2012-08-15

    Western blot analysis is a commonly used technique for determining specific protein levels in clinical samples. For normalization of protein levels in Western blot, a suitable loading control is required. On account of its relatively high and constant expression, β-actin has been widely employed in Western blot of cell cultures and tissue extracts. However, β-actin's presence in human plasma and this protein's putative role as a plasma-based loading control for Western blot analysis remain unknown. In this study, an enzyme-linked immunosorbent assay was used to determine the concentration of β-actin in human plasma, which is 6.29±0.54 ng/ml. In addition, the linearity of β-actin immunostaining and loaded protein amount was evaluated by Western blot, and a fine linearity (R²=0.974±0.012) was observed. Furthermore, the expression of plasma β-actin in major depressive disorder subjects and healthy controls was compared. The data revealed no statistically significant difference between these two groups. Moreover, the total coefficient of variation for β-actin expression in the two groups was 9.2±1.2%. These findings demonstrate that β-actin is present in human plasma and may possibly be used as a suitable loading control for plasma-based Western blot analysis in major depressive disorder. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Western blot assay for quantitative and qualitative antigen detection in vaccine development.

    Science.gov (United States)

    Kumar, Sanjai; Zheng, Hong; Mahajan, Babita; Kozakai, Yukiko; Morin, Merribeth; Locke, Emily

    2014-05-01

    Immunological methods for quantitative measurement, antigenic characterization, and monitoring the stability of active immunogenic component(s) are a critical need in the vaccine development process. This unit describes an enhanced chemiluminescence-based western blot for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP), a major malaria candidate vaccine antigen. The most salient features of this assay are its high sensitivity and reproducibility; it can reliably detect ∼5 to 10 pg PfCSP expressed on native parasites or recombinantly expressed in Escherichia coli. Although described for a specific vaccine antigen, this assay should be applicable for any antigen-antibody combination for which relevant detection reagents are available. Detailed stepwise experimental procedures and methods for data acquisition and analysis are described. Copyright © 2014 John Wiley & Sons, Inc.

  6. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein.

    Science.gov (United States)

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F; Nam, Ho-Woo

    2016-04-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.

  7. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein

    Science.gov (United States)

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F.; Nam, Ho-Woo

    2016-01-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection. PMID:27180586

  8. TSE strain differentiation in mice by immunohistochemical PrP(Sc) profiles and triplex Western blot.

    Science.gov (United States)

    van Keulen, Lucien J M; Langeveld, Jan P M; Dolstra, Corry H; Jacobs, Jorg; Bossers, Alex; van Zijderveld, Fred G

    2015-10-01

    TSE strains are routinely identified by their incubation period and vacuolation profile in the brain after intracerebral inoculation and serial passaging in inbred mouse lines. There are some major drawbacks to this method that are related to the variation in vacuolation that exists in the brains of mice infected with the same TSE strain and to variation between observers and laboratories in scoring vacuolation and determining the final incubation period. We investigated the potential of PrP(Sc) immunohistochemistry and triplex Western blotting as possible alternative methods to differentiate between TSE strains. TSE reference strains ME7, 87A/87V, 22A/22C, 79A/79V and 301C/301V were intracerebrally inoculated in RIII or VM inbred mice that differ in their PrP genotype. Immunohistochemical PrP(Sc) profiles were drawn up by scanning light microscopy both on coronal and sagittal sections. On the basis of the localization of PrP(Sc) in the cerebral cortex, hippocampus, and cerebellar cortex and the overall type of PrP(Sc) staining, all TSE strains could be well differentiated from each other through their typical strain dependent characteristics. In addition, Western blot showed that the combination of glycosylation profile and 12B2 epitope content of PrP(Sc) allowed to distinguish between all reference strains except for ME7 and 22A in VM mice. TSE strains in mice can be identified on the basis of their PrP(Sc) profile alone. The potential to identify TSE strains in ruminants with these PrP(Sc) profiles after a single primary passage in mice will be the topic of future studies. © 2014 British Neuropathological Society.

  9. Western blot diagnosis of vivax malaria with multiple stage-specific antigens of the parasite.

    Science.gov (United States)

    Son, E S; Kim, T S; Nam, H W

    2001-06-01

    Western blot analysis was performed to diagnose vivax malaria using stage-specific recombinant antigens. Genomic DNA from the whole blood of a malaria patient was used as templates to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1) of Plasmodium vivax. Each amplified DNA fragment was inserted into a pGEX-4T plasmid to induce the expression of GST fusion protein in Escherichia coli by IPTG. The bacterial cell extracts were separated on 10% SDS-PAGE followed by western blot analysis with patient sera which was confirmed by blood smear examination. When applied with patient sera, 147 (91.9%) out of 160 vivax malaria, 12 (92.3%) out of 13 falciparum malaria, and all 9 vivax/falciparum mixed malaria reacted with at least one antigen, while no reactions occurred with 20 normal uninfected sera. In the case of vivax malaria, CSP-1 reacted with 128 (80.0%) sera, MSP-1 with 102 (63.8%), AMA-1 with 128 (80.0%), SERA with 115 (71.9%), and EXP-1 with 89 (55.6%), respectively. We obtained higher detection rates when using 5 antigens (91.9%) rather than using each antigen solely (55.6-80%), a combination of 2 (76.3-87.5%), 3 (85.6-90.6%), or 4 antigens (89.4-91.3%). This method can be applied to serological diagnosis, mass screening in endemic regions, or safety test in transfusion of prevalent vivax malaria.

  10. Evaluation of the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test as an alternative to Western blot for confirmation of HIV infection.

    Science.gov (United States)

    Cárdenas, Ana María; Baughan, Eleonore; Hodinka, Richard L

    2013-12-01

    In the United States, a new HIV diagnostic algorithm has been proposed that uses an HIV-1/HIV-2 antibody differentiation immunoassay instead of Western blot or immunofluoresence for confirmatory testing. To evaluate the Multispot HIV-1/HIV-2 Rapid Test (Multispot) as an alternative to Western blot analysis for confirmation of HIV infection. A series of 205 serum and plasma specimens positive for HIV-1 or HIV-2 were used to compare the performance of Multispot to a standard HIV-1 Western blot. Positive samples included 63 specimens from patients>18 months of age, 33 proficiency survey specimens, and 109 specimens from nine commercial seroconversion and performance panels. In addition, 63 specimens from 51 HIV-exposed, uninfected children≤18 months of age in various stages of seroreversion and 192 HIV-negative samples were tested. Specimens were initially screened using a 4th generation HIV Ag/Ab Combo assay. Multispot readily discriminated between individuals with HIV-1 or HIV-2 infection and those who were uninfected. Of the 205 samples repeatedly reactive by the 4th generation screening assay, infection status was correctly confirmed by Multispot in 83.9% (172/205) compared to 68.8% (141/205) for Western blot. Multispot detected HIV-1 earlier in 27.6% of low-titer antibody specimens called indeterminate by Western blot, and effectively reduced the number of indeterminate results in seroreverting HIV-1 exposed, uninfected infants and for HIV-2 infections misinterpreted as indeterminate or positive by HIV-1 Western blot. Multispot offers speed and simplicity over Western blot and has an excellent performance for differentiation and confirmation of antibodies to HIV-1 and HIV-2. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Affinity and Western blotting reveal homologies between ovine intervertebral disc serine proteinase inhibitory proteins and bovine pancreatic trypsin inhibitor.

    Science.gov (United States)

    Melrose, J; Shen, B; Ghosh, P

    2001-12-01

    The objective of this study was to assess any similarities between ovine intervertebral disc (IVD) serine proteinase inhibitory proteins (SPIs) and known mammalian IVD SPIs. Ovine IVDs were dissected into the annulus fibrosus and nucleus pulposus and the tissue finely diced then extracted with 4 M guanidine hydrochloride. The tissue extracts were subjected to caesium chloride density gradient ultracentrifugation to separate the large high buoyant density (rho > 1.5 g/mL) proteoglycans from the SPI proteins of low buoyant density (rho top two ultracentrifuge fractions containing the SPIs of interest were subjected to enzyme linked immunosorbent analysis (ELISA) and also examined by Western and Affinity blotting using an antibody to bovine pancreatic trypsin inhibitor and biotinylated trypsin respectively for detection and an alkaline phosphatase 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium system for visualisation. The major SPI proteins present in the Western and Affinity blots were 34-36 kDa species, minor 12 and 16, and 85 and 120 kDa species were also present. Qualitatively similar results were obtained for each respective tissue zone of the lumbar and lumbosacral disc specimens examined. Densitometric analysis of the major 34-36 kDa SPI bands visualised on Western and Affinity blots using NIH 1.61.1 image analysis software indicated that lumbar IVD samples contained higher levels of this SPI species than lumbosacral IVD samples. ELISA confirmed that lumbar IVD extracts contained quantitatively higher levels of BPTI equivalents per g of tissue extracted than lumbosacral IVDs. This study therefore has demonstrated that the ovine disc contains a range of SPI species which share some homology with bovine pancreatic trypsin inhibitor and in this respect are similar to SPIs previously demonstrated in canine IVDs.

  12. Discrepancies between a new highly sensitive Toxoplasma gondii ELISA assay and other reagents: interest of Toxo IgG Western blot.

    Science.gov (United States)

    Leslé, F; Touafek, F; Fekkar, A; Mazier, D; Paris, L

    2011-10-01

    Immunodiagnostic assays are commonly used to screen for maternal toxoplasmic seroconversion during pregnancy. The introduction to the market of a new highly sensitive IgG assay, the Elecsys Toxo IgG test, has resulted in discrepancy issues with other immunoassays because of a lack of standardisation. Western blot appears to be a good alternative gold standard to the dye test, as the latter is not routinely available. For the present prospective study, we compared the analytical performances of two immunoassays, Elecsys Toxo IgG (Roche Diagnostics) and Platelia Toxo IgG (Bio-Rad, Marnes la Coquette, France), to Toxo II IgG Western blot (LDBio, Lyon, France) using 231 consecutive sera with low or equivocal IgG titres. Of these 231 sera, 213 presented discrepancies, which showed the importance of a confirmation test. Of the Elecsys Toxo IgG-positive results, 100% were confirmed by the Western blot with a positive threshold of 30 IU/ml for Elecsys; in the equivocal area (1-30 IU/ml), Western blot is negative in 54% of cases. Our results suggest that the lower diagnostic cut-off of Platelia Toxo IgG should be further reduced. Our study indirectly confirms that monitoring, especially for pregnant women, must be done in the same laboratory using the same technique. The ability to diagnose very early seroconversion using Western blot merits further study.

  13. Identification of α1-Antitrypsin as a Potential Candidate for Internal Control for Human Synovial Fluid in Western Blot.

    Science.gov (United States)

    Wang, Shaowei; Zhou, Jingming; Wei, Xiaochun; Li, Pengcui; Li, Kai; Wang, Dongming; Wei, Fangyuan; Zhang, Jianzhong; Wei, Lei

    Western blot of synovial fluid has been widely used for osteoarthritis (OA) research and diagnosis, but there is no ideal loading control for this purpose. Although β-actin is extensively used as loading control in western blot, it is not suitable for synovial fluid because it is not required in synovial fluid as a cytoskeletal protein. A good loading control for synovial fluid in OA studies should have unchanged content in synovial fluids from normal and OA groups, because synovial fluid protein content can vary with changes in synovial vascular permeability with OA onset. In this study, we explore the potential of using α1-antitripsin (A1AT) as loading control for OA synovial fluid in western blot. A1AT level is elevated in inflammatory conditions such as rheumatoid arthritis (RA). Unlike RA, OA is a non-inflammation disease, which does not induce A1AT. In this study, we identified A1AT as an abundant component of synovial fluid by Mass Spectrometry and confirmed that the level of A1AT is relative constant between human OA and normal synovial fluid by western blot and ELISA. Hence, we proposed that A1AT may be a good loading control for western blot in human OA synovial fluid studies provided that pathological conditions such as RA or A1AT deficiency associated liver or lung diseases are excluded.

  14. Analysis of Sperm Membrane Protein Relevant to Antisperm Antibody by Two-Dimensional Gel Electrophoresis and Western Blotting

    Institute of Scientific and Technical Information of China (English)

    Hao-fei WANG; Zhu-qiong XIANG; Yi-xing WANG

    2003-01-01

    Objective To identify the sperm membrane proteins that are associated with antisperm antibodyMethods Using antisperm antibody positive serum through unidimensional polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis followed by Western blot analysis to determine the molecular weights (MW) and isoelectric points (pI) of sperm membrane proteins that are associated with antisperm antibody.Results Eight kinds of MW with more than ten sperm membrane proteins can be recognized by antisperm antibody positive serum, of which the MWs and pI were 23 kD, 31 kD, 32 kD, 34 kD, 41 kD, 51 kD, 60 kD, 78 kD and 5.3, 5.5,5.7, 5.0, 5.3, 5.8, 6.0, 5.5~6.2, 4.6,5.1,5.5~5.8 respectively. The identification ratios of the sperm membrane proteins on 78 kD (60.7%), 60 kD (71.4%), 51 kD (14.9%) and 23 kD (14.29%) were higher.Conclusion The sperm membrane proteins with MW of 78 kD, 60 kD, 51 kD and 23 kD were associated with antisperm antibody and immunological infertility. Two-dimensional gel electrophoresis and Western blotting can precisely identify the sperm membrane proteins that are associated with antisperm antibody.

  15. Serological diagnosis of North American Paragonimiasis by Western blot using Paragonimus kellicotti adult worm antigen.

    Science.gov (United States)

    Fischer, Peter U; Curtis, Kurt C; Folk, Scott M; Wilkins, Patricia P; Marcos, Luis A; Weil, Gary J

    2013-06-01

    Abstract. We studied the value of an IgG Western blot (WB) with Paragonimus kellicotti (Pk) antigen for diagnosis of North American paragonimiasis. The test was evaluated with sera from patients with Pk and Paragonimus westermani infections, with control sera from patients with other helminth infections, and sera from healthy Americans. All 11 proven Pk infection sera and two samples from suspected cases that were negative by P. westermani WB at the Centers for Disease Control and Prevention (CDC) contained antibodies to antigens at 34 kDa and at 21/23 kDa. Seven of 7 P. westermani sera contained antibodies to the 34 kDa antigen, but only 2 recognized the 21/23 kDa doublet. No control samples were reactive with these antigens. Antibody reactivity declined after praziquantel treatment. Thus, the P. kellicotti WB appears to be superior to P. westermani WB for diagnosing Pk infections, and it may be useful for assessing responses to treatment.

  16. Characterization of excretory-secretory antigens of adult Toxocara canis by western blotting.

    Science.gov (United States)

    Sudhakar, N R; Samanta, S; Sahu, Shivani; Raina, O K; Gupta, S C; Goswami, T K; Lokesh, K M; Kumar, Ashok

    2014-06-01

    Toxocara canis is one of the most common helminth worm of dogs which continues to stimulate both public health concern alongside the higher scientific interest. It may cause visceral and ocular damage in humans especially in children. The identification of specific antigens of T. canis is important so as to develop better diagnostic techniques. Excretory-secretory (ES) antigens were prepared by culturing the adult T. canis worms in RPMI 1640 medium without serum supplementation followed by ammonium sulphate precipitation. These antigens were separated using sodium dodecyl sulphate-electrophoresis (SDS-PAGE). Recovered proteins ranged from 30 to 384 kDa. The specific reactivity of the T. canis excretory-secretory (TC-ES) proteins was checked by western blotting. The immuno-reactivity of the naturally infected dog sera with the TC-ES antigens showed five bands at 43, 57,105, 139 and 175 kDa. The immuno-reactivity of the hyper immune serum raised in rabbits against TC-ES antigens was observed with ten polypeptides of 21, 25, 30, 37, 45, 50, 57, 69, 77 and 105 kDa. Common antigens band were observed at 57 and 105 KDa. These antigens merit further evaluation as candidate for use in diagnosis of toxocariasis in humans and adult dogs.

  17. Enrichment of PrPSc in formalin-fixed, paraffin-embedded tissues prior to analysis by Western blot.

    Science.gov (United States)

    Nicholson, Eric M

    2011-07-01

    Diagnosis of prion disease is primarily through immunodetection of the infectious agent. Typically, 2 distinct procedures are recommended for a definitive diagnosis, with immunohistochemistry and Western blot providing the most information as to the specific isolate in question. In the past, these approaches required formalin-fixed, paraffin-embedded tissue and fresh or frozen tissue, respectively; however, methods have been developed that allow for use of fixed tissue for Western blot. The present study describes a method of enriching PrP(Sc) in formalin-fixed, paraffin-embedded tissues prior to Western blot analysis for the detection of PrP(Sc). With this modified procedure, 5 times the previously reported sample size may be used for analysis, greatly enhancing the sensitivity of this procedure.

  18. Qualitative and quantitative evaluation of Simon™, a new CE-based automated Western blot system as applied to vaccine development.

    Science.gov (United States)

    Rustandi, Richard R; Loughney, John W; Hamm, Melissa; Hamm, Christopher; Lancaster, Catherine; Mach, Anna; Ha, Sha

    2012-09-01

    Many CE-based technologies such as imaged capillary IEF, CE-SDS, CZE, and MEKC are well established for analyzing proteins, viruses, or other biomolecules such as polysaccharides. For example, imaged capillary isoelectric focusing (charge-based protein separation) and CE-SDS (size-based protein separation) are standard replacement methods in biopharmaceutical industries for tedious and labor intensive IEF and SDS-PAGE methods, respectively. Another important analytical tool for protein characterization is a Western blot, where after size-based separation in SDS-PAGE the proteins are transferred to a membrane and blotted with specific monoclonal or polyclonal antibodies. Western blotting analysis is applied in many areas such as biomarker research, therapeutic target identification, and vaccine development. Currently, the procedure is very manual, laborious, and time consuming. Here, we evaluate a new technology called Simple Western™ (or Simon™) for performing automated Western analysis. This new technology is based on CE-SDS where the separated proteins are attached to the wall of capillary by a proprietary photo activated chemical crosslink. Subsequent blotting is done automatically by incubating and washing the capillary with primary and secondary antibodies conjugated with horseradish peroxidase and detected with chemiluminescence. Typically, Western blots are not quantitative, hence we also evaluated the quantitative aspect of this new technology. We demonstrate that Simon™ can quantitate specific components in one of our vaccine candidates and it provides good reproducibility and intermediate precision with CV <10%. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Evaluation of a Western Blot Test in an Outbreak of Acute Pulmonary Histoplasmosis

    Science.gov (United States)

    Pizzini, Claudia V.; Zancopé-Oliveira, Rosely M.; Reiss, Errol; Hajjeh, Rana; Kaufman, Leo; Peralta, José Mauro

    1999-01-01

    A western blot (WB) test was evaluated for detection of antibodies against native glycosylated and chemically deglycosylated M and H antigens of Histoplasma capsulatum in serum obtained from patients during the acute phase of pulmonary histoplasmosis that occurred during an outbreak. Of 275 serum samples tested by immunodiffusion and complement fixation (CF) samples from 40 patients affected during this outbreak and from 37 negative controls were tested by WB test. A group of patients whose sera were negative for CF antibodies and precipitins early in the acute stage of histoplasmosis but who all seroconverted during convalescence 6 weeks later were tested with the WB test. Antibodies against untreated H and M antigens were detected at a 1:100 dilution by WB test in 45% of the 20 acute-phase serum samples and in all 20 of the convalescent-phase specimens. The WB test’s sensitivity for acute-phase specimens increased to 90% (18 of 20 specimens) when H and M antigens were treated by periodate oxidation to inactivate susceptible carbohydrate epitopes. When native glycosylated antigens were used in the WB test, positive reactions were observed in negative control serum specimens (3 of 37 specimens; 8%) and in serum specimens obtained from asymptomatic persons screened as part of the outbreak investigation (13 of 20 specimens; 65%). These positive reactions were also attributed to glycosidic epitopes since the specificity of the WB test increased from 78 to 100% when periodate-treated H and M antigens were used. WB test with deglycosylated H and M antigens of histoplasmin provides a rapid, sensitive, and specific test to diagnose acute pulmonary histoplasmosis before precipitins can be detected. PMID:9874658

  20. Electrostatic protein immobilization using charged polyacrylamide gels and cationic detergent microfluidic Western blotting.

    Science.gov (United States)

    Kim, Dohyun; Karns, Kelly; Tia, Samuel Q; He, Mei; Herr, Amy E

    2012-03-06

    We report a novel protein immobilization matrix for fully integrated microfluidic Western blotting (WB). The electrostatic immobilization gel (EIG) enables immobilization of all proteins sized using cetyl trimethylammonium bromide polyacrylamide gel electrophoresis (CTAB-PAGE), for subsequent electrophoretic probing with detection affinity reagents (e.g., labeled antibodies). The "pan-analyte" capture strategy introduced here uses polyacrylamide gel grafted with concentrated point charges (zwitterionic macromolecules), in contrast to existing microfluidic WB strategies that rely on a sandwich immunoassay format for analyte immobilization and detection. Sandwich approaches limit analyte immobilization to capture of only a priori known targets. A charge interaction mechanism study supports the hypothesis that electrostatic interaction plays a major role in analyte immobilization on the EIG. We note that protein capture efficiency depends on both the concentration of copolymerized charges and ionic strength of the gel buffer. We demonstrate pan-analyte immobilization of sized CTAB-laden model proteins (protein G, ovalbumin, bovine serum albumin, β-galactosidase, lactoferrin) on the EIG with initial capture efficiencies ranging from 21 to 100%. Target proteins fixed on the EIG (protein G, lactoferrin) are detected using antibody probes with signal-to-noise ratios of 34 to 275. The approach advances protein immunoblotting performance through 200× reduction on sample consumption, 12× reduction in assay duration, and automated assay operation, compared to slab-gel WB. Using the microfluidic WB assay, assessment of lactoferrin in human tear fluid is demonstrated with a goal of advancing toward nonbiopsy-based diagnosis of Sjögren's Syndrome, an autoimmune disease.

  1. Evaluation of immunodominant proteins of Mycobacterium avium paratuberculosis cell wall by Western blot analysis.

    Science.gov (United States)

    Hashemi, Maryam; Madani, Rasool; Razmi, Nematollah

    2014-04-01

    Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow growing mycobactin, whose dependence on mycobacterial species is known to be the causative agent of Johne's disease (paratuberculosis) in all species of domestic ruminants worldwide. The organism is transmitted via close contact, ingestion, or transplacentally from mother to fetus and occurs commonly in grazing domestic animals. Johne's disease (JD) is characterized by gradual weight loss, decreased milk production, and diarrhea due to the chronic, progressive, granulomatous enteritis and lymphadenitis. The disease can cause serious economic damage to the dairy industry due to the loss of milk production and early culling of infected animals. In recent years, researchers have focused on the identification of a specific antigen of M. paratuberculosis to use in diagnosis test and preparation of effective vaccine. The goal of this study is evaluation of the immunodominant proteins of M. paratuberculosis cell wall. The amount of protein was determined with a Lowry assay (22.68 μg/100 μL). For production of polyclonal antibody against proteins of M. paratuberculosis cell wall, a New Zealand white rabbit was immunized with antigen and Freund's adjuvant. After immunization, the rabbit was bled to produce enriched serum. Antibodies were purified from serum with ion exchange chromatography. In the Ouchterlony test, the reactions between antigen and antibodies were seen in dilutions of one quarter for serum, one quarter for Ig, and one half for IgG by clear precipitation lines due to the well immunization of the rabbit. Electrophoresis and Western blot analysis were used and subsequently a sharp band appeared in nitrocellulose paper; these bands were about 25, 37, 50, 75, and 150 kDa molecular weight, which indicated immunodominant proteins.

  2. Lectin staining and Western blot data showing differential sialylation of nutrient-deprived cancer cells to sialic acid supplementation.

    Science.gov (United States)

    Badr, Haitham A; AlSadek, Dina M M; Mathew, Mohit P; Li, Chen-Zhong; Djansugurova, Leyla B; Yarema, Kevin J; Ahmed, Hafiz

    2015-12-01

    This report provides data that are specifically related to the differential sialylation of nutrient deprived breast cancer cells to sialic acid supplementation in support of the research article entitled, "Nutrient-deprived cancer cells preferentially use sialic acid to maintain cell surface glycosylation" [1]. Particularly, breast cancer cells, when supplemented with sialic acid under nutrient deprivation, display sialylated glycans at the cell surface, but non-malignant mammary cells show sialylated glycans intracellularly. The impact of sialic acid supplementation under nutrient deprivation was demonstrated by measuring levels of expression and sialylation of two markers, EGFR1 and MUC1. This Data in Brief article complements the main manuscript by providing detailed instructions and representative results for cell-level imaging and Western blot analyses of changes in sialylation during nutrient deprivation and sialic acid supplementation. These methods can be readily generalized for the study of many types of glycosylation and various glycoprotein markers through the appropriate selection of fluorescently-labeled lectins.

  3. Enrichment of PrPSc in Formalin Fixed Paraffin Embedded Tissues Prior to Analysis by Western Blot

    Science.gov (United States)

    Diagnosis of prion disease is primarily through immunodetection of the infectious agent. Typically, 2 distinct procedures are recommended for a definitive diagnosis with immunohistochemistry and Western blot providing the most information as to the specific isolate in question. In the past these app...

  4. False-Positive Results in a Recombinant Severe Acute Respiratory Syndrome-Associated Coronavirus (SARS-CoV) Nucleocapsid-Based Western Blot Assay Were Rectified by the Use of Two Subunits (S1 and S2) of Spike for Detection of Antibody to SARS-CoV

    Science.gov (United States)

    Maache, Mimoun; Komurian-Pradel, Florence; Rajoharison, Alain; Perret, Magali; Berland, Jean-Luc; Pouzol, Stéphane; Bagnaud, Audrey; Duverger, Blandine; Xu, Jianguo; Osuna, Antonio; Paranhos-Baccalà, Glaucia

    2006-01-01

    To evaluate the reactivity of the recombinant proteins expressed in Escherichia coli strain BL21(DE3), a Western blot assay was performed by using a panel of 78 serum samples obtained, respectively, from convalescent-phase patients infected with severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (30 samples) and from healthy donors (48 samples). As antigen for detection of SARS-CoV, the nucleocapsid protein (N) showed high sensitivity and strong reactivity with all samples from SARS-CoV patients and cross-reacted with all serum samples from healthy subjects, with either those obtained from China (10 samples) or those obtained from France (38 serum samples), giving then a significant rate of false positives. Specifically, our data indicated that the two subunits, S1 (residues 14 to 760) and S2 (residues 761 to 1190), resulted from the divided spike reacted with all samples from SARS-CoV patients and without any cross-reactivity with any of the healthy serum samples. Consequently, these data revealed the nonspecific nature of N protein in serodiagnosis of SARS-CoV compared with the S1 and S2, where the specificity is of 100%. Moreover, the reported results indicated that the use of one single protein as a detection antigen of SARS-CoV infection may lead to false-positive diagnosis. These may be rectified by using more than one protein for the serodiagnosis of SARS-CoV. PMID:16522785

  5. MDR-TB Antibody Response (Western Blot) to Fractions of Isoniazid and Rifampicin Resistant Antigens of Mycobacterium tuberculosis.

    Science.gov (United States)

    Hadizadeh Tasbiti, Alireza; Yari, Shamsi; Ghanei, Mostafa; Shokrgozar, Mohammad Ali; Bahrmand, Ahmadreza

    2015-12-01

    Drug-resistant TB poses a major threat to control of TB worldwide. Despite progress in the detection of Multidrug-resistant TB (MDR-TB) cases, a major diagnostic gap remains: 55% of reported TB patients estimated to have MDR-TB were not detected in 2013. MDR-TB antigens were conjugated to CNBr-activated Sepharose 4B. Specific polyclonal antibodies against MDR-TB Ags were prepared in rabbits using two boosted injections of the MDR-TB antigen. The antibodies were purified and treated with susceptible TB to remove any non-specific and cross-reactive antibodies. In the present study, comparative analysis of electrophoretic pattern of different antigens of INH/RIF-resistant TB were studied for identifying protein profiles. A RIF-resistant TB antigen was shown here to have different protein profiles from INH-resistant TB isolate. The results of Western blotting analysis showed that in the RIF- and INH-resistant antigenic fractions some bands of 14.4 and 45 kDa as immunogenic were common. Moreover, four bands of RIF-resistant TB antigen fractions (16, 19, 21, and 45 KDa) and one band of INH-resistant TB (about 26 KDa) were detected as diagnostic antigens. This study suggests that the Western blot is an accurate test to survey INH- and RIF-resistant TB antigens of M. tuberculosis infection. These findings indicate that MDR-TB diagnosis (based on Ag detection) could be useful in the identification of disease stages that precede symptomatic and microbiologically positive TB, such as subclinical and incipient TB.

  6. Applications of an Automated and Quantitative CE-Based Size and Charge Western Blot for Therapeutic Proteins and Vaccines.

    Science.gov (United States)

    Rustandi, Richard R; Hamm, Melissa; Lancaster, Catherine; Loughney, John W

    2016-01-01

    Capillary Electrophoresis (CE) is a versatile and indispensable analytical tool that can be applied to characterize proteins. In recent years, labor-intensive SDS-PAGE and IEF slab gels have been replaced with CE-SDS (CGE) and CE-IEF methods, respectively, in the biopharmaceutical industry. These two CE-based methods are now an industry standard and are an expectation of the regulatory agencies for biologics characterization. Another important and traditional slab gel technique is the western blot, which detects proteins using immuno-specific reagents after SDS-PAGE separation. This technique is widely used across industrial and academic laboratories, but it is very laborious, manual, time-consuming, and only semi-quantitative. Here, we describe the applications of a relatively new CE-based western blot technology which is automated, fast, and quantitative. We have used this technology for both charge- and size-based CE westerns to analyze biotherapeutic and vaccine products. The size-based capillary western can be used for fast antibody screening, clone selection, product titer, identity, and degradation while the charge-based capillary western can be used to study product charge heterogeneity. Examples using this technology for monoclonal antibody (mAb), Enbrel, CRM197, and Clostridium difficile (C. difficile) vaccine proteins are presented here to demonstrate the utility of the capillary western techniques. Details of sample preparation and experimental conditions for each capillary western mode are described in this chapter.

  7. Analysis of Gene and Protein Expression in Atherosclerotic Mouse Aorta by Western Blot and Quantitative Real-Time PCR.

    Science.gov (United States)

    Rivera-Torres, José

    2015-01-01

    Atherosclerosis involves changes in gene and protein expression patterns in affected arteries. Quantification of these alterations is essential for understanding the molecular mechanisms underlying this pathology. Western blot and real-time PCR-used to quantify protein and messenger RNA levels, respectively-are invaluable molecular biology tools, particularly when material is limited. The availability of many genetically modified mouse models of atherosclerosis makes the mouse aorta an ideal tissue in which to carry out these expression pattern analyses. In this chapter, protocols are presented for mRNA and protein extraction from mouse aorta and for the accurate quantification of mRNA expression by RT-PCR and of proteins by western blot.

  8. Western blot confirmation of the H+/K+-ATPase proton pump in the human larynx and submandibular gland.

    Science.gov (United States)

    Altman, Kenneth W; Kinoshita, Yayoi; Tan, Melin; Burstein, David; Radosevich, James A

    2011-11-01

    The authors have previously demonstrated the H(+)/K(+)-ATPase (proton pump) in human larynx and lung glands via immunohistochemistry (IHC). The present hypothesis is that the proton pump is expressed in other seromucinous glands of the digestive tract that can be confirmed by IHC and Western blot analysis. Prospective controlled tissue analysis study. Academic medical institution. Ten anonymous fresh-frozen donor specimens were obtained, comprising 3 submandibular glands, 4 larynges, and 3 normal stomach specimens for control. Submandibular gland sections were immunostained with 2 monoclonal antibodies selectively reactive with α or β subunits of the H(+)/K(+)-ATPase. Western blot analysis was performed on all specimens. Consistent IHC staining was observed in the submandibular gland specimens for both α and β subunits. Western blot analysis revealed very strong expression for the stomach at 100 kDa, corresponding to the α protein, and weak but notable banding for all larynx and submandibular gland specimens. Similar findings were noted for the 60- to 80-kDa glycosylated β subunit protein, as well as the 52-kDa β subunit precursor for all specimens. The H(+)/K(+)-ATPase (proton) pump is present in the human larynx and submandibular gland although in much lower concentrations than in the stomach. Proton pump involvement in human aerodigestive seromucinous glands may have a role in protecting mucosa from acid environments (local or systemic), explain heightened laryngeal sensitivity in those patients with laryngopharyngeal reflux, and be a site of action for proton pump inhibitor pharmacotherapy.

  9. Investigation of Anti-Toxocara Antibodies in Epileptic Patients and Comparison of Two Methods: ELISA and Western Blotting

    Directory of Open Access Journals (Sweden)

    Mohammad Zibaei

    2013-01-01

    Full Text Available The relationship between Toxocara infection and epilepsy was previously demonstrated by several case-control studies and case reports. These previous studies were often based on the enzyme-linked immunosorbent assay (ELISA using Toxocara excretory-secretory antigens, which are not specific due to cross-reactivity with other parasitic infections such as ascariasis, trichuriasis, and anisakiasis. An immunoblot analysis is highly specific and can detect low levels of Toxocara antibodies. Therefore, this assay may be useful in the identification of toxocariasis in epileptic patients. We examined patients who had epilepsy and healthy subjects for seropositivity for Toxocara infection by ELISA and Western blotting. Out of 85 epileptic patients, 10 (11.8% and 3 (3.5% persons exhibited Toxocara immunoglobulin G (IgG antibodies responses by ELISA and by both techniques, respectively. Moreover, in the healthy group (, 3 (3.5% persons were positive by ELISA, but none was detected by Western blotting. This study indicates that Toxocara infection is a risk factor for epilepsy in Iran. These findings strongly suggest the need to perform Western blotting immunodiagnosis, as well as the ELISA using Toxocara excretory-secretory antigens, to improve diagnosis of human toxocariasis in patients with epilepsy.

  10. Western blotting using Strongyloides ratti antigen for the detection of IgG antibodies as confirmatory test in human strongyloidiasis

    Directory of Open Access Journals (Sweden)

    Luciana Pereira Silva

    2003-07-01

    Full Text Available The present study was conducted to evaluate the frequency of antigenic components recognized by serum IgG antibodies in Western blotting (WB using a Strongyloides ratti larval extract for the diagnosis of human strongyloidiasis. In addition, the WB results were compared to the enzyme-linked immunosorbent assay (ELISA and the indirect immunofluorescence antibody test (IFAT results. Serum samples of 180 individuals were analyzed (80 with strongyloidiasis, 60 with other intestinal parasitoses, and 40 healthy individuals. S. ratti was obtained from fecal culture of experimentally infected Rattus rattus. For IFAT, S. ratti larvae were used as antigen and S. ratti larval antigenic extracts were employed in WB and ELISA. Eleven S. ratti antigenic components were predominantly recognized by IgG antibodies in sera of patients with strongyloidiasis. There was a positive concordance for the three tests in 87.5% of the cases of strongyloidiasis. The negative concordance in the three tests was 94% and 97.5%, in patients with other intestinal parasitoses and healthy individuals, respectively. In cases of positive ELISA and negative IFAT results, diagnosis could be confirmed by WB. ELISA, IFAT, and WB using S. ratti antigens showed a high rate of sensitivity and specificity. In conclusion, WB using S. ratti larval extract was able to recognize 11 immunodominant antigenic components, showing to be a useful tool to define the diagnosis in cases of equivocal serology.

  11. [Optimization of the immunoelectrophoresis technic (western blot) for the confirmation of human immunodeficiency virus infection (HIV) in Panama].

    Science.gov (United States)

    Pascale, J M; de Austin, E; de Moreno, N O; Ledezma, C; de Márquez, E; Blanco, R; Quiroz, E; Calzada, J; Vincensini, A R; de Martin, M C

    1995-01-01

    The purpose of this study is to report the results of the authors' investigation to apply the western blot technique (WB UP-LCS) in the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection. To do this, the authors separated the proteins of the HIV-1 virus by electrophoresis, based on their molecular weight, in poliacilamide gel with SDS (SDS-PAGE) during 3 hours at 200 volts. Then they electrotransferred these proteins to nitrocellulose paper during four hours at 200 milliamperes, with the aid of external cooling. The nitrocellulose strips were evaluated considering the incubation time (1 and 16 hours), two conjugates (human anti IgG with Peroxidase and human anti IgG Biotin plus Streptatividine with Peroxidase) and two dilutions of the patients' sera (1/50 and 1/100). Based on their results the Authors conclude that, in the first place, the optimal conditions for the test include a dilution of 1/100 of the patients serum, incubation of the serum for 16 hours and the use of the conjugate of anti human IgG with Biotin and Streptavidine with Peroxidase; secondary, that the immunologic reactivity against proteins p24 and gp 160/120 is the most important diagnostic criterion for the confirmation of infection with HIV-1 and that they obtained a diagnostic correlation of 100% at a cost which was 5 to 7 times less than that of the commercial system.

  12. Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development.

    Science.gov (United States)

    Rocha-Martins, Maurício; Njaine, Brian; Silveira, Mariana S

    2012-01-01

    Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.

  13. Exposure to Sarcocystis spp. in horses from Spain determined by Western blot analysis using Sarcocystis neurona merozoites as heterologous antigen.

    Science.gov (United States)

    Arias, M; Yeargan, M; Francisco, I; Dangoudoubiyam, S; Becerra, P; Francisco, R; Sánchez-Andrade, R; Paz-Silva, A; Howe, D K

    2012-04-30

    Horses serve as an intermediate host for several species of Sarcocystis, all of which utilize canids as the definitive host. Sarcocystis spp. infection and formation of latent sarcocysts in horses often appears to be subclinical, but morbidity can occur, especially when the parasite burden is large. A serological survey was conducted to determine the presence of antibodies against Sarcocystis spp. in seemingly healthy horses from the Galicia region of Spain. Western blot analyses using Sarcocystis neurona merozoites as heterologous antigen suggested greater than 80% seroprevalance of Sarcocystis spp. in a sample set of 138 horses. The serum samples were further tested with enzyme-linked immunosorbent assays (ELISAs) based on recombinant S. neurona-specific surface antigens (rSnSAGs). As expected for horses from the Eastern Hemisphere, less than 4% of the serum samples were positive when analyzed with either the rSnSAG2 or the rSnSAG4/3 ELISAs. An additional 246 horses were tested using the rSnSAG2 ELISA, which revealed that less than 3% of the 384 samples were seropositive. Collectively, the results of this serologic study suggested that a large proportion of horses from this region of Spain are exposed to Sarcocystis spp. Furthermore, the anti-Sarcocystis seroreactivity in these European horses could be clearly distinguished from anti-S. neurona antibodies using the rSnSAG2 and rSnSAG4/3 ELISAs. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Lectin staining and Western blot data showing differential sialylation of nutrient-deprived cancer cells to sialic acid supplementation

    Directory of Open Access Journals (Sweden)

    Haitham A. Badr

    2015-12-01

    Full Text Available This report provides data that are specifically related to the differential sialylation of nutrient deprived breast cancer cells to sialic acid supplementation in support of the research article entitled, “Nutrient-deprived cancer cells preferentially use sialic acid to maintain cell surface glycosylation" [1]. Particularly, breast cancer cells, when supplemented with sialic acid under nutrient deprivation, display sialylated glycans at the cell surface, but non-malignant mammary cells show sialylated glycans intracellularly. The impact of sialic acid supplementation under nutrient deprivation was demonstrated by measuring levels of expression and sialylation of two markers, EGFR1 and MUC1. This Data in Brief article complements the main manuscript by providing detailed instructions and representative results for cell-level imaging and Western blot analyses of changes in sialylation during nutrient deprivation and sialic acid supplementation. These methods can be readily generalized for the study of many types of glycosylation and various glycoprotein markers through the appropriate selection of fluorescently-labeled lectins.

  15. Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

    Directory of Open Access Journals (Sweden)

    NUNES Cáris Maroni

    1997-01-01

    Full Text Available Visceral larva migrans (VLM is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA using the larval excretory-secretory antigen of T. canis (TES, the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa. Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

  16. Mycoplasma agassizii strain variation and distinct host antibody responses explain differences between enzyme-linked immunosorbent assays and Western blot assays.

    Science.gov (United States)

    Wendland, Lori D; Klein, Paul A; Jacobson, Elliott R; Brown, Mary B

    2010-11-01

    The precarious status of desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises has resulted in conservation efforts that now include health assessment as an important component of management decision-making. Mycoplasmal upper respiratory tract disease (URTD) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. In this study, serum samples obtained from eight Gopherus tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with Mycoplasma agassizii as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of mycoplasmal URTD were used to evaluate four distinct clinical isolates of M. agassizii as antigens for ELISA and Western blot analyses. Each animal sample reacted in the Western blot with its homologous M. agassizii strain, but recognition of heterologous M. agassizii strains was variable. Further, individual animals varied significantly with respect to the specific proteins recognized by the humoral immune response. An additional 114 Gopherus serum samples were evaluated using ELISA antigens prepared from the four distinct M. agassizii strains; A₄₀₅ values were significantly correlated (r² goodness of fit range, 0.708 to 0.771; P Western blot binding patterns. Thus, reliance on a single M. agassizii strain as an antigen in Western blot assays may provide false-negative results. This could have adverse consequences for the well-being of these environmentally sensitive hosts if false-negative animals were relocated to sites consisting of true-negative populations.

  17. Expansion of HIV screening to non-clinical venues is aided by the use of dried blood spots for Western blot confirmation.

    Science.gov (United States)

    Sullivan, Timothy J; Antonio-Gaddy, Mara San; Richardson-Moore, April; Styer, Linda M; Bigelow-Saulsbery, Deborah; Parker, Monica M

    2013-12-01

    HIV rapid testing programs in New York State (NYS) are required to collect a specimen for confirmation of a preliminary positive result; however, some venues have limited capacity to collect venous blood, and confirmation using oral fluid is restricted by cost and availability. To evaluate the feasibility of using dried blood spots (DBS) at non-clinical HIV rapid testing sites for Western blot testing. The New York State Department of Health facilitated registration of 48 non-clinical HIV test sites and provided training on DBS procedures. Following a reactive rapid test, DBS were collected by fingerstick onto filter paper cards, dried and mailed to the NYS public health laboratory for Western blot testing. From October 2010 to December 2012, 280 DBS specimens were submitted for confirmation. Four (1.4%) were unsatisfactory for testing and 276 (98.6%) DBS were tested. Of these, 235 (85.1%) were positive, 37 (13.4%) were negative and 4 (1.4%) were indeterminate. During this period, the laboratory also received 1033 venous blood specimens for rapid test confirmation, and 35 (3.4%) were unsatisfactory. Of the 998 tested by Western blot, 784 (78.6%) were positive, 197 (19.7%) were negative and 17 (1.7%) were indeterminate. Compared to venous blood, the percentage of rapid test referral specimens with a positive Western blot was significantly greater for DBS specimens and the frequency of unsatisfactory specimens did not differ significantly. These results indicate that DBS are a suitable alternative to venous blood for confirmation of HIV rapid tests conducted at non-clinical sites. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Standard loading controls are not reliable for Western blot quantification across brain development or in pathological conditions.

    Science.gov (United States)

    Goasdoue, Kate; Awabdy, Doreen; Bjorkman, Stella Tracey; Miller, Stephanie

    2016-02-01

    A frequently utilized method of data quantification in Western blot analysis is comparison of the protein of interest with a house keeping gene or control protein. Commonly used proteins include β-actin, glyceraldehyde 3 phosphate dehydrogenase (GAPDH), and α-tubulin. Various reliability issues have been raised when using this technique for data analysis-particularly when investigating protein expression changes during development and in disease states. In this study, we have demonstrated that β-actin, GAPDH, and α-tubulin are not appropriate controls in the study of development and hypoxic-ischemic induced damage in the piglet brain. We have also shown that using an in-house pooled standard, loaded on all blots is a reliable method for controlling interassay variability and data normalization in protein expression analysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Western blot analysis of a limited number of cells: a valuable adjunct to proteome analysis of paraffin wax-embedded, alcohol-fixed tissue after laser capture microdissection.

    Science.gov (United States)

    Martinet, Wim; Abbeloos, Vanessa; Van Acker, Nathalie; De Meyer, Guido R Y; Herman, Arnold G; Kockx, Mark M

    2004-03-01

    In recent years, laser capture microdissection (LCM) has been used successfully to obtain distinct populations of cells for subsequent molecular analysis. Because of the limited sample availability and the absence of in vitro amplification steps for proteins, the use of LCM for proteome analysis largely depends on highly sensitive protein detection methods. In this study, a western blot protocol was developed and validated for the detection of beta-actin and the moderately expressed cell death protein caspase-3 in small numbers of cells. Initially, cultured human U937 monocytes and whole sections of paraffin wax-embedded, alcohol-fixed human tonsils were used to optimize protein electrophoresis and western blotting conditions. High-performance NuPAGE Bis-Tris gels in combination with high-quality transfer membranes, optimized antibody concentrations, and a sensitive chemiluminescent substrate provided a strong signal for beta-actin with approximately 500 U937 cells. In the same way, procaspase-3 could be identified with approximately 1000 cells. Similar results were obtained with germinal centre cells that were procured from paraffin wax-embedded, alcohol-fixed human tonsils by LCM. Treatment of U937 cells with etoposide rapidly induced cell death and allowed the detection of active caspase-3 with approximately 2500 cells (0.8 pg of protein). The findings of this study suggest that western blotting is a valuable adjunct to proteome analysis of LCM procured cells.

  20. Transcription Factors Downstream of IL-4 and TGF-β Signals: Analysis by Quantitative PCR, Western Blot, and Flow Cytometry.

    Science.gov (United States)

    Sugimoto, Atsushi; Kawakami, Ryoji; Mikami, Norihisa

    2017-01-01

    IL-9-producing Th9 cell is a novel Th cell subset involved in type II allergic inflammations such as asthma. Th9 cells can be induced from naïve Th cells in the presence of IL-4 and TGF-β. It is also well established that downstream signals of IL-4 and TGF-β, including STAT6, IRF4, Smad, and PU.1, directly mediate IL-9 production in Th9 cells. In this chapter we describe the methods of flow cytometry, qPCR and western blot analysis to determine the expression or activation of these transcription factors downstream of IL-4 and TGF-β.

  1. How to Distinguish Between the Activity of HDAC1-3 and HDAC6 with Western Blot.

    Science.gov (United States)

    Beyer, Mandy; Kiweler, Nicole; Mahboobi, Siavosh; Krämer, Oliver H

    2017-01-01

    Histone deacetylases (HDACs) catalyze the deacetylation of lysine residues in their target proteins. This biochemical modification can have profound effects on the functions of these proteins and a dysregulation of HDAC activity contributes to severe diseases, including neoplastic transformation. In the following chapter, we present a strategy that allows to distinguish between the inhibition of the class I HDACs HDAC1, 2, and 3 and of the class IIb HDAC HDAC6. This method is based on Western blot and relies on the detection of hyperacetylated substrates of class I or class IIb HDACs in lysates from cells that were treated with histone deacetylase inhibitors (HDACi).

  2. Evaluation of western blotting for the diagnosis of enzootic bovine leukemia Avaliação da técnica de western blot no diagnóstico da leucose enzoótica bovina

    Directory of Open Access Journals (Sweden)

    E.T. Gonzalez

    1999-08-01

    Full Text Available A western blotting (WB procedure has been developed for detecting antibodies to bovine leukosis virus (BLV in cattle sera. Two hundred and thirty three serum samples from naturally infected cattle with BLV virus and serial bleedings from experimentally BLV infected cows were used. An agar gel immunodiffusion test (AGID was used for comparing with the results obtained by WB. The AGID positive sera showed a different degree of reactivity by WB test against the two most important viral antigens (gp51 and p24, or against one of them. Other proteins (gp30, p15, p12 and p10 were not detected with any AGID positive sera, being observed occasionally three bands corresponding to the p24 protein. Using sera obtained by BLV experimental inoculation, the antibodies directed to p24 appeared early (between the 2nd and 4th week post inoculation and thereafter antibodies to gp51were detected in some animals. The analysis of field serum samples by AGID as compared to WB showed an agreement of 90.9%. Only 1.7% of sera were negative by AGID and positive by WB and 7.2% that were not conclusive by AGID and were defined by WB (4.2% as positive and 3.0% as negative.Um sistema de western blotting (WB foi desenvolvido para detecção de anticorpos contra o vírus da leucose em soros de bovinos. Foram utilizadas amostras de soros de 233 animais naturalmente infectados e soros de vacas experimentalmente infectadas. O teste de imunodifusão em ágar (AGID foi usado para comparação dos resultados. Graus diferentes de reatividade foram observados em soros positivos ao AGID, quando testados em WB frente a um ou aos dois antígenos mais importantes (gp51 e p24. Outras proteínas (gp30, p15, p12 e p10 não foram detectadas por nenhum soro positivo ao AGID, sendo que três bandas correspondentes à proteína p24 foram observadas ocasionalmente. Em soros obtidos por inoculação experimental, anticorpos contra a proteína p24 foram detectados entre a segunda e a quarta semanas

  3. Evaluation of ELISA coupled with Western blot as a surveillance tool for Trichinella infection in wild boar (Sus scrofa).

    Science.gov (United States)

    Cuttell, Leigh; Gómez-Morales, Maria Angeles; Cookson, Beth; Adams, Peter J; Reid, Simon A; Vanderlinde, Paul B; Jackson, Louise A; Gray, C; Traub, Rebecca J

    2014-01-31

    Trichinella surveillance in wildlife relies on muscle digestion of large samples which are logistically difficult to store and transport in remote and tropical regions as well as labour-intensive to process. Serological methods such as enzyme-linked immunosorbent assays (ELISAs) offer rapid, cost-effective alternatives for surveillance but should be paired with additional tests because of the high false-positive rates encountered in wildlife. We investigated the utility of ELISAs coupled with Western blot (WB) in providing evidence of Trichinella exposure or infection in wild boar. Serum samples were collected from 673 wild boar from a high- and low-risk region for Trichinella introduction within mainland Australia, which is considered Trichinella-free. Sera were examined using both an 'in-house' and a commercially available indirect-ELISA that used excretory-secretory (E/S) antigens. Cut-off values for positive results were determined using sera from the low-risk population. All wild boar from the high-risk region (352) and 139/321 (43.3%) of the wild boar from the low-risk region were tested by artificial digestion. Testing by Western blot using E/S antigens, and a Trichinella-specific real-time PCR was also carried out on all ELISA-positive samples. The two ELISAs correctly classified all positive controls as well as one naturally infected wild boar from Gabba Island in the Torres Strait. In both the high- and low-risk populations, the ELISA results showed substantial agreement (k-value=0.66) that increased to very good (k-value=0.82) when WB-positive only samples were compared. The results of testing sera collected from the Australian mainland showed the Trichinella seroprevalence was 3.5% (95% C.I. 0.0-8.0) and 2.3% (95% C.I. 0.0-5.6) using the in-house and commercial ELISA coupled with WB respectively. These estimates were significantly higher (P<0.05) than the artificial digestion estimate of 0.0% (95% C.I. 0.0-1.1). Real-time PCR testing of muscle from

  4. Estrogen receptors alpha and beta in rat placenta: detection by RT-PCR, real time PCR and Western blotting

    Directory of Open Access Journals (Sweden)

    Al-Bader Maie D

    2006-03-01

    Full Text Available Abstract Background High levels of estrogens during pregnancy not only retard placental and fetal growth but can lead to reproductive tract abnormalities in male progeny. Estrogens act through estrogen receptors (ER to modulate the transcription of target genes. These ER exist in two isoforms, ER alpha and ER beta and recently several variants of these isoforms have been identified. Methods The expressions of ER isoforms and variants have been studied in rat placenta at 16, 19 and 21 days gestation (dg. Gene expression was assessed using RT-PCR and real time PCR while protein expression was studied using Western blotting followed by immunodetection. Placental homogenates were probed with: a monoclonal antibody raised against the steroid binding domain of the ER alpha (ER alpha -S, a monoclonal antibody raised against the hinge region of ER alpha (ER alpha -H and a polyclonal antibody raised against the amino terminus of ER beta. Results ER alpha and ER beta mRNA and protein were detected from as early as 16 dg. Two PCR products were detected for ER alpha, one for the wild type ER alpha, and a smaller variant. Real time PCR results suggested the presence of a single product for ER beta. The antibodies used for detection of ER alpha protein both identified a single 67 kDa isoform; however a second 54 kDa band, which may be an ER alpha variant, was identified when using the ER alpha -H antibody. The abundance of both ER alpha bands decreased significantly between 16 and 19 dg. As for ER beta, four bands (76, 59, 54 and 41 kDa were detected. The abundance of the 59 and 54 kDa bands decreased significantly between 16 and 19 dg. Conclusion This study shows that both ER protein isoforms and their variants are present in rat placenta. The decrease in their expression near parturition suggests that the placenta may be relatively unresponsive to estrogens at this stage.

  5. The combination of quantitative PCR and western blot detecting CP4-EPSPS component in Roundup Ready soy plant tissues and commercial soy-related foodstuffs.

    Science.gov (United States)

    Xiao, Xiao; Wu, Honghong; Zhou, Xinghu; Xu, Sheng; He, Jian; Shen, Wenbiao; Zhou, Guanghong; Huang, Ming

    2012-06-01

    With the widespread use of Roundup Ready soy (event 40-3-2) (RRS), the comprehensive detection of genetically modified component in foodstuffs is of significant interest, but few protein-based approaches have been found useful in processed foods. In this report, the combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different RRS plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing both meat and plant protein concentrates. The validity of the 2 methods was confirmed first. We also showed that the CP4-EPSPS protein existed in different RRS plant tissues. In certain cases, the results from the western blot and the qPCR were not consistent. To be specific, at least 2 degraded fragments of CP4-EPSPS protein (35.5 and 24.6 kDa) were observed. For dried bean curd crust and deep-fried bean curd, a degraded protein fragment with the size of 24.6 kDa appeared, while cp4-epsps gene could not be traced by qPCR. In contrast, we found a signal of cp4-epsps DNA in 3 foodstuffs, including soy-containing ham cutlet product, meat ball, and sausage by qPCR, while CP4-EPSPS protein could not be detected by western blot in such samples. Our study therefore concluded that the combination of DNA- and protein-based methods would compensate each other, thus resulting in a more comprehensive detection from nucleic acid and protein levels. The combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different Roundup Ready soy (event 40-3-2) plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing a combination of both meat and plant protein concentrates. This study indicated that the combination of DNA- and protein-based methods

  6. Quantum dot bio-conjugate: as a western blot probe for highly sensitive detection of cellular proteins

    Science.gov (United States)

    Kale, Sonia; Kale, Anup; Gholap, Haribhau; Rana, Abhimanyu; Desai, Rama; Banpurkar, Arun; Ogale, Satishchandra; Shastry, Padma

    2012-03-01

    In the present study, we report a quantum dot (QD)-tailored western blot analysis for a sensitive, rapid and flexible detection of the nuclear and cytoplasmic proteins. Highly luminescent CdTe and (CdTe)ZnS QDs are synthesized by aqueous method. High resolution transmission electron microscopy, Raman spectroscopy, fourier transform infrared spectroscopy, fluorescence spectroscopy and X-ray diffraction are used to characterize the properties of the quantum dots. The QDs are functionalized with antibodies of prostate apoptosis response-4 (Par-4), poly(ADP-ribose) polymerases and β actin to specifically bind with the proteins localized in the nucleus and cytoplasm of the cells, respectively. The QD-conjugated antibodies are used to overcome the limitations of conventional western blot technique. The sensitivity and rapidity of protein detection in QD-based approach is very high, with detection limits up to 10 pg of protein. In addition, these labels provide the capability of enhanced identification and localization of marker proteins in intact cells by confocal laser scanning microscopy.

  7. [Western blot technique standardization for specific diagnosis of Chagas disease using excretory-secretory antigens of Trypanosoma cruzi epimastigotes].

    Science.gov (United States)

    Escalante, Hermes; Jara, César; Davelois, Kelly; Iglesias, Miguel; Benites, Adderly; Espinoza, Renzo

    2014-01-01

    Evaluate the effectiveness of Western Blot for the specific diagnosis of Chagas disease using excretory-secretory antigens of Trypanosoma cruzi epimastigotes. Antigens were obtained after twenty hours of incubation in Eagle’s Minimum Essential Medium, which were prepared at a protein concentration of 0.2 ug/uL to be faced with 10 mL pool of serum from patients with Chagas disease and a conjugated anti-IgG labeled with peroxidase. The presence of the following antigens was observed: 10, 12, 14, 15, 19, 20, 23, 26, 30, 33, 36, 40, 42, 46, 58, 63, 69, 91, 100, and 112 kDa; of which antigens of 10, 12, 14, 15, 19, 20, 23, and 26 kDa were considered to be specific using pools of serum from patients with other parasitosis and serum from people with no parasites. The sensitivity of the technique was assessed using individual serum from 65 patients with Chagas disease; and the specificity with serum from 40 patients with other parasitosis, and serums from five people who did not have parasites. The technique has a sensitivity of 95.4% in the detection of one to eight specific bands, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 93.7%. Western Blot technique with excretory-secretory antigens of T. cruzi epimastigotes is effective in the diagnosis of Chagas disease in Peru; therefore, it can be used as a confirmatory test.

  8. Interaction of murine intestinal mast cell proteinase with inhibitors (serpins) in blood; analysis by SDS-PAGE and western blotting.

    Science.gov (United States)

    Irvine, J; Newlands, G F; Huntley, J F; Miller, H R

    1990-01-01

    The interaction of mouse intestinal mast cell proteinase (IMCP) with serine proteinase inhibitors (serpins) in blood was analysed: (i) by examining the capacity of the inhibitors in blood to block the binding of the irreversible serine esterase inhibitor [3H]diisopropyl fluorophosphate (DFP); (ii) by Western blotting. The binding of [3H]DFP to IMCP was blocked very rapidly by inhibitors in mouse serum and, by Western blotting, this inhibition was associated with the appearance of a 73,000 MW proteinase/inhibitor complex together with a series of higher (greater than 100,000) MW complexes. IMCP was not dissociated from these complexes when electrophoresed under reducing conditions, although prior heat treatment of mouse serum (60 for 30-160 min) abolished the formation of all proteinase/inhibitor complexes. Similarly, the activity of a 48,000 MW inhibitor of chymotrypsin was abolished by heat treatment. A titration experiment established that between 0.5 and 5 mg IMCP were inhibited per ml of serum. The properties and MW of the IMCP inhibitor complexes are typical of serpins and suggest that IMCP secreted during intestinal immunological reactions would be rapidly and irreversibly inactivated by plasma-derived inhibitors.

  9. Analyses of the Results of HIV-1 Western Blot Confirmative Test and NAT Test in Voluntary Blood Donors%无偿献血者HIV-1抗体蛋白印迹确认与核酸检测结果分析

    Institute of Scientific and Technical Information of China (English)

    吕蓉; 王伦善; 盛琪琪; 赵阳; 蒋菲菲; 李敏; 刘忠

    2012-01-01

    Objective To explore the performance of confirmative Western blot (WB) and TMA-chemical luminescence test in detecting the reactive-samples for HIV antibody ELISA assay. Methods 117 reactive specimens detected by ELISA were repeatedly tested by two methods of primary screening assay and TMA-luminescence nucleic acid amplification testing(NAT). All samples of S/CO>0. 8 were analysed by the WB confirmative test. Serological tests participated in the external quality assessment (EQA) were surported by China CDC and Australian international CITIC as well as NAT participated in the EQA of Clinical Laboratory Center of the Ministry of Health and Australian CITIC. Results The results of all 117 tested samples were as followed, in primary screening test:S/CO>1,0. 8result in false positive results. WB method can bring uncertain results. For the uncertain specimens, for instance, only positive in gpl60/120, P17, P24, NAT method can be used to confirm the HIV infection.%目的 对酶联免疫检测HIV抗体呈反应性标本进行蛋白印迹(WB法)确认和TMA-化学发光法对照检测,以探讨其应用特点.方法 将本中心检验科ELISA法检测结果呈HIV抗体反应性的117份标本,重新进行ELISA法检测,结果为S/CO>0.8的标本做蛋白印迹(WB法)确认试验.同时,对117份标本采用TMA-化学发光法检测核酸作为对照试验.血清学检测参加国家CDC及澳大利亚(CITIC)室间质评;核酸检测参加卫生部临检中心和澳大利亚(CITIC)室间质评.结果 ELISA初筛试验:117份标本中,S/CO>1的为37份;0.8<S/CO<1的为11份,S/CO<0.8的为69份;抗体确认试验:HIV抗体确认阳性7份,不确定4

  10. Application of Western blot analysis for the diagnosis of Encephalitozoon cuniculi infection in rabbits: example of a quantitative approach.

    Science.gov (United States)

    Desoubeaux, Guillaume; Pantin, Ana; Peschke, Roman; Joachim, Anja; Cray, Carolyn

    2017-02-01

    Diagnosis of Encephalitozoon cuniculi infection in rabbits remains a major veterinary issue. ELISA or immunofluorescence assays are the current reference standards of serological tests. However, these conventional techniques suffer from a lack of accuracy for distinguishing active from past infections, as a positive serostatus is common in clinically normal rabbits. In this study, we assessed the diagnostic performance of Western blot (WB) to detect both anti-E. cuniculi immunoglobulin G (IgG) and immunoglobulin M (IgM) in comparison with ELISA and to address the intensity of the immune response through a quantitative approach. Positive WB results were highly correlated with the E. cuniculi-related diseased status (P < 0.0001). Although it was more labor intensive and less standardized, quantitative WB provided detailed comparable analysis regarding the humoral response and diagnostic performance similar to ELISA testing with statistically higher sensitivity (88.4 vs. 76.1% for IgG detection and 84.3 vs. 70.4% for IgM, P < 0.01). Several specific WB bands were shown to be significantly associated with concomitant clinical signs, like the one located at 50 kDa (OR = 8.2, [2.4-27.7], P = 0.0008) for IgG and (OR = 27.9, [4.2-187.9], P = 0.0006) for IgM. Therefore, the quantitative WB may have application in veterinary diagnostic laboratories to increase the accuracy of the clinical diagnosis of E. cuniculi infection. In addition, this tool may help to further understand the development and function of the humoral immune response to this infectious agent.

  11. Development of enzyme immunoassays (ELISA and Western blot) for the serological diagnosis of dermatophytosis in symptomatic and asymptomatic cats.

    Science.gov (United States)

    Santana, Aline Elisa; Taborda, Carlos Pelleschi; Severo, Julia So; Rittner, Glauce Mary Gomes; Muñoz, Julian Esteban; Larsson, Carlos Eduardo; Larsson, Carlos Eduardo

    2017-03-11

    Dermatophytosis is the most common fungal infection in cats worldwide and plays an important role in both animal and human health due to their high zoonotic potential. Effective screening is a strong preventive measure and the fungal culture is quite useful but requires full laboratorial experience and it takes a long time to obtain the result. A rapid and accurate screening test for dermatophytosis in cats is crucial for the effective control of disease outbreaks. The aim of this study was to develop and evaluate the diagnostic efficacy of enzyme immunoassays (ELISA and Western blot [WB]) for the rapid and precise diagnosis of dermatophytosis in cats. Seventy cats of various ages were divided into three groups: S (symptomatic, n = 20), AS (asymptomatic, n = 30), and N (negative, n = 20). All animals were submitted to fungal culture and blood samples for carrying out the serological tests. A significant difference (P < 0.05) was found between IgG-specific levels of sera of Microsporum canis positive and negative animals. There was no statistic difference between groups symptomatic and asymptomatic. The ELISA test showed sensitivity of 94% and specificity of 75%. Receiver operating characteristic (ROC) analysis also showed higher diagnostic accuracy (AUC 0.925). The WB technique detected 13 bands, and the 50 kDa protein was considered the most immunogenic protein, observing reactivity in 83.3% in the symptomatic group and 66.6% in the asymptomatic group. The study concluded that ELISA and WB were useful tools to reliably detect cats that have been exposed to M. canis. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. Quantitative analysis of a biopharmaceutical protein in cell culture samples using automated capillary electrophoresis (CE) western blot.

    Science.gov (United States)

    Xu, Dong; Marchionni, Kentaro; Hu, Yunli; Zhang, Wei; Sosic, Zoran

    2017-10-25

    An effective control strategy is critical to ensure the safety, purity and potency of biopharmaceuticals. Appropriate analytical tools are needed to realize such goals by providing information on product quality at an early stage to help understanding and control of the manufacturing process. In this work, a fully automated, multi-capillary instrument is utilized for size-based separation and western blot analysis to provide an early readout on product quality in order to enable a more consistent manufacturing process. This approach aims at measuring two important qualities of a biopharmaceutical protein, titer and isoform distribution, in cell culture harvest samples. The acquired data for isoform distribution can then be used to predict the corresponding values of the final drug substance, and potentially provide information for remedy through timely adjustment of the downstream purification process, should the expected values fall out of the accepted range. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Evaluation of two sets of immunohistochemical and Western blot confirmatory methods in the detection of typical and atypical BSE cases

    Directory of Open Access Journals (Sweden)

    Greenlee Justin J

    2011-09-01

    Full Text Available Abstract Background Three distinct forms of bovine spongiform encephalopathy (BSE, defined as classical (C-, low (L- or high (H- type, have been detected through ongoing active and passive surveillance systems for the disease. The aim of the present study was to compare the ability of two sets of immunohistochemical (IHC and Western blot (WB BSE confirmatory protocols to detect C- and atypical (L- and H-type BSE forms. Obex samples from cases of United States and Italian C-type BSE, a U.S. H-type and an Italian L-type BSE case were tested in parallel using the two IHC sets and WB methods. Results The two IHC techniques proved equivalent in identifying and differentiating between C-type, L-type and H-type BSE. The IHC protocols appeared consistent in the identification of PrPSc distribution and deposition patterns in relation to the BSE type examined. Both IHC methods evidenced three distinct PrPSc phenotypes for each type of BSE: prevailing granular and linear tracts pattern in the C-type; intraglial and intraneuronal deposits in the H-type; plaques in the L-type. Also, the two techniques gave comparable results for PrPSc staining intensity on the C- and L-type BSE samples, whereas a higher amount of intraglial and intraneuronal PrPSc deposition on the H-type BSE case was revealed by the method based on a stronger demasking step. Both WB methods were consistent in identifying classical and atypical BSE forms and in differentiating the specific PrPSc molecular weight and glycoform ratios of each form. Conclusions The study showed that the IHC and WB BSE confirmatory methods were equally able to recognize C-, L- and H-type BSE forms and to discriminate between their different immunohistochemical and molecular phenotypes. Of note is that for the first time one of the two sets of BSE confirmatory protocols proved effective in identifying the L-type BSE form. This finding helps to validate the suitability of the BSE confirmatory tests for BSE

  14. Development, validation, and pilot application of a semiquantitative Western blot analysis and an ELISA for bovine adiponectin.

    Science.gov (United States)

    Mielenz, M; Mielenz, B; Singh, S P; Kopp, C; Heinz, J; Häussler, S; Sauerwein, H

    2013-04-01

    Adiponectin is an adipose tissue-derived glycoprotein circulating as highly abundant multimers. It regulates glucose metabolism and insulin sensitivity. In ruminants, valid data about serum concentrations and tissue-specific protein expression are lacking, and we, therefore, aimed to generate a polyclonal antibody against bovine adiponectin to apply it in immunodetection. The specificity of the purified anti-adiponectin antibody was established by Western blot analysis with the use of reducing and denaturing conditions applied to both the purified protein and the bovine serum samples. Besides bovine serum, the applicability of the antibody for immunodetection of adiponectin was confirmed for the supernatant fluid of in vitro-differentiated bovine adipocytes, for protein extracts from bovine adipose tissue, and also in a multispecies comparison: bands comparable in size with monomeric bovine adiponectin were obtained under denaturing conditions in serum of camel, horse, human, mouse, pig, roe deer, and sheep. In addition, when used in immunohistochemistry on bovine adipose tissue sections, a characteristic adipocyte-specific staining pattern was obtained with this antibody. The antibody was used for establishing a semiquantitative Western blot procedure and the development of an ELISA. Both methods were extensively validated and were first applied to characterize the serum adiponectin concentrations in multiparous dairy cows during the transition from pregnancy to lactation, that is, 3 wk before until 5 wk after calving. With both assays a time effect (P = 0.017, P = 0.026, respectively) with lowest values at the day of parturition was observed. We thus established 2 useful tools to validly assess bovine adiponectin at the protein level. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Western blot analysis of BK channel β1-subunit expression should be interpreted cautiously when using commercially available antibodies.

    Science.gov (United States)

    Bhattarai, Yogesh; Fernandes, Roxanne; Kadrofske, Mark M; Lockwood, Lizbeth R; Galligan, James J; Xu, Hui

    2014-10-01

    Large conductance Ca(2+)-activated K(+) (BK) channels consist of pore-forming α- and accessory β-subunits. There are four β-subunit subtypes (β1-β4), BK β1-subunit is specific for smooth muscle cells (SMC). Reduced BK β1-subunit expression is associated with SMC dysfunction in animal models of human disease, because downregulation of BK β1-subunit reduces channel activity and increases SMC contractility. Several anti-BK β1-subunit antibodies are commercially available; however, the specificity of most antibodies has not been tested or confirmed in the tissues from BK β1-subunit knockout (KO) mice. In this study, we tested the specificity and sensitivity of six commercially available antibodies from five manufacturers. We performed western blot analysis on BK β1-subunit enriched tissues (mesenteric arteries and colons) and non-SM tissue (cortex of kidney) from wild-type (WT) and BK β1-KO mice. We found that antibodies either detected protein bands of the appropriate molecular weight in tissues from both WT and BK β1-KO mice or failed to detect protein bands at the appropriate molecular weight in tissues from WT mice, suggesting that these antibodies may lack specificity for the BK β1-subunit. The absence of BK β1-subunit mRNA expression in arteries, colons, and kidneys from BK β1-KO mice was confirmed by RT-PCR analysis. We conclude that these commercially available antibodies might not be reliable tools for studying BK β1-subunit expression in murine tissues under the denaturing conditions that we have used. Data obtained using commercially available antibodies should be interpreted cautiously. Our studies underscore the importance of proper negative controls in western blot analyses. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  16. Standardisation of Western blotting to detect HTLV-1 antibodies synthesised in the central nervous system of HAM/TSP patients

    Directory of Open Access Journals (Sweden)

    Luiz Claudio Pereira Ribeiro

    2013-09-01

    Full Text Available Intrathecal synthesis of human T-lymphotropic virus type 1 (HTLV-1 antibodies (Abs represents conclusive evidence of a specific immune response in the central nervous system of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP patients. Western blotting (WB for HTLV Abs in serum is a confirmatory test for HTLV-1 infection. The aim of this study was to standardise the Western blot to demonstrate the intrathecal pattern of Abs against HTLV-1 proteins in HAM/TSP patients. Paired cerebrospinal fluid (CSF and serum samples were selected from 20 patients with definite HAM/TSP, 19 HTLV-1 seronegative patients and two HTLV-1 patients without definite HAM/TSP. The presence of reactive bands of greater intensity in the CSF compared to serum (or bands in only the CSF indicated the intrathecal synthesis of anti-HTLV-1 Abs. All definite HAM/TSP patients presented with an intrathecal synthesis of anti-HTLV-1 Abs; these Abs were not detected in the control patients. The most frequent intrathecal targets of anti-HTLV-1 Abs were GD21, rgp46-I and p24 and, to a lesser extent, p19, p26, p28, p32, p36, p53 gp21 and gp46. The intrathecal immune response against env (GD21 and rgp46-I and gag (p24 proteins represents the most important humoral pattern in HAM/TSP. This response may be used as a diagnostic marker, considering the frequent association of intrathecal anti-HTLV-1 Ab synthesis with HAM/TSP and the pathogenesis of this neurological disease.

  17. Utility of Western Blot Analysis for the Diagnosis of Cutaneous Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Marzieh ASHRAFMANSOURI

    2015-12-01

    Full Text Available Background: Cutaneous leishmaniasis (CL is a parasitic disease with a relatively wide distribution in different areas of the world, including Iran. The parasite is mainly diagnosed microscopically, but serological approaches might be useful for diagnosis as well.  This study aimed to assess the efficacy of an immunoblotting system for serodiagnosis of cutaneous leishmaniasis in Iran.Methods: Sixty-one sera samples from parasitologically confirmed CL patients and 50 sera samples from healthy controls along with 50 sera sample from non-CL patients were collected. Native strain of Leishmania major was cultured in Schnei­der medium and soluble Leishmania antigens were prepared from amastigotes-like parasites. All of sera samples were evaluated by an immunoblot­ting system.Results: Components of 14 to 135 kDa were detectable by the sera of CL pa­tients. From 61 sera of CL patients, 59 cases (96.7% detected a 63 kDa subunit and 51 cases (83.6% recognized a 32-35 kDa component. Among all subunits, the 63 kDa band showed the highest sensitivity (96.7% and a 75 kDa band had the highest (98% specificity.Conclusion: Immunoblotting has a satisfactory performance in diagnosis of CL and this test can be used, as an aid, for proper diagnosis of CL.

  18. Glycophospholipid Formulation with NADH and CoQ10 Significantly Reduces Intractable Fatigue in Western Blot-Positive ‘Chronic Lyme Disease’ Patients: Preliminary Report

    Directory of Open Access Journals (Sweden)

    Garth L. Nicolson

    2012-03-01

    Full Text Available Background: An open label 8-week preliminary study was conducted in a small number of patients to determine if a combination oral supplement containing a mixture of phosphoglycolipids, coenzyme Q10 and microencapsulated NADH and other nutrients could affect fatigue levels in long-term, Western blot-positive, multi-symptom ‘chronic Lyme disease’ patients (also called ‘post-treatment Lyme disease’ or ‘post Lyme syndrome’ with intractable fatigue. Methods: The subjects in this study were 6 males (mean age = 45.1 ± 12.4 years and 10 females (mean age = 54.6 ± 7.4 years with ‘chronic Lyme disease’ (determined by multiple symptoms and positive Western blot analysis that had been symptomatic with chronic fatigue for an average of 12.7 ± 6.6 years. They had been seen by multiple physicians (13.3 ± 7.6 and had used many other remedies, supplements and drugs (14.4 ± 7.4 without fatigue relief. Fatigue was monitored at 0, 7, 30 and 60 days using a validated instrument, the Piper Fatigue Scale.Results: Patients in this preliminary study responded to the combination test supplement, showing a 26% reduction in overall fatigue by the end of the 8-week trial (p< 0.0003. Analysis of subcategories of fatigue indicated that there were significant improvements in the ability to complete tasks and activities as well as significant improvements in mood and cognitive abilities. Regression analysis of the data indicated that reductions in fatigue were consistent and occurred with a high degree of confidence (R2= 0.998. Functional Foods in Health and Disease 2012, 2(3:35-47 Conclusions: The combination supplement was a safe and effective method to significantly reduce intractable fatigue in long-term patients with Western blot-positive ‘chronic Lyme disease.’

  19. Characterization of 65 epitope-specific dystrophin monoclonal antibodies in canine and murine models of duchenne muscular dystrophy by immunostaining and western blot.

    Science.gov (United States)

    Kodippili, Kasun; Vince, Lauren; Shin, Jin-Hong; Yue, Yongping; Morris, Glenn E; McIntosh, Mark A; Duan, Dongsheng

    2014-01-01

    Epitope-specific monoclonal antibodies can provide unique insights for studying cellular proteins. Dystrophin is one of the largest cytoskeleton proteins encoded by 79 exons. The absence of dystrophin results in Duchenne muscular dystrophy (DMD). Over the last two decades, dozens of exon-specific human dystrophin monoclonal antibodies have been developed and successfully used for DMD diagnosis. Unfortunately, the majority of these antibodies have not been thoroughly characterized in dystrophin-deficient dogs, an outstanding large animal model for translational research. To fill the gap, we performed a comprehensive study on 65 dystrophin monoclonal antibodies in normal and dystrophic dogs (heart and skeletal muscle) by immunofluorescence staining and western blot. For comparison, we also included striated muscles from normal BL10 and dystrophin-null mdx mice. Our analysis revealed distinctive species, tissue and assay-dependent recognition patterns of different antibodies. Importantly, we identified 15 antibodies that can consistently detect full-length canine dystrophin in both immunostaining and western blot. Our results will serve as an important reference for studying DMD in the canine model.

  20. The Multispot rapid HIV-1/HIV-2 differentiation assay is comparable with the Western blot and an immunofluorescence assay at confirming HIV infection in a prospective study in three regions of the United States.

    Science.gov (United States)

    Pandori, Mark W; Westheimer, Emily; Gay, Cindy; Moss, Nicholas; Fu, Jie; Hightow-Weidman, Lisa B; Craw, Jason; Hall, Laura; Giancotti, Francesca R; Mak, Mae Ling; Madayag, Carmela; Tsoi, Benjamin; Louie, Brian; Patel, Pragna; Owen, S Michele; Peters, Philip J

    2013-12-01

    A new HIV diagnostic algorithm has been proposed which replaces the use of the HIV-1 Western blot and HIV-1 immunofluorescence assays (IFA) as the supplemental test with an HIV-1/HIV-2 antibody differentiation assay. To compare an FDA-approved HIV-1/HIV-2 antibody differentiation test (Multispot) as a confirmatory test with the HIV-1 Western blot and IFA. Participants were screened with an HIV-1/HIV-2 combination Antigen/Antibody (Ag/Ab) screening assay. Specimens with repeatedly reactive results were tested with Multispot and either Western blot or IFA. Specimens with discordant screening and confirmatory results were resolved with HIV-1 RNA testing. Individuals (37,876) were screened for HIV infection and 654 (1.7%) had a repeatedly reactive Ag/Ab assay result. On Multispot, 554 (84.7%) were HIV-1 reactive, 0 (0%) were HIV-2 reactive, 1 (0.2%) was reactive for both HIV-1 and HIV-2 (undifferentiated), 9 (1.4%) were HIV-1 indeterminate, and 90 (13.8%) were non-reactive. HIV-1 RNA was detected in 47/90 Multispot non-reactive (52.2%) specimens. Among specimens confirmed to have HIV infection (true positives), Multispot and Western blot detected HIV-1 antibody in a similar proportion of cases (93.7% vs. 94.4% respectively) while Multispot and IFA also detected HIV-1 antibody in a similar proportion of cases (84.5% vs. 83.4% respectively). In this study, Multispot confirmed HIV infections at a similar proportion to Western blot and IFA. Multispot, Western blot, and IFA, however, did not confirm all of the reactive Ag/Ab assay results and underscores the importance of HIV NAT testing to resolve discordant screening and confirmatory results. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Determining the cleavage site for the mature antimicrobial peptide of Nile tilapia β-defensin using 2D electrophoresis, western blot, and mass spectrometry analysis.

    Science.gov (United States)

    Chang, Chin-I; Chen, Li-Hao; Hu, Yeh-Fang; Wu, Chia-Che; Tsai, Jyh-Ming

    2017-03-01

    Several proteomic techniques were used to determine the cleavage site of the mature antimicrobial peptide of Nile tilapia β-defensin. The computer-predicted Nile tilapia β-defensin ((25)ASFPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL(66)) composed of 42 amino acids was chemically synthesized and prepared to produce an antibody for Western blotting. Total proteins from the skin of the Nile tilapia were separated on two-dimensional electrophoresis, and the spot of Nile tilapia β-defensin was recognized using Western blot analysis. It was then excised and extracted from the gel. The precise molecular mass of this spot was determined by LC-MS/MS spectrometry. Four major peptides were discovered, with molecular weights of 4293.2 Da, 4306.5 Da, 4678.9 Da, and 4715.0 Da. The calculated mass of the 40-amino-acid sequence ((27)FPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL(66)) of Nile tilapia β-defensin starting from Phe27 and ending with Leu66 was 4293.18 Da, which completely matched the 4293.2 Da peptide that was obtained from the mass spectrometry analysis. This result confirmed that the cleavage site for the mature C-terminal Nile tilapia β-defensin is at residue Ser26-Phe27, not at Ala24-25 as predicted by computer analysis. This study provides a simple but reliable model to determine the cleavage site for a mature antimicrobial peptide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. The diagnosis of proventricular dilatation disease: use of a Western blot assay to detect antibodies against avian Borna virus.

    Science.gov (United States)

    Villanueva, Itamar; Gray, Patricia; Mirhosseini, Negin; Payne, Susan; Hoppes, Sharman; Honkavuori, Kirsi S; Briese, Thomas; Turner, Debra; Tizard, Ian

    2010-07-14

    Avian Borna virus (ABV) has recently been shown to be the causal agent of proventricular dilatation disease (PDD) a lethal neurologic disease of captive psittacines and other birds. An immunoblot assay was used to detect the presence of antibodies against avian Borna virus in the serum of affected birds. A lysate from ABV-infected duck embryo fibroblasts served as a source of antigen. The assay was used to test for the presence of antibodies to ABV in 117 birds. Thirty of these birds had biopsy or necropsy-confirmed proventricular dilatation disease (PDD), while the remaining 87 birds were apparently healthy or were suffering from diseases other than PDD. Sera from 27 of the 30 PDD cases (90%) contained antibodies to ABV. Seventy-three (84%) of the apparently "healthy" birds were seronegative. Additionally, sera from seven macaws and one parrot trapped in the Peruvian Amazon were seronegative. Positive sera recognized the bornaviral nucleoprotein (N-protein). While the presence of antibodies to ABV largely corresponded with the development of clinical PDD, 14 apparently healthy normal birds possessed detectable antibodies to ABV. The existence of a carrier state was confirmed when 13 of 15 apparently healthy cockatiels were shown by PCR to have detectable ABV RNA in their feces. Western blot assays may be of significant assistance in diagnosing proventricular dilatation disease. Many apparently healthy birds may however be seronegative while, at the same time, shedding ABV in their feces. (c) 2009 Elsevier B.V. All rights reserved.

  3. Serodiagnosis of grass carp reovirus infection in grass carp Ctenopharyngodon idella by a novel Western blot technique.

    Science.gov (United States)

    He, Yongxing; Jiang, Yousheng; Lu, Liqun

    2013-12-01

    Frequent outbreaks of grass carp hemorrhagic disease, caused by grass carp reovirus (GCRV) infection, pose as serious threats to the production of grass carp Ctenopharyngodon idella. Although various nucleic acids-based diagnostic methods have been shown effective, lack of commercial monoclonal antibody against grass carp IgM has impeded the development of any reliable immunoassays in detection of GCRV infection. The present study describes the preparation and screening of monoclonal antibodies against the constant region of grass carp IgM protein, and the development of a Western blot (WB) protocol for the specific detection of antibodies against outer capsid VP7 protein of GCRV that serves as antibody-capture antigen in the immunoassay. In comparison to a conventional RT-PCR method, validity of the WB is further demonstrated by testing on clinical fish serum samples collected from a grass carp farm in Jiangxi Province during disease pandemic in 2011. In conclusion, the WB technique established in this study could be employed for specific serodiagnosis of GCRV infection. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Relative performance of Organon kit in comparison to Du Pont for confirmatory serological testing of HIV infection by western blot test in sera from blood donors.

    Science.gov (United States)

    Aggarwal, R K; Chatterjee, R; Chattopadhya, D; Kumari, S

    1992-06-01

    A total of 32 specimens with different categories of reactivity by Du Pont Western Blot kit comprising of specimens showing full spectrum of HIV-I antigen specific bands, 19 specimens showing total absence of bands and four specimens showing non-specific bands (without any interpretative importance) were subjected to Western Blot testing by Organon test. Of the nine specimens showing full spectrum of bands by Du Pont the correlation with Organon kit was 100 per cent based on WHO criteria. Four specimens with non-specific indeterminate band pattern by Du Pont failed to show any band in Organon kit, indicating that latter to be more specific.

  5. A study of the technique of western blot for diagnosis of lyme disease caused by Borrelia afzelii in China.

    Science.gov (United States)

    Liu, Zhi Yun; Hao, Qin; Hou, Xue Xia; Jiang, Yi; Geng, Zhen; Wu, Yi Mou; Wan, Kang Lin

    2013-03-01

    To study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure. FP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and immunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples (159 patients with Lyme disease and 292 controls), all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index. Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively. Establishment of WB criteria for B. afzelii is important in validating the diagnostic assays for Lyme disease in China. Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  6. Detection of Potentially Diagnostic Leishmania Antigens with Western Blot Analysis of Sera from Patients with Cutaneous and Visceral Leishmaniases.

    Science.gov (United States)

    Seyyedtabaei, Seyyed Javad; Rostami, Ali; Haghighi, Ali; Mohebali, Mehdi; Kazemi, Bahram; Fallahi, Shirzad; Spotin, Adel

    2017-01-01

    Visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are important public health problems in Iran. We aimed to evaluate the diagnostic potential of Western blot (WB) compared with indirect immunofluorescence test (IFAT) to serodiagnosis of leishmaniasis. This study was performed from 2010-2014 and participants were different parts of Iran. Serum samples were obtained from 43 patients with proven CL, 33 patients with proven VL, 39 patients with other parasitic diseases and 23 healthy individuals. WB sensitivity for CL and VL was 100% and 91%, compared to IFA 4.6% and 87.8%, respectively. Sera from patients with CL and VL recognized numerous antigens with molecular weights ranging from 14 to 68 kDa and 12 to 94 kDa, respectively. The most sensitive antigens were 14 and 16 kDa for CL recognized by 100% of the sera from patients with proven CL and 12, 14 and 16 kDa for VL, recognized by 63.6%, 100% and 63.6% of the sera from patients with proven VL respectively. WB analysis is more sensitive than IFAT for the diagnosis of leishmaniasis particularly in cases of cutaneous leishmaniasis. The 12, 14 and 16 kDa can be valuable diagnostic molecules for serodiagnosis of leishmaniasis because at least two immunogenic molecules were simultaneously detected by all patient sera, as well as produced antibodies against these antigens have no cross-reactivity with other control groups. WB could be useful for screening and serodiagnosis of CL and VL in epidemiologic studies in endemic areas.

  7. A theoretical timeline for myocardial infarction: immunohistochemical evaluation and western blot quantification for Interleukin-15 and Monocyte chemotactic protein-1 as very early markers.

    Science.gov (United States)

    Turillazzi, Emanuela; Di Paolo, Marco; Neri, Margherita; Riezzo, Irene; Fineschi, Vittorio

    2014-07-02

    Experimental and human studies have demonstrated that innate immune mechanisms and consequent inflammatory reaction play a critical role in cardiac response to ischemic injury. Thus, the detection of immuno-inflammatory and cellular phenomena accompanying cardiac alterations during the early inflammatory phase of myocardial infarction (MI) may be an excellent diagnostic tool. Current knowledge of the chronology of the responses of myocardial tissue following the occurrence of ischemic insult, as well as the existence of numerous studies aiming to identify reliable markers in dating MI, induced us to investigate the myocardial specimens of MI fatal cases in order to better define the age of MI. We performed an immunohistochemical study and a Western blot analysis to evaluate detectable morphological changes in myocardial specimens of fatal MI cases and to quantify the effects of cardiac expression of inflammatory mediators (CD15, IL-1β, IL-6, TNF-α, IL-15, IL-8, MCP-1, ICAM-1, CD18, tryptase) and structural and functional cardiac proteins. We observed a biphasic course of MCP-1: it was strongly expressed in the very early phase (0-4 hrs), to diminish in the early period (after 6-8 hrs). Again, our choice of IL-15 is explained by the synergism with neutrophilic granulocytes (CD15) and our study shows the potential for striking cytokine synergy in promoting fast, local neutrophil response in damaged tissues. A progressively stronger immunoreaction for the CD15 antibody was visible in the areas where the margination of circulating inflammatory cells was detectable, up to very strong expression in the oldest ones (>12 hours). Further, the induction of CD15, IL-15, MCP-1 expression levels was quantified by Western blot analysis. The results were as follows: IL-15/β-actin 0.80, CD15/β-actin 0.30, and MCP-1/β-actin 0.60, matching perfectly with the results of immunohistochemistry. Control hearts from traumatic death cases did not show any immunoreactivity to the

  8. Serum detection of IgG antibodies against Demodex canis by western blot in healthy dogs and dogs with juvenile generalized demodicosis.

    Science.gov (United States)

    Ravera, Ivan; Ferreira, Diana; Gallego, Laia Solano; Bardagí, Mar; Ferrer, Lluís

    2015-08-01

    The aim of this study was to investigate the presence of canine immunoglobulins (Ig) G against Demodex proteins in the sera of healthy dogs and of dogs with juvenile generalized demodicosis (CanJGD) with or without secondary pyoderma. Demodex mites were collected from dogs with CanJGD. Protein concentration was measured and a western blot technique was performed. Pooled sera from healthy dogs reacted mainly with antigen bands ranging from 55 to 72 kDa. Pooled sera from dogs with CanJGD without secondary pyoderma reacted either with 10 kDa antigen band or 55 to 72 kDa bands. Pooled sera from dogs with CanJGD with secondary pyoderma reacted only with a 10 kDa antigen band. The results of this study suggest that both healthy dogs and dogs with CanJGD develop a humoral response against different proteins of Demodex canis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle

    DEFF Research Database (Denmark)

    Vigelsø Hansen, Andreas; Dybboe, Rie; Hansen, Christina Neigaard

    2015-01-01

    physiology where the intervention has been suggested to impede normalization (ageing, muscle atrophy, and different muscle fiber type composition). The linearity of signal and the methodological variation coefficient was obtained. Furthermore, the inter- and intraindividual variation in signals obtained from...... SF and RP was measured in relation to ageing, muscle atrophy, and different muscle fiber type composition, respectively. A stronger linearity of SF and β-actin compared with GAPDH and α-tubulin was observed. The methodological variation was relatively low in all four methods (4-11%). Protein level...... and differences in muscle fiber type. The novel SF technology adds lower variation to the results compared with the existing methods for correcting for loading inaccuracy in Western blotting of human skeletal muscle in applied physiology....

  10. Resolution and identification of major peanut allergens using a combination of fluorescence two-dimensional differential gel electrophoresis, western blotting and Q-TOF mass spectrometry.

    Science.gov (United States)

    Peanut allergy is triggered by several proteins known as allergens. The matching resolution and identification of major peanut allergens in 2D protein maps, was accomplished by the use of fluorescence two-dimensional differential gel electrophoresis (2D DIGE), Western blotting and quadrupole time-of...

  11. Carbonic anhydrase IX as a specific biomarker for clear cell renal cell carcinoma: comparative study of Western blot and immunohistochemistry and implications for diagnosis.

    Science.gov (United States)

    Giménez-Bachs, José M; Salinas-Sánchez, Antonio S; Serrano-Oviedo, Leticia; Nam-Cha, Syong H; Rubio-Del Campo, Antonio; Sánchez-Prieto, Ricardo

    2012-10-01

    This study aimed to evaluate the usefulness of carbonic anhydrase IX (CA-IX) expression in clear cell renal cell carcinoma (CCRCC) using two different techniques to detect protein expression. An experimental, cross-sectional, analytical study was conducted to analyse proteins in renal tumour and healthy tissue specimens from 38 consecutive patients who underwent nephrectomy for renal cancer. CA-IX protein expression was measured by immunohistochemistry and Western blot analysis and quantified. Statistical analysis was performed with the positive and negative specific agreements and kappa coefficient. The sensitivity and specificity of both techniques were assessed. Statistical tests were conducted to analyse the association between CA-IX expression quantitation and normal prognosis factors (TNM stage and Fuhrman nuclear grade), only in CCRCC. The mean patient age was 65 years, 78.9% of patients were men and 57.9% of tumours were CCRCC. CA-IX protein expression was positive in 63.2% of tumours by immunohistochemistry and in 60.5% by Western blot. Both techniques detected CA-IX expression only in CCRCC and unclassifiable tumours. High concordance indices were observed for CCRCC diagnosis. Western blot and immunohistochemistry had a sensitivity of 95.5% and 100%, respectively; the specificity was 100% in both techniques. CA-IX expression quantitation did not correlate with tumour stage or Fuhrman nuclear grade. Immunochemistry and Western blot techniques can be used to detect abnormal CA-IX protein expression in CCRCC and to support morphology-based diagnostic techniques.

  12. Evaluation of Western blot, ELISA and latex agglutination tests to detect Toxoplasma gondii serum antibodies in farmed red deer.

    Science.gov (United States)

    Patel, Kandarp Khodidas; Howe, Laryssa; Heuer, Cord; Asher, Geoffery William; Wilson, Peter Raymond

    2017-09-15

    Abortion due to Toxoplasma gondii has been suspected in New Zealand farmed red deer. However, knowledge around the epidemiology and prevalence of T. gondii in farmed red deer is limited. The aim of this study was to firstly, assess the sensitivity and specificity of two commercially available assays, ELISA and latex agglutination test (LAT), for use in deer and secondly, to estimate the sero-prevalence of T. gondii in red deer. A total of 252 sera from rising 2-year-old and adult hinds from 17 New Zealand red deer herds at early and late pregnancy scanning and from known aborted and/or non-aborted hinds were tested for the presence of T. gondii antibodies. Each assays' sensitivity and specificity was evaluated by both the Western Blot (WB) as a gold standard method and Bayesian latent class (BLC) analysis in the absence of a gold standard. The sensitivity and specificity for WB were 95.8% (95% credible interval: 89.5-99.2%) and 95.1% (95% credible interval: 90.6-98.1%), respectively. For the LAT at the manufacturer's recommended ≥1:32 cut-off titre, the sensitivity (88.7%, 95% credible interval: 80.8-94.7%) and specificity (74.3%, 95% credible interval: 67.5-80.5%) were lower and higher than the sensitivity (76.2%, 95% credible interval: 66.7-84.5%) and specificity (89.7%, 95% credible interval: 84.5-93.9%) at a ≥1:64 cut-off, using (BLC) analysis. Sensitivity and specificity of the LAT at cut-off titre of 1:32 were estimated to be 84.4% (95% CI: 74.9-90.9%) and 73.5% (95% CI: 65.8-79.9%) against WB. The LAT had better agreement with WB at cut-off titre of ≥1:64 than ≥1:32 (Kappa=0.63 vs 0.54). At optimised cut-off S/P of 15.5%, the sensitivity (98.8%, 95% credible interval 96.1-99.8%) and specificity (92.8%, 95% credible interval 88.9-95.7%) of the ELISA were higher and lower, respectively, than the sensitivity (85.1%, 95% credible interval 76.2-91.9%) and specificity (98.5%, 95% credible interval 96.9-99.4%) at manufacturer's cut-off S/P of 30%, from BLC

  13. Characterization of Sm14 related components in different helminths by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis

    Directory of Open Access Journals (Sweden)

    Nilton Thaumaturgo

    2002-10-01

    Full Text Available Sm14 was the first fatty acid-binding protein homologue identified in helminths. Thereafter, members of the same family were identified in several helminth species, with high aminoacid sequence homology between them. In addition, immune crossprotection was also reported against Fasciola hepatica infection, in animals previously immunized with the Schistosoma mansoni vaccine candidate, r-Sm14. In the present study, data on preliminary sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis of nine different helminth extracts focusing the identification of Sm14 related proteins, is reported. Out of these, three extracts - Ascaris suum (males and females, Echinostoma paraensei, and Taenia saginata - presented components that comigrated with Sm14 in SDS-PAGE, and that were recognized by anti-rSm14 policlonal serum, in Western blotting tests.

  14. Analysis of the Crude Antigen of Hymenolepis nana from Mice by SDS-PAGE and the Determination of Specific Antigens in Protein Structure by Western Blotting

    OpenAIRE

    GÖNENÇ, Bahadır

    2002-01-01

    Protein bands of crude antigens of Hymenolepis nana were determined by SDS-PAGE and Western blotting. Thirty Swiss albino mice were allotted into two groups of 15 each as positive (infected with H. nana) and negative (non-infected with H. nana) groups. The natural infections of H. nana and other helminths were determined by centrifugal flotation of faeces. After bleeding, the mice were necropsied and their guts were examined for H. nana and other intestinal helminths. Sera from mice were test...

  15. The epidemiology of tick-borne haemoparasites as determined by the reverse line blot hybridization assay in an intensively studied cohort of calves in western Kenya.

    Science.gov (United States)

    Njiiri, Nyawira E; Bronsvoort, B Mark deC; Collins, Nicola E; Steyn, Helena C; Troskie, Milana; Vorster, Ilse; Thumbi, S M; Sibeko, Kgomotso P; Jennings, Amy; van Wyk, Ilana Conradie; Mbole-Kariuki, Mary; Kiara, Henry; Poole, E Jane; Hanotte, Olivier; Coetzer, Koos; Oosthuizen, Marinda C; Woolhouse, Mark; Toye, Philip

    2015-05-30

    The development of sensitive surveillance technologies using PCR-based detection of microbial DNA, such as the reverse line blot assay, can facilitate the gathering of epidemiological information on tick-borne diseases, which continue to hamper the productivity of livestock in many parts of Africa and elsewhere. We have employed a reverse line blot assay to detect the prevalence of tick-borne parasites in an intensively studied cohort of indigenous calves in western Kenya. The calves were recruited close to birth and monitored for the presence of infectious disease for up to 51 weeks. The final visit samples from 453 calves which survived for the study period were analyzed by RLB. The results indicated high prevalences of Theileria mutans (71.6%), T. velifera (62.8%), Anaplasma sp. Omatjenne (42.7%), A. bovis (39.9%), Theileria sp. (sable) (32.7%), T. parva (12.9%) and T. taurotragi (8.5%), with minor occurrences of eight other haemoparasites. The unexpectedly low prevalence of the pathogenic species Ehrlichia ruminantium was confirmed by a species-specific PCR targeting the pCS20 gene region. Coinfection analyses of the seven most prevalent haemoparasites indicated that they were present as coinfections in over 90% of the cases. The analyses revealed significant associations between several of the Theileria parasites, in particular T. velifera with Theileria sp. sable and T. mutans, and T. parva with T. taurotragi. There was very little coinfection of the two most common Anaplasma species, although they were commonly detected as coinfections with the Theileria parasites. The comparison of reverse line blot and serological results for four haemoparasites (T. parva, T. mutans, A. marginale and B. bigemina) indicated that, except for the mostly benign T. mutans, indigenous cattle seem capable of clearing infections of the three other, pathogenic parasites to below detectable levels. Although the study site was located across four agroecological zones, there was

  16. Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis.

    Science.gov (United States)

    Schüchner, Stefan; Andorfer, Peter; Mudrak, Ingrid; Ogris, Egon

    2016-08-17

    Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film.

  17. Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

    Science.gov (United States)

    Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

    2017-09-11

    The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG1, kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

  18. Effects of tetracyclines on neutrophil, gingival, and salivary collagenases. A functional and western-blot assessment with special reference to their cellular sources in periodontal diseases.

    Science.gov (United States)

    Sorsa, T; Ding, Y; Salo, T; Lauhio, A; Teronen, O; Ingman, T; Ohtani, H; Andoh, N; Takeha, S; Konttinen, Y T

    1994-09-06

    The characterization and regulation of matrix metalloproteinases (MMPs) have been studied to determine their role(s) in periodontal tissue destruction. Progress in elucidating the roles of MMPs in periodontal tissue destruction has led to a new concept involving the chemotherapeutic inhibition on MMPs, a therapeutic strategy which less than a decade ago was considered "a difficult and perhaps impossible task." Tetracyclines/doxycycline (DOXY) and their chemically modified nonantimicrobial derivatives (CMTs) are known to inhibit the matrix metalloproteinases, especially preferring human neutrophil collagenase (MMP-8), and prevent the oxidative activation of procollagenases. We characterized by Western blotting the molecular forms and cellular sources of gingival tissue, dental plaque, gingival crevicular fluid (GCF), and salivary MMPs associated with periodontitis. Also the molecular forms of tissue inhibitors of matrix metalloproteinases (TIMP-1 and TIMP-2) in periodontitis were studied by Western blot. Neutrophil (PMN)-derived MMPs were found to predominate in periodontitis, and phospholipase C present in increased amounts in periodontitis sites was found to be a potential inducer of PMN degranulation. We further studied the effects of DOXY on molecular forms of different latent and active MMPs purified from different cellular sources (PMNs, fibroblasts, keratinocytes) and present in vivo in oral exudates (gingival extracts, GCF, and saliva). DOXY inhibition of activated (oxidatively or proteolytically) MMPs were not associated with MMP fragmentation. Michaelis-Menten plots of initial rates of degradation of soluble type I collagen revealed an apparent Km value of 0.3-0.6 microM for MMP-8, and 75 microM DOXY inhibited MMP-8 in a manner which did not result in changes in apparent Km value but did prevent the initial degradation reaching Vmax providing evidence for noncompetitive inhibition. Treatment of patients with long-term DOXY medication results in decreased

  19. Estandarización de la técnica de Western blot para el diagnóstico de la fasciolosis humana utilizando antígenos de excreción-secreción de Fasciola hepática Western blot technique standardization of the diagnosis of human fasciolosis using Fasciola hepatica excreted-secreted antigens

    Directory of Open Access Journals (Sweden)

    Hermes Escalante

    2011-09-01

    finding of parasite eggs in the stool microscopy. Antigens of 10, 12, 17, 23, 27, 30, 36, 43, 66 and 136 kDa were detected and used to develop the Western blot technique. The sensitivity was evaluated using sera from 67 fasciolosis patients, and the specificity using sera from 57 patients with other parasitic diseases, and 10 from healthy individuals. Results. Out of the 67 sera, 64 reacted with the 23 kDa band and 61 with the one of 17 kDa. These two bands were not detected in sera from patients with other parasitic diseases or in those from healthy volunteers and thus could be considered specific and diagnostic. Conclusions. The sensitivity of the test, using the bands of 17 and 23 kDa, was 95.5% for positive reactions to at least one of these two bands, being its specificity 100% with a positive predictive value of 100% and negative predictive value of 95.71%.

  20. Immunohistochemical and Western blot analysis of two protein tyrosine phosphatase receptors, R and Z1, in colorectal carcinoma, colon adenoma and normal colon tissues.

    Science.gov (United States)

    Woźniak, Marta; Gamian, Elżbieta; Łaczmańska, Izabela; Sąsiadek, Maria M; Duś-Szachniewicz, Kamila; Ziółkowski, Piotr

    2014-05-01

    Two classes of proteins, namely tyrosine kinases (PTK) and phosphatases (PTP), play an important role in cell proliferation and differentiation, thus leading to an acceleration or inhibition of tumour growth. The role of the above proteins in colorectal carcinoma (CRC) growth is a well-known event. In this study we carried out immunohistochemical and Western blot analysis of colorectal carcinoma, adenoma and normal colon tissue in relation to two protein tyrosine phosphatase receptors, R and Z1. Twenty-five cases of CRC were analyzed and the results were compared with similar data obtained in non-malignant tissues. High expression of both PTP receptors was observed in all examined cases of CRC, adenoma and normal colon tissue in this study. These results are not in line with recently published data, showing that genetic coding for PTPRR and PTPRZ1 were hypermethylated in CRC's. We presume that the protein tyrosine phosphatase overexpression in colorectal carcinoma is not enough to protect from the progression of disease.

  1. A Western blot-based investigation of the yeast secretory pathway designed for an intermediate-level undergraduate cell biology laboratory.

    Science.gov (United States)

    Hood-Degrenier, Jennifer K

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in two distinct steps of protein secretion were differentiated using a genetic reporter designed specifically to identify defects in the first step of the pathway, the insertion of proteins into the endoplasmic reticulum (Vallen, 2002). We have developed two versions of a Western blotting assay that serves as a second way of distinguishing the two secretory mutants, which we pair with the genetic assay in a 3-wk laboratory module. A quiz administered before and after students participated in the lab activities revealed significant postlab gains in their understanding of the secretory pathway and experimental techniques used to study it. A second survey administered at the end of the lab module assessed student perceptions of the efficacy of the lab activities; the results of this survey indicated that the experiments were successful in meeting a set of educational goals defined by the instructor.

  2. Cytomegalovirus Destruction of Focal Adhesions Revealed in a High-Throughput Western Blot Analysis of Cellular Protein Expression† ▿

    OpenAIRE

    Stanton, Richard James; McSharry, Brian Patrick; Rickards, Carole Ruth; Wang, Edward Chung Yern; Tomasec, Peter; Wilkinson, Gavin William Grahame

    2007-01-01

    Human cytomegalovirus (HCMV) systematically manages the expression of cellular functions, rather than exerting the global shutoff of host cell protein synthesis commonly observed with other herpesviruses during the lytic cycle. While microarray technology has provided remarkable insights into viral control of the cellular transcriptome, HCMV is known to encode multiple mechanisms for posttranscriptional and posttranslation regulation of cellular gene expression. High-throughput Western blotti...

  3. A comparison of the immune parameters of dogs infected with visceral leishmaniasis using Western blot and neutralization techniques Comparação dos parâmetros imunológicos de cães infectados com leishmaniose visceral usando as técnicas de Western blot e neutralização

    Directory of Open Access Journals (Sweden)

    Yeda L. Nogueira

    2007-12-01

    Full Text Available The Western blot technique was used to demonstrate the presence of antibodies in the blood of dogs that presented canine visceral leishmaniasis. This technique was used against some specific molecules present in the lysate of the promastigote form of Leshmania chagasi.Through the association of the results of the Western blot technique with the morphological alterations seen as a result of the serum neutralization technique performed in McCoy cells (which mimetizes the macrophage it was possible to observe the role of some molecules of great relevance in determining the disease in symptomatic dogs as well as that of some other molecules associated with asymptomatic infected dogs that may become transmitters as well as differentiating them as asymptomatic resistant dogs. In the sera analyses carried out during the immunobloting a variation of 9 to 27 immunoreacting bands was observed, which were then compared using Dice's similarity coefficient. In the dendrogram constructed on the basis of the coefficient, 50% similarity was observed among the total number of reagent bands with the promastigote lysate, thus creating five groups. The main difference observed related to the clinical condition of the dogs: symptomatic and asymptomatic dogs were found in separate groups. The asymptomatic group of dogs was distributed in two different places in the dendrogram because they presented two different behavior patterns regarding the cellular morphology in the serum neutralization reaction: the presence or absence of cellular lysis. According to this analysis it is possible to evaluate the immune status and associate it with specific markers observed in the reaction found in the Western blot strips.A técnica de Western blot foi utilizada para demonstrar a presença de anticorpos do soro de cães, que apresentavam leishmaniose visceral canina, contra algumas moléculas específicas no lisado da forma promastigota de Leshmania chagasi.Através da associa

  4. Study of a viral-dual infection in rainbow trout (Oncorhynchus mykiss) by seroneutralization, western blot and polymerase chain reaction assays.

    Science.gov (United States)

    Rodríguez, S; Vilas, M P; Alonso, M; Pérez, S I

    1995-12-01

    Viral-dual infections in fish are of interest to aquaculture practices but they are rarely described and studied. Several methods were applied in this work to demonstrate a case of coinfection in a reared rainbow trout (Oncorhynchus mykiss) population. Inoculation in cell cultures and cross-neutralization tests were the standard procedures that made it possible to isolate and identify a birnavirus, the infectious pancreatic necrosis virus (IPNV), and suspect of a second virus. Western blotting with both polyclonal and monoclonal antibodies, and reverse transcriptional-polymerase chain reaction (RT-PCR) demonstrate coexistence of both, IPNV and a rhabdovirus.

  5. Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

    Science.gov (United States)

    Yamamoto, Norio; Yamashita, Yoko; Yoshioka, Yasukiyo; Nishiumi, Shin; Ashida, Hitoshi

    2016-08-01

    Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  6. APPLICATION OF WESTERN BLOTTING TECHNIQUE FOR EVALUATING THE EXPRESSION OF VASOPRESSIN RECEPTORS IN THE HEART CELLS; IMPORTANCE IN THE CARDIOVASCULAR SYSTEM

    Directory of Open Access Journals (Sweden)

    Manoj G Tyagi

    2012-08-01

    Full Text Available Vasopressin, a posterior pituitary hormone is responsible for water reabsorption by the kidneys and maintenance of cardio-vascular homeostasis. Vasopressin receptors are characterized as VR 1 (V1a, VR2 (V2, and VR3 (V1b. VR1, which is abundant in vascular smooth muscles, causes vasoconstriction by increasing intracellular calcium via the phosphatidylinositol bisphosphonate pathway and a positive inotropic effect in cardiac muscle. VR2 has also been shown to be expressed in the heart. There is emerging role for vasopressin receptors in health and disease. This study describes the application of Western blotting to elucidate the importance of vasopressin receptors in the heart cells.

  7. A comparative study of Toxoplasma gondii seroprevalence in mink using a modified agglutination test, a Western blot, and enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Gu, Yi; Wang, Zedong; Cai, Yufeng; Li, Xiaoxing; Wei, Feng; Shang, Limin; Li, Jiping; Liu, Quan

    2015-09-01

    Toxoplasma gondii can infect almost all warm-blooded animals, and many serological methods have been developed to detect T. gondii infection in a variety of animal species. In the present study, the seroprevalence of T. gondii infection in farmed mink in northeast China was determined using the modified agglutination test (MAT), a Western blot (WB), and 3 enzyme-linked immunosorbent assays (ELISAs) with protein A/G conjugate, using either of 2 recombinant dense granule antigens, GRA1 and GRA7, or Toxoplasma soluble antigens (TSA). There was no significant difference between the detection results of the GRA1-, GRA7-, and TSA-ELISAs and WB (McNemar chi-square, P > 0.05), but a significant difference was observed between MAT and WB (P < 0.05). A near perfect agreement (97.0%) was found between the GRA7-ELISA and WB (κ = 0.83), and a substantial agreement (92.4-93.1%) was observed in the TSA- and GRA1-ELISAs (κ = 0.68-0.73). The GRA7-ELISA showed the highest sensitivity and specificity, and the lowest false-positive and negative rates, while the MAT gave both a low sensitivity and frequent false positives in comparison to the WB. Receiver operating characteristic analysis revealed the largest area under curve of 0.85 (95% confidence interval: 0.74-0.96), and the highest relative sensitivity (72.7%) and specificity (99.0%) for a cutoff value of 0.19 in the GRA7-ELISA. These results indicate that the GRA7-ELISA is suitable for detection of T. gondii infection in mink and that MAT should be used with caution.

  8. A research design for the quantification of the neuropeptides substance p and calcitonin gene-related Peptide in rat skin using Western blot analysis.

    Science.gov (United States)

    Lapin, Guilherme Abbud Franco; Hochman, Bernardo; Nishioka, Michele Akemi; Maximino, Jessica Ruivo; Chadi, Gerson; Ferreira, Lydia Masako

    2015-06-01

    To describe and standardize a protocol that overcomes the technical limitations of Western blot (WB) analysis in the quantification of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) following nociceptive stimuli in rat skin. Male Wistar rats (Rattus norvegicus albinus) weighing 250 to 350 g were used in this study. Elements of WB analysis were adapted by using specific manipulation of samples, repeated cycles of freezing and thawing, more thorough maceration, and a more potent homogenizer; increasing lytic reagents; promoting greater inhibition of protease activity; and using polyvinylidene fluoride membranes as transfer means for skin-specific protein. Other changes were also made to adapt the WB analysis to a rat model. University research center. Western blot analysis adapted to a rat model. This research design has proven effective in collecting and preparing skin samples to quantify SP and CGRP using WB analysis in rat skin. This study described a research design that uses WB analysis as a reproducible, technically accessible, and cost-effective method for the quantification of SP and CGRP in rat skin that overcomes technical biases.

  9. Identification of reference proteins for Western blot analyses in mouse model systems of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD toxicity.

    Directory of Open Access Journals (Sweden)

    Stephenie D Prokopec

    Full Text Available Western blotting is a well-established, inexpensive and accurate way of measuring protein content. Because of technical variation between wells, normalization is required for valid interpretation of results across multiple samples. Typically this involves the use of one or more endogenous controls to adjust the measured levels of experimental molecules. Although some endogenous controls are widely used, validation is required for each experimental system. This is critical when studying transcriptional-modulators, such as toxicants like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD.To address this issue, we examined hepatic tissue from 192 mice representing 47 unique combinations of strain, sex, Ahr-genotype, TCDD dose and treatment time. We examined 7 candidate reference proteins in each animal and assessed consistency of protein abundance through: 1 TCDD-induced fold-difference in protein content from basal levels, 2 inter- and intra- animal stability, and 3 the ability of each candidate to reduce instability of the other candidates. Univariate analyses identified HPRT as the most stable protein. Multivariate analysis indicated that stability generally increased with the number of proteins used, but gains from using >3 proteins were small. Lastly, by comparing these new data to our previous studies of mRNA controls on the same animals, we were able to show that the ideal mRNA and protein control-genes are distinct, and use of only 2-3 proteins provides strong stability, unlike in mRNA studies in the same cohort, where larger control-gene batteries were needed.

  10. Low Proviral Load is Associated with Indeterminate Western Blot Patterns in Human T-Cell Lymphotropic Virus Type 1 Infected Individuals: Could Punctual Mutations be Related?

    Science.gov (United States)

    Cánepa, Camila; Salido, Jimena; Ruggieri, Matías; Fraile, Sindy; Pataccini, Gabriela; Berini, Carolina; Biglione, Mirna

    2015-10-28

    indeterminate Western blot (WB) patterns are a major concern for diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1) infection, even in non-endemic areas. (a) to define the prevalence of indeterminate WB among different populations from Argentina; (b) to evaluate if low proviral load (PVL) is associated with indeterminate WB profiles; and (c) to describe mutations in LTR and tax sequence of these cases. Among 2031 samples, 294 were reactive by screening. Of them, 48 (16.3%) were WB indeterminate and of those 15 (31.3%) were PCR+. Quantitative real-time PCR (qPCR) was performed to 52 HTLV-1+ samples, classified as Group 1 (G1): 25 WB+ samples from individuals with pathologies; Group 2 (G2): 18 WB+ samples from asymptomatic carriers (AC); and Group 3 (G3): 9 seroindeterminate samples from AC. Median PVL was 4.78, 2.38, and 0.15 HTLV-1 copies/100 PBMCs, respectively; a significant difference (p=0.003) was observed. Age and sex were associated with PVL in G1 and G2, respectively. Mutations in the distal and central regions of Tax Responsive Elements (TRE) 1 and 2 of G3 were observed, though not associated with PVL.The 8403A>G mutation of the distal region, previously related to high PVL, was absent in G3 but present in 50% of WB+ samples (p = 0.03). indeterminate WB results confirmed later as HTLV-1 positive may be associated with low PVL levels. Mutations in LTR and tax are described; their functional relevance remains to be determined.

  11. Low Proviral Load is Associated with Indeterminate Western Blot Patterns in Human T-Cell Lymphotropic Virus Type 1 Infected Individuals: Could Punctual Mutations be Related?

    Directory of Open Access Journals (Sweden)

    Camila Cánepa

    2015-10-01

    Full Text Available Background: indeterminate Western blot (WB patterns are a major concern for diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1 infection, even in non-endemic areas. Objectives: (a to define the prevalence of indeterminate WB among different populations from Argentina; (b to evaluate if low proviral load (PVL is associated with indeterminate WB profiles; and (c to describe mutations in LTR and tax sequence of these cases. Results: Among 2031 samples, 294 were reactive by screening. Of them, 48 (16.3% were WB indeterminate and of those 15 (31.3% were PCR+. Quantitative real-time PCR (qPCR was performed to 52 HTLV-1+ samples, classified as Group 1 (G1: 25 WB+ samples from individuals with pathologies; Group 2 (G2: 18 WB+ samples from asymptomatic carriers (AC; and Group 3 (G3: 9 seroindeterminate samples from AC. Median PVL was 4.78, 2.38, and 0.15 HTLV-1 copies/100 PBMCs, respectively; a significant difference (p=0.003 was observed. Age and sex were associated with PVL in G1 and G2, respectively. Mutations in the distal and central regions of Tax Responsive Elements (TRE 1 and 2 of G3 were observed, though not associated with PVL.The 8403A>G mutation of the distal region, previously related to high PVL, was absent in G3 but present in 50% of WB+ samples (p = 0.03. Conclusions: indeterminateWBresults confirmed later as HTLV-1 positive may be associated with low PVL levels. Mutations in LTR and tax are described; their functional relevance remains to be determined.

  12. Evaluation of Line Immunoassay to Detect HTLV-1 Infection in an Endemic Area, Southwestern Japan; Comparison with Polymerase Chain Reaction and Western Blot.

    Science.gov (United States)

    Umeki, Kazumi; Umekita, Kunihiko; Hashikura, Yuuki; Yamamoto, Ikuo; Kubo, Kazuyoshi; Nagatomo, Yasuhiro; Okayama, Akihiko

    2017-02-01

    Human T-lymphotropic virus type 1 (HTLV-1) has been recognized as a cause of adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and HTLV-1-associated uveitis. HTLV-1 infection is normally detected by screening for HTLV-1 antibodies, and positive samples are confirmed by Western blot (WB). However, WB fails to confirm some samples that were positive for HTLV-1 antibodies on screening. Line immunoassay (LIA) is commonly used in Europe and Brazil, but not in Japan. Therefore, we evaluated the performance of LIA as a method of confirming HTLV-1 antibodies using samples in Japan. LIA was compared with polymerase chain reaction (PCR) and WB using 50 negative and 70 positive samples tested by chemiluminescent enzyme immunoassay (CLEIA) in Miyazaki, Japan, an HTLV-1 endemic area. LIA (INNO-LIA HTLVI/II Score) and WB (Problot HTLV-I) were performed according to the manufacturer's instructions. Real-time PCR for HTLV-1 pX region was performed using DNA derived from white blood cells. The samples that tested negative by real-time PCR were further tested by nested PCR. All 50 CLEIA negative samples were determined to be negative by LIA and PCR. Of the 70 positive samples, 66 tested positive by both of LIA and PCR. Three samples tested negative by LIA and PCR, and the remaining sample (PCR negative) showed non-specific staining in LIA and WB. WB showed more indeterminate results than LIA. Gp21 antibody in LIA demonstrated a high ability to discriminate between positive and negative PCR results. Furthermore, the degree of gp21 antibody reaction by LIA showed correlation with HTLV-1 proviral loads (PVLs). Our results indicate that LIA performs well in confirming HTLV-1 seropositivity by showing a low incidence of indeterminate results and good agreement with PCR using samples in Japan, although the number of samples tested was small. In addition, semi-quantitative antibody titer to gp21 correlated well with HTLV-1 PVLs. Further study

  13. Evaluation of two sets of immunohistochemical and Western blot confirmatory methods in the detection of typical and atypical BSE cases

    Science.gov (United States)

    Aims: To compare the ability of the Italian and the U.S. bovine spongiform encephalopathy (BSE) confirmatory protocols in the detection of classical (C-) and atypical - low (L-) and high (H-) type- BSE forms. Methods and Results: Obex samples from U.S. and Italian C-type BSE cases, a U.S. H-type and...

  14. A comparison of antigenic peptides in muscle larvae of several Trichinella species by two-dimensional western-blot analysis with monoclonal antibodies

    Directory of Open Access Journals (Sweden)

    Dea-Ayuela M.A.

    2001-06-01

    Full Text Available The antigens recognised by mAb US5 specific to 53 kDa glycoprotein (gp 53 in T. spiralis L-1 muscle larvae (TSL1 antigens, mAb US9 specific to gp 53 in TSL1 from all encapsulated species and mAb US4 specific to a tyvelose containing tetrasaccharide present in TSL1, were investigated in crude extracts from muscle larvae of T. spiralis, T. nativa and T. britovi by 2D-electrophoresis and western-blot. At least four proteins of different pI were recognised by mAb US5 on T. spiralis antigens. Recognition profile of mAb US9 on T. spiralis antigens exhibited some variation with regard to that of the US5. Polymorphism was apparent in gp 53. High reactivity was shown by the mAb US4 with the three species.

  15. Comparison of the efficiency of two commercial kits - ELFA and Western blot in estimating the phase of Toxoplasma gondii infection in pregnant women.

    Science.gov (United States)

    Sroka, Jacek; Wójcik-Fatla, Angelina; Zając, Violetta; Sawczyn, Anna; Cisak, Ewa; Karamon, Jacek; Dutkiewicz, Jacek; Bojar, Iwona

    2016-12-23

    Sera of 89 pregnant women were selected according to the results of ELFA IgM, IgG and avidity IgG, and tested with commercial tests IgM, IgG and avidity IgG Western Blot (WB) to compare the efficacy of both techniques in determining the phase of T. gondii infection. In total, 81 of 89 tested sera (91.0%) were classified as positive, both in the ELFA and WB tests for the presence of anti-Toxoplasma antibodies of class IgG, indicating a past infection, while the prevalence of anti-Toxoplasma positive reactions associated with the antibodies of class IgM indicating a recent infection was much lower - 31.5% and 20.2%, respectively. Sera of 81 women were also tested in the ELFA and WB tests for avidity, e.g. ability of forming high-molecular IgG antibody complexes. Low or medium results in these tests (in this study all classified as low), indicating a recent infection, were detected by ELFA and WB in 22.2% and 45.7% of the total examined samples, respectively. The Spearman's rank test for correlation, performed for recognition of quantitative data of the ELFA and WB tests (index, units or points), revealed a highly significant correlation between the ELFA and WB tests for homologous classes of antibodies, both for IgM and IgG (p0.05), except for the WB test for IgM antibodies, which showed a significant correlation with the ELFA test for IgG antibodies (p<0.01). A highly significant negative correlation between the ELFA and WB test for IgM antibodies and ELFA and WB tests for IgG avidity was demonstrated (p<0.01), except for a relationship between the WB test for IgM and WB for avidity, which was not significant. Such negative correlations are theoretically expected, as strong complexes with the participation of IgG antibodies are absent in the early phase of toxoplasmosis when early antibodies of IgM class are present. Summarizing, this study indicates the high usefulness of the commercial ELFA and WB tests in serodiagnostics of toxoplasmosis in pregnant women

  16. A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis.

    Science.gov (United States)

    Lin, Wen-Wei; Chen, I-Ju; Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R; Cheng, Tian-Lu

    2016-01-01

    Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15-120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.

  17. Pacientes neurológicos del noroeste del Perú con serología positiva por Western Blot a la larva de Taenia solium

    Directory of Open Access Journals (Sweden)

    Hermes Escalante A

    2004-04-01

    Full Text Available Objetivos: Determinar la frecuencia de pacientes con sintomatología neurológica de la zona noroeste del Perú que presentan serología positiva por Western Blot a la larva de Taenia solium. Material y Métodos: El estudio se realizó en 3515 pacientes de cero a noventa años de edad y de ambos sexos con sintomatología neurológica, procedentes de los departamentos de Piura, Lambayeque, Cajamarca, Ancash, La Libertad y algunas poblaciones de la zona selvática (5 a 10° LS; 77 a 82° LO. Dichos pacientes fueron atendidos en consultorios privados y en los servicios de Neurología de los Hospitales Belén y Regional de Trujillo desde enero de 1997 hasta diciembre de 2000. De cada uno de los pacientes se obtuvo una muestra sanguínea, cuyo suero fue procesado por la técnica de Western Blot, la cual tiene una sensibilidad de 91% y una especificidad de 100%. Resultados: Se detectó serología positiva en 562 pacientes, la cual representa una prevalencia de 16%, siendo el sexo masculino el que presentó mayor porcentaje (58,4%. Los grupos etáreos con mayor frecuencia de serología positiva fueron los de 41 a 50 años (18,7% y 31 a 40 años (17,4%. Los mayores porcentajes de serología positiva se obtuvo en pacientes procedentes de Piura y Lambayeque. Conclusiones: Los pacientes estudiados presentan una alta frecuencia de serología positiva a la larva de T. solium.

  18. [Recombinant expression of hantaan virus protein N with application of Western-blot in detecting anti-hantavirus antibody].

    Science.gov (United States)

    Yao, P P; Xu, F; Sun, Y S; Yang, Z R; Zhang, Y; Yue, M; Zhu, H P

    2017-04-10

    Objective: S gene of hantavirus(HV) was expressed in insect cells by genetic engineering technology. The expression product of S gene was used as antigen to detect anti-HV specific antibody IgG in serum. Methods: Gene encoding NP of the strain HV-Z10 was amplified by PCR and then its eukaryotic expression system rBAC-Z10S-TN was constructed by using the routine genetic engineering method. SDS-PAGE was applied to measure the expression of rNP.Ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product. Indirect immuno-fluorescence assay (IFA) was used to determine the specific immune-reactivity of rNP. WB assay was established to detect the serum samples from 95 confirmed HFRS patients. Parameters related to the outcomes of detection were compared with the routine HV-IgG IFA method. Results: rBAC-Z10S-TN was able to express rNP with high efficiency. The purified rNP only showed a single protein fragment in the gel after SDS-PAGE. HV IgG could efficiently recognize rNP and hybridize with the recombinant protein. 97.67% of the serum samples from the HFRS patients were positive confirmed by WB. Conclusions: We successfully constructed a high efficient prokaryotic expression system of NP encoding gene from hantavirus strain HV-Z10. WB assay which was established in this study could be used as a new serological test for HFRS diagnosis, thanks to the simplicity, safety, sensitivity and specificity of this method.

  19. Rendimiento diagnóstico del Western Blot para detectar simultáneamente anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana

    Directory of Open Access Journals (Sweden)

    Kelly Davelois

    Full Text Available Objetivo. Determinar el rendimiento diagnóstico de la técnica de Western Blot para detectar simultáneamente anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana. Materiales y métodos. Estudio transversal de evaluación de prueba diagnóstica. Se obtuvieron los antígenos de excreción-secreción de las larvas de Taenia solium, quistes de Echinococcus granulosus; y la forma adulta de Fasciola hepática; que luego fueron separados electroforéticamente en geles de poliacrilamida individuales, transferidos y fijados a una membrana de nitrocelulosa para ser enfrentados con sueros de pacientes con las tres parasitosis. La sensibilidad de la técnica se evaluó empleando 300 sueros individuales, 60 pools de dos parasitosis y 20 pools de tres parasitosis y la especificidad con 75 sueros de pacientes con otras parasitosis, 10 de pacientes con otras enfermedades y 15 sueros de personas no parasitadas. Resultados. La técnica reconoció trece glicoproteínas (GP: GP 35, 31, 24, 23, 18, 17, 14 y 13 kDa para cisticercosis, GP 8,16 y 21 kDa para hidatidosis y GP: 17 y 23 kDa para fascioliasis. La prueba detectó la presencia de anticuerpos alcanzando una sensibilidad de 96% (IC95%: 94,62-98,54% en la detección de una o las trece bandas, una especificidad de 100% (IC95%: 99,50 - 100,00%; individualmente, se tuvo una sensibilidad para cisticercosis de 97% (IC95%: 93,16-100%, para hidatidosis de 94% (IC95%: 88,85-99,15% y para fascioliasis de 96% (IC95%: 91,66-100%. Conclusiones. La prueba de Western blot es eficaz en la detección, simultanea de anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana, y puede ser utilizada como prueba de descarte o confirmatoria en zonas endémicas.

  20. An optimized xylene-free protein extraction method adapted to formalin-fixed paraffin embedded tissue sections for western blot analysis.

    Science.gov (United States)

    Mansour, Anthony G; Khalil, Pamela Abou; Bejjani, Noha; Chatila, Rajaa; Dagher-Hamalian, Carole; Faour, Wissam H

    2017-03-01

    Deparaffinization of formalin-fixed paraffin embedded (FFPE) tissues with xylene currently remains a major challenge to the biomedical community. We developed an efficient xylene-free protocol to isolate proteins from archived FFPE human tissue sections. A total of 79 different types of FFPE tissue sections of 8 µm thickness were obtained from various archived FFPE specimens. Deparaffinization was conducted by gently washing each section with around 1 ml of hot distilled water (≈80°C). The deparaffinized tissues were homogenized in lysis buffer, and the isolated proteins were quantified and efficiently resolved using western blot analysis for the presence of Protein kinase B (PKB/AKT) and β-actin. Moreover, a significant amount of proteins was successfully isolated with an average of 2.31 µg/µl. The migration pattern of AKT and β-actin obtained from the specimens was similar to the positive control obtained from protein lysates prepared from in vitro cultured MDA231 cancer cell lines. AKT was successfully identified in all specimens, and β-actin protein was resolved with an efficiency higher than 80%. The entire extraction procedure requires only 20 minutes. This newly developed technique is an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissue sections adequate for molecular analysis.

  1. Re-purposing of histological tissue sections for corroborative western blot analysis of hypothalamic metabolic neuropeptide expression following delineation of transactivated structures by Fos immuno-mapping.

    Science.gov (United States)

    Alenazi, Fahaad S H; Ibrahim, Baher A; Briski, Karen P

    2015-04-01

    Fos immunocytochemistry is a valuable anatomical mapping tool for distinguishing cells within complex tissues that undergo genomic activation, but it is seldom paired with corroborative molecular analytical techniques. Due to preparatory requirements that include protein cross-linking for specimen sectioning, histological tissue sections are regarded as unsuitable for those methods. Our studies show that pharmacological activation of the hindbrain energy sensor AMPK by AICAR elicits estradiol (E)-dependent patterns of Fos immunolabeling of hypothalamic metabolic loci. Here, Western blotting was applied to hypothalamic tissue removed from histological sections of E- versus oil (O)-implanted ovariectomized (OVX) female rat brain to measure levels of metabolic transmitters associated with Fos-positive structures. In both E and O rats, AICAR treatment elicited alterations in pro-opiomelanocortin, neuropeptide Y, SF-1, and orexin-A neuropeptide expression that coincided with patterns of Fos labeling of structures containing neurons that synthesize these neurotransmitters, e.g. arcuate and ventromedial nuclei and lateral hypothalamic area. O, but not E animals also exhibited parallel augmentation of tissue corticotropin-releasing hormone neuropeptide levels and paraventricular nucleus Fos staining. Data demonstrate the utility of immunoblot analysis as a follow-through technique to capitalize on Fos mapping of transactivation sites in the brain. Findings that induction of Fos immunoreactivity coincides with adjustments in hypothalamic metabolic neuropeptide expression affirms that this functional indicator reflects changes in neurotransmission in pathways governing metabolic outflow. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Immunohistochemical and western blot analysis suggest that the soluble forms of FGF1-2 and FGFR1-2 sustain tail regeneration in the lizard.

    Science.gov (United States)

    Alibardi, Lorenzo

    2017-08-18

    Fibroblast Growth Factors 1-2 (FGF1-2) stimulate tail regeneration in lizards and therefore the distribution of their receptors, FGFR1-2, in the regenerating tail of the lizard. Podarcis muralis has been studied using immunofluorescence and western blotting. Immunoreactive protein bands at 15-16kDa for FGF1-2 in addition to those at 50-65kDa are detected in the regenerating epidermis, but weak bands at 35, 45 and 50kDa appear from the regenerating connective tissues. Strongly immunolabeled bands for FGFR1 at 32, 60, and 80kDa and less intense for FGFR2 only appear in the regenerating tail. In normal tail epidermis and dermis, higher MW forms are present at 80 and 115-140kDa, respectively, but they disappear in the regenerating epidermis and dermis where low MW forms of FGFR1-2 are found at 50-70kDa. Immunolocalization confirms that most FGFR1-2 are present in the wound epidermis, Apical Epidermal Peg, ependymal tube while immunolabeling lowers in regenerating muscles, blastema cells, cartilage and connectives tissues. The likely release of FGFs from the Apical Epidermal Peg and ependyma and the presence of their receptors in these tissues may determine the autocrine stimulation of proliferation and a paracrine stimulation of the blastema cells through their FGF Receptors. Copyright © 2017 Elsevier GmbH. All rights reserved.

  3. Western blot seroindeterminate individuals for Human T-lymphotropic Virus 1/2 (HTLV-1/2 in Fortaleza (Brazil: a serological and molecular diagnostic and epidemiological approach

    Directory of Open Access Journals (Sweden)

    Terezinha de Jesus Teixeira Santos

    2003-06-01

    Full Text Available How to handle Western blot (WB seroindeterminate individuals for Human T-lymphotropic Virus 1/2 (HTLV-1/2 constitutes a challenge for blood banks and fam ilies. We made a cross-sectional study of 191 enzyme linked immunoassay (EIA reactive individuals from the hematological center (HEMOCE of Fortaleza (Brazil, examining their serological (WB and molecular (PCR diagnosis, and demographic profiles, as well as a possible association of their condition with other infectious pathologies and risk factors. Ethical institutional approval and personal consent were obtained. Out of 191 EIA reactive individuals, 118 were WB seroindeterminate and 73 were seropositive for HTLV-1/2. In the PCR analysis of 41 WB seroindeterminate individuals, 9 (22% were positive and 32 (78% were negative for HTLV-1/2. The demographic analysis indicated a trend towards a predominance of males among the seroindeterminate individuals and females in the seropositive ones. The seroindeterminate individuals were younger than the seropositive ones. We did not find any association of these conditions with syphilis, Chagas disease or HIV or hepatitis, and with risk factors such as breast-feeding, blood transfusion, STD (syphilis and IDU.

  4. Evaluación de la técnica Western blot para la detección de antígenos de Hymenolepis nana

    Directory of Open Access Journals (Sweden)

    Flora Chávez-Salas

    2013-04-01

    Full Text Available Este trabajo tuvo como objetivo evaluar la técnica de inmunoelectrotransferencia (Western Blot para detectar los antígenos específicos de excreción/secreción de Hymenolepis nana en sueros de pacientes con himenolepiosis y con otras helmintiosis confirmadas. Se utilizó a Mesocricetus auratus “hamster” para obtener ejemplares adultos de H. nana. Los antígenos de excreción/secreción fueron obtenidos en el medio MEM (Minimum Essential Medium Eagle, y enfrentados con un grupo de sueros de pacientes con himenolepiosis confirmada para evaluar su calidad inmunológica y con sueros individuales de pacientes con himenolepiosis y con otras helmintiosis confirmadas para detectar mediante la técnica de “Western Blot”, los antígenos específicos de este cestode. El grupo de sueros de pacientes con himenolepiosis confirmada reconoció las bandas antigénicas de 50,1; 42,6; 38,9; 32,9; 26,3; 22,4 y 18,6 kDa; sin embargo, los sueros individuales reconocieron diferente número de bandas, siendo la de 50,1 KDa la que fue reconocida por todos ellos. Los sueros de pacientes con helmintiosis confirmadas no reconocieron la banda de 50,1 kDa; sin embargo, dieron reacción cruzada con algunas de las demás bandas, a excepción de los sueros de pacientes con cisticercosis que no reconocieron a ninguna de las bandas de estos antígenos. Se concluye que el antígeno de excreción/secreción de H. nana de 50,1 kDa es específico de este cestode por ser reconocido por todos los sueros de pacientes con himenolepiosis confirmada y no por sueros de pacientes con otras helmintiosis utilizando la técnica de “Western Blot”.

  5. Reconnaissance paleomagnetic results from western Cuba

    Science.gov (United States)

    Bazhenov, Mikhail L.; Pszczolkowski, Andrzej; Shipunov, Stanislav V.

    1996-03-01

    A paleomagnetic study of Mesozoic rocks from the Sierra de Los Organos and Sierra del Rosario fold belts of western Cuba revealed postfolding magnetisation in diabases of the Late Jurassic El Sábalo Formation and carbonates of the middle Cretaceous Pons and the Late Cretaceous Carmita and Moreno formations. Steep components with inclinations of about 70° were isolated from all three formations; at the same time, postfolding shallow components were also found in a few samples of the Pons limestones. We rule out a possibility to account for these results by either horizontal movements or non-dipole field anomaly. Neither very appealing is a hypothesis of a post-remagnetization tilt of the entire region. All the components appear to be confined to a plane perpendicular to the main structural trends; we hypothesize that the remanences might have been distorted or re-aligned during deformation; this assumption, however, is far from being proven. In contrast, well-defined characteristic components were isolated from basalts of the Aptian-Albian Encrucijada ( {D}/{I} = {247°}/{23°}, K = 14, a95 = 9.0°) and the Late Cretaceous Orozco ( {D}/{I} = {228°}/{22°}, K = 110, a95 = 4.7) formations from the Bahia Honda zone in the north of western Cuba; the remanence in the Encrucijada Formation is shown to predate deformation. Mean inclinations in both formations match those in Cretaceous volcanics from central Cuba, and all the results show lower latitudes than expected from the reference data for the North American plate thus implying that volcanic domains of Cuba were displaced northward by about 1000 km prior to the Middle Eocene. Cretaceous declinations in western and central Cuba differ by about the same amount as the major structural trends of these two areas suggesting oroclinal bending of Cuba. At the same time, both areas are rotated counterclockwise with respect to North America thus implying movements on a broader scale.

  6. Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment

    Directory of Open Access Journals (Sweden)

    Tetsuya Okuda

    2017-02-01

    Full Text Available Protein modification by O-linked N-acetylglucosamine (O-GlcNAcylation is one of the post transcriptional modifications occurring on cellular proteins. This paper provides a data set relating to the O-GlcNAcylation of cellular proteins detected by RL2 and CTD110.6 antibodies, which are commonly used for detection of protein O-GlcNAcylation, in 2-deoxy-d-glucose (2DG-treated human teratocarcinoma NCCIT cells in support of the research article entitled “A novel, promoter-based, target-specific assay identifies 2-deoxy-d-glucose as an inhibitor of globotriaosylceramide biosynthesis” (Okuda et al., 2009 [1]. The main article described a suppressive effect of 2DG on an Sp1 target gene in NCCIT cells and discussed the relationship between the effect of 2DG and O-GlcNAcylation status of Sp1. The data in this paper complements this relationship by Western blotting and clearly showed that the 2DG treatment increased O-GlcNAcylation of cellular proteins in NCCIT cells, whereas the RL2 and CTD110.6 epitopes were detected in a different manner. The RL2 epitope was detected on Sp1 during 2DG treatment, and the level was transiently increased at 24 h. In contrast, the CTD110.6 epitope became detectable on Sp1 over 72 h after 2DG treatment, and then the other proteins containing CTD110.6 epitopes also appeared in the cell lysates and the anti-Sp1 antibody precipitates.

  7. Comparison analysis between Western blot method and Magnetic particle Chemiluminescence method detection SLE autoantibody%免疫印迹法和磁微粒化学发光法检测SLE自身抗体的比对分析

    Institute of Scientific and Technical Information of China (English)

    周厚清

    2016-01-01

    目的:比较免疫印迹法(Western blot)和磁微粒化学发光法(Magnetic particle Chemiluminescence method)检测系统性红斑狼疮(SLE)自身抗体的一致性。方法:用免疫印迹法和磁微粒化学发光法对123份血清进行检测,其中系统性红斑狼疮阳性患者63例,健康体检者60例,用卡方检验对两种方法结果进行比较分析。结果:免疫印迹法和磁微粒化学发光法中抗Sm抗体阳性率分别为27.0%和14.3%、抗RNP/Sm阳性率分别为54.0%和42.9%、dsDNA阳性率分别为41.3%和30.2%、抗核小体抗体阳性率分别为31.7%和20.6%、抗核糖体P蛋白抗体阳性率分别为28.6%和11.1%。两种方法的准确性无显著性差异(>0.05)总符合率为80.6%,Kappa值为0.669。结论:免疫印迹法和磁微粒化学发光法检测系统性红斑狼疮自身抗体的检测结果具有可靠性,较好的符合率和准确性,磁微粒化学发光法可以作为简便、快速、经济的检测方法应用于系统性红斑狼疮自身抗体的检测。%Objective: The present study aims to compare Western blot and Magnetic particle Chemiluminescence method consistency on autoimmune antibody detection of systemic lupus erythematosus. Methods: Western blot assay and Magnetic particle Chemiluminescence method were used to detect autoimmune antibody from 123 serum samples, of which 63 patients with systemic lupus erythematosus, the others from 60 healthy persons. The results of the two methods were compared by X2 test. Results: Anti-Sm antibody positive rate of Western blot and Magnetic particle Chemiluminescence method were 27.0 and 14.3 percent respectively. Anti- RNP/Sm antibody positive rates were 39.7% and 42.9% respectively. Anti- ds- DNA antibody positive rates were 41.3% and 30.2% respectively. Anti- nucleosome antibody positive rates were 31.7% and 20.6% respectively. Anti- ribosomal protein antibody positive rates were 28.6% and 11.1% respectively. The accuracy

  8. Northern blotting analysis

    DEFF Research Database (Denmark)

    Josefsen, Knud; Nielsen, Henrik

    2011-01-01

    is analysed by hybridization to one or more specific probes that are labelled for subsequent detection. Northern blotting is relatively simple to perform, inexpensive, and not plagued by artefacts. Recent developments of hybridization membranes and buffers have resulted in increased sensitivity closing...

  9. Quantitative analysis of the IgG and IgG subclass immune responses to chromosomal Pseudomonas aeruginosa beta-lactamase in serum from patients with cystic fibrosis by western blotting and laser scanning densitometry

    DEFF Research Database (Denmark)

    Petersen, T D; Ciofu, O; Pressler, T

    1996-01-01

    lung infection with P aeruginosa was further investigated by correlating the a beta ab IgG subclasses with pulmonary function in patients with cystic fibrosis. METHODS: Immunoglobulin G (IgG) and IgG subclass a beta ab were investigated by western blotting and quantified by laser scanning densitometry...

  10. Delineation of pulmonary airway fluid protein fractions with HRPO binding-avidity by far-Western ligand blot and mass spectrometry analyses: a model methodology for detecting mannose-binding protein expression profiles.

    Science.gov (United States)

    Coyne, Cody P; Rashmir-Raven, Ann; Jones, Toni; Mochal, Cathleen; Linford, Robert L; Brashier, Michael; Eddy, Alison

    2009-01-01

    Limited research to date has characterized the potential for HRPO to function as a primary molecular probe. Pulmonary airway fluid was developed by non-reducing far-Western (ligand) blot analyses utilizing conjugated HRPO-strepavidin or non-conjugated HRPO without the presence of primary immunoglobulin. Endogenous esterase-like biochemical activity of fractions within pulmonary airway fluid was inactivated to determine if they were capable of biochemically converting HRPO chemiluminescent substrate. Complementary analyses modified pulmonary fluid and HRPO with beta-galactosidase and alpha-mannosidase respectively, in addition to determining the influence of mannose and maltose competitive binding on HRPO far-Western (ligand) blot analyses. Identification of pulmonary fluid fractions detected by HRPO far-Western blot analyses was determined by mass spectrometry. Modification of pulmonary fluid with beta-galactosidase, and HRPO with alpha-mannosidase in concert with maltose and mannose competitive binding analyses altered the intensity and spectrum of pulmonary fluid fractions detected by HRPO far-Western blot analysis. Identity of pulmonary airway fluid fractions detected by HRPO far-Western (ligand) blot analysis were transferrin, dynein, albumin precursor, and two 156 kDa equine peptide fragments. HRPO can function as a partially-selective primary molecular probe when applied in either a conjugated or non-conjugated form. Some protein fractions can form complexes with HRPO through molecular mechanisms that involve physical interactions at the terminal alpha-mannose-rich regions of HRPO glycan side-chains. Based on its known molecular composition and structure, HRPO provides an opportunity for the development of diagnostics methodologies relevant to disease biomarkers that possess mannose-binding avidity.

  11. Testing UK blood donors for exposure to human parvovirus 4 using a time-resolved fluorescence immunoassay to screen sera and Western blot to confirm reactive samples.

    Science.gov (United States)

    Maple, Peter A C; Beard, Stuart; Parry, Ruth P; Brown, Kevin E

    2013-10-01

    Human parvovirus 4 (ParV4), a newly described member of the family Parvoviridae, like B19V, has been found in pooled plasma preparations. The extent, and significance, of ParV4 exposure in UK blood donors remain to be determined and reliable detection of ParV4 immunoglobulin (Ig)G, using validated methods, is needed. With ParV4 virus-like particles a ParV4 IgG time-resolved fluorescence immunoassay (TRFIA) was developed. There is no gold standard or reference assay for measuring ParV4 IgG and the utility of the TRFIA was first examined using a panel of sera from people who inject drugs (PWIDS)--a high-prevalence population for ParV4 infection. Western blotting was used to confirm the specificity of TRFIA-reactive sera. Two cohorts of UK blood donor sera comprising 452 sera collected in 1999 and 156 sera collected in 2009 were tested for ParV4 IgG. Additional testing for B19V IgG, hepatitis C virus antibodies (anti-HCV), and ParV4 DNA was also undertaken. The rate of ParV4 IgG seroprevalence in PWIDS was 20.7% and ParV4 IgG was positively associated with the presence of anti-HCV with 68.4% ParV4 IgG-positive sera testing anti-HCV-positive versus 17.1% ParV4 IgG-negative sera. Overall seropositivity for ParV4 IgG, in 608 UK blood donors was 4.76%. The ParV4 IgG seropositivity for sera collected in 1999 was 5.08%, compared to 3.84% for sera collected in 2009. No ParV4 IgG-positive blood donor sera had detectable ParV4 DNA. ParV4 IgG has been found in UK blood donors and this finding needs further investigation. © 2013 American Association of Blood Banks.

  12. Diagnostic performance of ELISA, IFAT and Western blot for the detection of anti-Leishmania infantum antibodies in cats using a Bayesian analysis without a gold standard.

    Science.gov (United States)

    Persichetti, Maria Flaminia; Solano-Gallego, Laia; Vullo, Angela; Masucci, Marisa; Marty, Pierre; Delaunay, Pascal; Vitale, Fabrizio; Pennisi, Maria Grazia

    2017-03-13

    Anti-Leishmania antibodies are increasingly investigated in cats for epidemiological studies or for the diagnosis of clinical feline leishmaniosis. The immunofluorescent antibody test (IFAT), the enzyme-linked immunosorbent assay (ELISA) and western blot (WB) are the serological tests more frequently used. The aim of the present study was to assess diagnostic performance of IFAT, ELISA and WB to detect anti-L. infantum antibodies in feline serum samples obtained from endemic (n = 76) and non-endemic (n = 64) areas and from cats affected by feline leishmaniosis (n = 21) by a Bayesian approach without a gold standard. Cut-offs were set at 80 titre for IFAT and 40 ELISA units for ELISA. WB was considered positive in presence of at least a 18 KDa band. Statistical analysis was performed through a written routine with MATLAB software in the Bayesian framework. The latent data and observations from the joint posterior were simulated in the Bayesian approach by an iterative Markov Chain Monte Carlo technique using the Gibbs sampler for estimating sensitivity and specificity of the three tests. The median seroprevalence in the sample used for evaluating the performance of tests was estimated at 0.27 [credible interval (CI) = 0.20-0.34]. The median sensitivity of the three different methods was 0.97 (CI: 0.86-1.00), 0.75 (CI: 0.61-0.87) and 0.70 (CI: 0.56-0.83) for WB, IFAT and ELISA, respectively. Median specificity reached 0.99 (CI: 0.96-1.00) with WB, 0.97 (CI: 0.93-0.99) with IFAT and 0.98 (CI: 0.94-1.00) with ELISA. IFAT was more sensitive than ELISA (75 vs 70%) for the detection of subclinical infection while ELISA was better for diagnosing clinical leishmaniosis when compared with IFAT (98 vs 97%). The overall performance of all serological techniques was good and the most accurate test for anti-Leishmania antibody detection in feline serum samples was WB.

  13. ANALYSIS OF Treponema pallidum RECOMBINANT ANTIGENS FOR DIAGNOSIS OF SYPHILIS BY WESTERN BLOTTING TECHNIQUE Análise de antígenos recombinantes de Treponema pallidum no diagnóstico da sífilis utilizando a técnica de Western Blotting

    Directory of Open Access Journals (Sweden)

    Neuza Satomi SATO

    1999-03-01

    Full Text Available Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rTp17 antigen. Some samples were non-reactive or showed a weak reaction with GST-rTp47 and/or GST-rTp15, and apparently there was no correlation with the stage of disease. There was no seropositivity among blood donors. No sample reacted with purified GST. We concluded that due to their specificity these recombinant antigens can be used as GST fusion protein for development of syphilis diagnostic assays.Os antígenos recombinantes de Treponema pallidum GST-rTp47, GST-rTp17 e GST-rTp15, produzidos em fusão com glutationa S-transferase (GST em E. coli, foram analisados quanto ao potencial diagnóstico da sífilis pela técnica de Western blotting. Foram testadas 53 amostras, sendo 25 de pacientes em diferentes estágios clínicos da sífilis, com resultados positivos no teste treponêmico clássico; 25 amostras procedentes de doadores de banco de sangue, com sorologia negativa e 3 de pacientes com doença sexualmente transmissível não relacionado à sífilis. Todas as amostras de pacientes com sífilis apresentaram alta reatividade com o antígeno GST-rTp17. Quanto aos antígenos GST-rTp47 e GST-Tp15 verificou-se uma variação na presença ou na intensidade da reação em diferentes amostras de pacientes com sífilis, sem mostrar correlação com o estágio da doença. Nenhuma reatividade contra quaisquer desses antígenos foi observada com as amostras do grupo controle. Nenhuma das amostras testadas apresentaram reatividade com a GST purificada. A

  14. Comparison of T24H-his, GST-T24H and GST-Ts8B2 recombinant antigens in western blot, ELISA and multiplex bead-based assay for diagnosis of neurocysticercosis.

    Science.gov (United States)

    Hernández-González, Ana; Noh, John; Perteguer, María Jesús; Gárate, Teresa; Handali, Sukwan

    2017-05-15

    Currently, the reference standard assay for the serodiagnosis of neurocysticercosis (NCC) is the lentil lectin-bound glycoproteins/enzyme-linked immunoelectrotransfer blot (LLGP-EITB). The main disadvantage of this technique is the complexity of obtaining and purifying the LLGP extract. This could be solved by replacement with highly specific recombinant antigens from Taenia solium. Based on previous studies, we selected and produced the recombinant Ts8B2 and T24H proteins and applied them to three diagnostic techniques: western blot (WB), enzyme-linked immunosorbent assay (ELISA) and the multiplex bead-based assay (MBA). The Ts8B2 and T24H cDNA sequences were expressed in a prokaryotic system and the corresponding expression products purified; three recombinant proteins were further characterized: T24H-his, GST-T24H and GST-Ts8B2. The proteins on WB, ELISA and MBA were tested against 149 sera from patients with NCC confirmed by brain imaging, 40 sera from patients with other parasitic diseases, and 131 sera from US. individuals without evidence of neurocysticercosis (clinical/serological/brain imaging). The sensitivity and specificity of each antigen by WB were calculated by counting the number of true positive, false positive, true negative and false negative results. Using the receiver operating characteristic (ROC) curves, the cut-off values for the ELISA and MBA were established as well as the sensitivity and specificity of each assay. All three antigens showed a high sensitivity on WB in active NCC cases with two or more viable cysts and low sensitivity for cases with single viable cyst or calcified lesions and inactive NCC. WB showed the highest specificity and sensitivity out of the three diagnostic techniques. The recombinant T24H-his was the best diagnostic reagent in WB (100% sensitivity, 99.4% specificity), exhibiting similar results to the LLGP-EITB, against the same panel of NCC sera. The GST-T24H antigen worked better than the others in ELISA and MBA

  15. Northern blotting analysis

    DEFF Research Database (Denmark)

    Josefsen, Knud; Nielsen, Henrik

    2011-01-01

    Northern blotting analysis is a classical method for analysis of the size and steady-state level of a specific RNA in a complex sample. In short, the RNA is size-fractionated by gel electrophoresis and transferred by blotting onto a membrane to which the RNA is covalently bound. Then, the membran...

  16. Problem-Solving Test: Southwestern Blotting

    Science.gov (United States)

    Szeberényi, József

    2014-01-01

    Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA,…

  17. Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

    Science.gov (United States)

    Methogo, Ruth Menque; Dansette, Patrick M.; Klarskov, Klaus

    2007-12-01

    A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

  18. 亚洲带绦虫囊尾蚴特异性抗原筛选和鉴定%Screen and detection of specific antigens of Taenia asiatica cysticercus with two dimensional electrophoresis, western blotting and MS/MS spectrometry

    Institute of Scientific and Technical Information of China (English)

    方文; 肖靓靓; 包怀恩; 牟荣

    2011-01-01

    Objective To screen and identify specific antigens of Taenia asiatica cysticercus and to predict the function of target proteins using bioinformatics method. Methods Six three-way cross hybrid pigs were randomly divided into experimental group and control group and each experimental pig was infected with 84 000 eggs. Serum samples were collected 40 days after the infection. The total protein of Taenia asiatica cysticercus was separated by two dimensional electrophoresis and western blotting was performed to find out distinct antigens. Proteins from the two groups were identified by electrospray ion trap mass spectrometry(ESI-Trap MS). Results Totally 204 ± 11 spots were detected through Coomassie brilliant blue stained gels with relative molecular weight( Mr) of 14 400 -94 000 kDa and isoelectric point(pl) of 3. 0 - 10. 0. Western-blotting showed 6 specific antigen spots with pool sera of infected pigs. Two of the 6 antigens with known functions were ascribed to ATPase family gene 3-like protein 2 and ATP synthase beta subunit. Conclusion Two specific antigens of Taenia asiatica cysticercus have been identified.%目的 对亚洲带绦虫囊尾蚴的特异性抗原进行筛选、鉴定和生物信息学分析.方法 6头20d龄三元杂交乳猪随机分为实验组和对照组,实验组每头灌胃亚洲带绦虫虫卵悬液8.4万个;20 d后,分别制备实验组及对照组心脏血混合血清;收集、制备实验组亚洲带绦虫囊尾蚴蛋白并进行双向电泳分析,将凝胶蛋白斑点转移至聚偏氟乙烯膜,用实验组及对照组混合血清(均为1∶10)为一抗,羊抗猪辣根过氧化物酶- IgG(1∶200)为二抗,进行蛋白质印迹分析和质谱鉴定.结果 双向电泳凝胶共检测到( 204±11)个蛋白质斑点,相对分子质量为14 400 ~94 000 kPa,等电点(pI)为3.0~10.0;筛选出6个特异性抗原,经质谱鉴定出其中2个蛋白,分别为ATP酶管家基因3-类蛋白2(AT-Pase family gene 3-like protein 2)

  19. Analysis of glabrous canary seeds by ELISA, mass spectrometry, and Western blotting for the absence of cross-reactivity with major plant food allergens.

    Science.gov (United States)

    Boye, Joyce Irene; Achouri, Allaoua; Raymond, Nancy; Cleroux, Chantal; Weber, Dorcas; Koerner, Terence B; Hucl, Pierre; Patterson, Carol Ann

    2013-06-26

    Glabrous (hairless) canary seed belongs to the Poaceae (Gramineae) family and could serve as an alternative source of gluten-free cereal grain. In this study, allergenic cross-reactivities between hairless, dehulled canary seeds (Phalaris canariensis) and major allergenic proteins from gluten, soy, peanuts, tree nuts, sesame, and mustard were studied using commercial enzyme-linked immune sorbent assay (ELISA) kits specific for these target allergens. Mass spectrometry (MS) and immunoblotting were further used to assess for the presence of gluten-specific protein fragments. MS results revealed the likely presence of proteins homologous with rice, oat, corn, carrot, tomato, radish, beet, and chickpea. However, no presence of celiac-related gluten fragments from wheat, rye, barley, or their derivatives was found. Immunoblotting studies yielded negative results, further confirming the absence of gluten in the canary seed samples tested. No cross-reactivities were detected between canary seeds and almond, hazelnut, mustard, peanut, sesame, soy, walnut, and gluten using ELISA.

  20. Detection of mu opioid receptor (MOPR) and its glycosylation in rat and mouse brains by western blot with anti-μC, an affinity-purified polyclonal anti-MOPR antibody.

    Science.gov (United States)

    Huang, Peng; Chen, Chongguang; Liu-Chen, Lee-Yuan

    2015-01-01

    Our experience demonstrates that it is difficult to identify MOPR in rat and mouse brains by western blot, in part due to low abundance of the receptor and a wide relative molecular mass (Mr) range of the receptor associated with its heterogeneous glycosylation states. Here, we describe generation and purification of anti-μC (a rabbit polyclonal anti-MOPR antibody), characterization of its specificity in immunoblotting of HA-tagged MOPR expressed in a cell line, and ultimately, unequivocal detection of the MOPR in brain tissues by western blot with multiple rigorous controls. In particular, using brain tissues from MOPR knockout (K/O) mice as the negative controls allowed unambiguous identification of the MOPR band, since the anti-MOPR antibody, even after affinity purification, recognizes nonspecific protein bands. The MOPR was resolved as a faint, broad, and diffuse band with a wide Mr range of 58-84 kDa depending on brain regions and species. Upon deglycosylation to remove N-linked glycans by PNGase F (but not Endo H), the MOPR became a dense and sharp band with Mr of ~43 kDa, close to the theoretical Mr of its deduced amino acid sequences. Thus, MOPRs in rodent brains are differentially glycosylated by complex type of N-linked glycans in brain region- and species-specific manners. Furthermore, we characterized the MOPR in an A112G/N38D-MOPR knockin mouse model that possesses the equivalent substitution of the A118G/N40D SNP in the human MOPR gene. The substitution removes one of the four and five N-linked consensus glycosylation sites of the mouse and human MOPR, respectively. We demonstrated that the Mr of the MOPR in A112G mouse brains was lower than that in wild-type mouse brains, and that the difference was due to lower degrees of N-linked glycosylation.

  1. Interrogation of multidrug resistance (MDR1) P-glycoprotein (ABCB1) expression in human pancreatic carcinoma cells: correlation of 99mTc-Sestamibi uptake with western blot analysis.

    Science.gov (United States)

    Harpstrite, Scott E; Gu, Hannah; Natarajan, Radhika; Sharma, Vijay

    2014-10-01

    Histopathological studies indicate that ∼63% of pancreatic tumors express multidrug resistance (MDR1) P-glycoprotein (Pgp) and its polymorphic variants. However, Pgp expression detected at the mRNA or protein level does not always correlate with functional transport activity. Because Pgp transport activity is affected by specific mutations and the phosphorylation state of the protein, altered or less active forms of Pgp may also be detected by PCR or immunohistochemistry, which do not accurately reflect the status of tumor cell resistance. To interrogate the status of the functional expression of MDR1 Pgp in MiaPaCa-2 and PANC-1 cells, cellular transport studies using Tc-Sestamibi were performed and correlated with western blot analysis. Biochemical transport assays in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells, human epidermal carcinoma drug-sensitive KB-3-1 cells, and human breast carcinoma MCF-7 cells (negative controls), and human epidermal carcinoma drug-resistant KB-8-5 cells, human breast carcinoma stably transfected with Pgp MCF-7/MDR1Pgp cells, and liver carcinoma HepG2 cells (positive controls) were performed. Protein levels were determined using a monoclonal antibody C219. Tc-Sestamibi demonstrates accumulation in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells. Uptake profiles are not affected by treatment with LY335979, a Pgp inhibitor, and correlate with western blot analysis. These cellular transport studies indicate an absence of Pgp at a functional level in MiaPaCa-2 and PANC-1 cells. Because major pancreatic tumors originate from the pancreatic duct and Tc-Sestamibi undergoes a dominant hepatobiliary mode of excretion, it would not be a sensitive probe for imaging pancreatic adenocarcinomas. Following interrogation of the functional status of Pgp in other pancreatic carcinoma cells, chemotherapeutic drugs that are also MDR1 substrates could offer alternative therapeutics for treating pancreatic adenocarcinomas.

  2. 新孢子虫和弓形虫ELISA及western blot检测方法的建立及应用%Establishment and application of indirect ELISA assays and western blot to detect antibodies agaist Neospora caninum and Toxoplasma gondii

    Institute of Scientific and Technical Information of China (English)

    陈亮; 闫双; 刘启生; 曹雯丽; 王真; 巴音查汗

    2012-01-01

    为建立牛新孢子虫和弓形虫的免疫学检测方法,并调查新疆部分地区牛新孢子虫和弓形虫的感染情况,本研究应用纯化的新孢子虫重组蛋白SRS2 (NcSRS2)和弓形虫重组蛋白SAG2 (TgSAG2)作为包被抗原,分别建立新孢子虫和弓形虫的ELISA和western blot血清学检测方法,并进行特异性和重复性试验,以其检测662份疑似样品,并与商品化试剂盒检测结果比较验证.特异性和重复性试验结果表明,建立的方法特异性强、重复性良好.采用建立的两种ELISA方法对662份临床样品的检测结果表明,新孢子虫和弓形虫的抗体阳性率分别为13.44%(89/662)和5.29%(35/662);与IDEXX试剂盒和永辉试剂盒的符合率分别为94.11%和95.92%.此外,western blot检测的新孢子虫和弓形虫抗体阳性率分别为5.14%(34/662)和3.17%(21/662);与建立的ELISA检测方法的符合率分别为91.69%和97.89%.本研究为分析奶牛流产的原因提供了一定的依据.%Neospora caninum and Toxoplasma gondii are closely related protozoan parasites which causes of abortion and congenital disease in ruminants. To establish the methods for detection of the antibodies against N. caninum and T. gondii in bovine, The indirect ELISAs and western blot assay were developed based on purified recombinant protein SRS2 of N. cammim and SAG2 of T. gondii, respectively. The results show that these methods were highly specificity and repeatability. A total of 662 bovine serum samples were tested and the positive rates for N. caninum and T. gondii were 13.44% (89/662) and 5.29% (35/662) detected by the developed ELISAs, which were 94.11% and 95.92% agreements with TDEXX' N. caninum Antibody Test Kit and Yong hui' T. gondii Antibody Test Kit, respectively. In addition, the positive rates for N. caninum and T. gondii were 5.14% (34/662) and 3.17% (21/662) using western blot, and the agreements with the developed ELISAs were 91.69% and 97

  3. Comparison of GP5+/6+-PCR and SPF10-line blot assays for detection of high-risk human papillomavirus in samples from women with normal cytology results who develop grade 3 cervical intraepithelial neoplasia.

    NARCIS (Netherlands)

    Hesselink, A.T.; Ham, M.A.P.C. van; Heideman, D.A.; Groothuismink, Z.M.; Rozendaal, L.; Berkhof, J.; Kemenade, F.J. van; Massuger, L.A.; Melchers, W.J.G.; Meijer, C.J.; Snijders, P.J.L.M.

    2008-01-01

    Using a case control approach, we performed a two-way comparison study between GP5+/6+-PCR and HPV SPF(10)-Line Blot 25 (SPF(10)) assays for detection of 14 types of high-risk human papillomavirus (hrHPV) in samples from women with normal cytology results who had or developed grade 3 cervical

  4. Results and perspectives of tectonomagnetic investigations in the Western Antarctic

    Directory of Open Access Journals (Sweden)

    Ihor O. Chobotok

    2009-06-01

    Full Text Available The results of long-term (1998-2005 yrs. tectonomagnetic investigations in the Western Antarctic near the location
    of Ukrainian Antarctic Station «Academic Vernadsky» are reviewed. The peculiarities of the Earth’s
    anomalous magnetic field and its dynamic temporal variations (tectonomagnetic anomalies were studied using
    the newly founded tectonomagnetic polygon. Near the Argentine Archipelago intensive tectonomagnetic effects
    up to -2.8 nT/year were determined. Their spatial-temporal structure agrees with tectonic structure elements. We
    suggest that the nature of such effects is caused by a piezomagnetic effect under the influence of stretching tectonic
    forces (few bars per year in sub-latitudinal direction. Perspectives of tectonomagnetic investigations in the
    region are discussed.

  5. 孕产妇HIV抗体筛查阳性与免疫印迹试验对比%Comparison of Positive of HIV Antibody Screening and Western Blot Test in Pregnant Women

    Institute of Scientific and Technical Information of China (English)

    朱厚宏; 王晨笛; 刘杨; 张祁; 孟建彤; 黄薇

    2011-01-01

    Objective To explore the relationship between the results of HIV antibody screening test and Westem Blot test ( WB) in pregnant women. Methods Thirty three positive samples from pregnant women were tested with the ELISA and Westem blot test (WB). Those with indefinite result by WB test were re-tested 3 and 6 months later, and HIV-1 viral load testing ( VL) was carried out at 6th month. The results of ELISA and the WB test were analyzed. Result The 33 samples were all positive by ELISA and 20 were HIV-1 positive by WB test. The ELISA and the WB test positive coincidence rate was 60. 61 % (20/33). Of 20 samples 0were with S/CO value more than 8, the WB test results of HIV-1 antibody were all positive. Of 13 samples were with S/CO value less than 1 , 5 were negative and 8 were indefinite in WB test. The HIV-1 viral load test result at 6th month was less than the minimum detection limit (50 IU/ml ) . Conclusion The false positive in HIV antibody screening test (ELISA) was observed. The false positive sample were in the WB test negative and indefinite ones. ELISA and the WB test positive coincidence rate increased with the S/CO value of ELISA. The HIV WB test indefinite result in pregnant women was mostly due to non-specific immune response.%目的 探讨孕产妇人类免疫缺陷病毒(HIV)抗体筛查试验阳性与免疫印迹试验(WB)结果的关系.方法 对33份孕产妇HIV抗体待复查样本,进行酶联免疫吸附试验(ELISA )及免疫印迹试验(WB),对WB试验结果不确定者进行3个月、6个月随访检测,6个月随访样本补充HIV-1病毒载量检测,分析ELISA检测与WB试验检测结果.结果 33份样本ELISA复检均呈阳性,免疫印迹试验(WB)中20份确认为HIV-1抗体阳性,ELISA检测与WB试验阳性符合率为60.61%(20/33); S/CO值>8的20份样本,WB试验结果均为HIV-1抗体阳性,I < S/CO值<8的13份样本,WB试验5份为HIV抗体阴性,8份为HIV抗体不确定;6个月随访样本HIV-1病毒载量检测,

  6. The Use of Biotin to Demonstrate Immunohistochemistry, Western Blotting, and Dot Blots in University Practical Classes

    Science.gov (United States)

    Millar, Thomas James; Knighton, Ronald; Chuck, Jo-Anne

    2012-01-01

    Immunological detection of proteins is an essential method to demonstrate to undergraduate biology students, however, is often difficult in resource and time poor student laboratory sessions. This method describes a failsafe method to rapidly and economically demonstrate this technique using biotinylated proteins or biotin itself as targets for…

  7. The Use of Biotin to Demonstrate Immunohistochemistry, Western Blotting, and Dot Blots in University Practical Classes

    Science.gov (United States)

    Millar, Thomas James; Knighton, Ronald; Chuck, Jo-Anne

    2012-01-01

    Immunological detection of proteins is an essential method to demonstrate to undergraduate biology students, however, is often difficult in resource and time poor student laboratory sessions. This method describes a failsafe method to rapidly and economically demonstrate this technique using biotinylated proteins or biotin itself as targets for…

  8. Automated design of genomic Southern blot probes

    Directory of Open Access Journals (Sweden)

    Komiyama Noboru H

    2010-01-01

    Full Text Available Abstract Background Sothern blotting is a DNA analysis technique that has found widespread application in molecular biology. It has been used for gene discovery and mapping and has diagnostic and forensic applications, including mutation detection in patient samples and DNA fingerprinting in criminal investigations. Southern blotting has been employed as the definitive method for detecting transgene integration, and successful homologous recombination in gene targeting experiments. The technique employs a labeled DNA probe to detect a specific DNA sequence in a complex DNA sample that has been separated by restriction-digest and gel electrophoresis. Critically for the technique to succeed the probe must be unique to the target locus so as not to cross-hybridize to other endogenous DNA within the sample. Investigators routinely employ a manual approach to probe design. A genome browser is used to extract DNA sequence from the locus of interest, which is searched against the target genome using a BLAST-like tool. Ideally a single perfect match is obtained to the target, with little cross-reactivity caused by homologous DNA sequence present in the genome and/or repetitive and low-complexity elements in the candidate probe. This is a labor intensive process often requiring several attempts to find a suitable probe for laboratory testing. Results We have written an informatic pipeline to automatically design genomic Sothern blot probes that specifically attempts to optimize the resultant probe, employing a brute-force strategy of generating many candidate probes of acceptable length in the user-specified design window, searching all against the target genome, then scoring and ranking the candidates by uniqueness and repetitive DNA element content. Using these in silico measures we can automatically design probes that we predict to perform as well, or better, than our previous manual designs, while considerably reducing design time. We went on to

  9. Western blot analysis of IgG responses of sheep infected with Fasciola hepatica and F.gigantica%免疫印迹分析试验感染肝片吸虫和大片吸虫绵羊的IgG应答

    Institute of Scientific and Technical Information of China (English)

    张为宇; MOREAU Emmanuelle; 黄维义; CHAUVIN Alain

    2003-01-01

    免疫印迹(Western blot)分析试验感染肝片吸虫和大片吸虫绵羊的抗片形吸虫分泌排泄产物(FhESP和FgESP)IgG应答,结果表明,FhESP中分子质量在17.1~21.6 ku之间的4条主要蛋白带仅被肝片吸虫感染以后(0WPI以后)的血清识别且不被对照组血清识别; FgESP中分子质量在19.0~71.1 ku之间的6条主要蛋白带仅被大片吸虫感染以后(0WPI以后)的血清识别且不被对照组血清识别.

  10. Western blot expression of 5-lipoxygenase in the brain from striped dolphins (stenella coeruleoalba) and bottlenose dolphins (tursiops truncatus) with or without encephalitis/meningo-encephalitis of infectious nature.

    Science.gov (United States)

    Di Guardo, G; Falconi, A; Di Francesco, A; Mazzariol, S; Centelleghe, C; Casalone, C; Pautasso, A; Cocumelli, C; Eleni, C; Petrella, A; Di Francesco, C E; Sabatucci, A; Leonardi, L; Serroni, A; Marsili, L; Storelli, M M; Giacominelli-Stuffler, R

    2015-01-01

    Dolphin Morbillivirus (DMV), Toxoplasma gondii and Brucella ceti are pathogens of major concern for wild cetaceans. Although a more or less severe encephalitis/meningo-encephalitis may occur in striped dolphins (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus) infected by the aforementioned agents, almost no information is available on the neuropathogenesis of brain lesions, including the neuronal and non-neuronal cells targeted during infection, along with the mechanisms underlying neurodegeneration. We analyzed 5-lipoxygenase (5-LOX) expression in the brain of 11 striped dolphins and 5 bottlenose dolphins, affected or not by encephalitic lesions of various degrees associated with DMV, T. gondii and B. ceti. All the 8 striped dolphins with encephalitis showed a more consistent 5-LOX expression than that observed in the 3 striped dolphins showing no morphologic evidence of brain lesions, with the most prominent band intensity being detected in a B. ceti-infected animal. Similar results were not obtained in T. gondii-infected vs T. gondii-uninfected bottlenose dolphins. Overall, the higher 5-LOX expression found in the brain of the 8 striped dolphins with infectious neuroinflammation is of interest, given that 5-LOX is a putative marker for neurodegeneration in human patients and in experimental animal models. Therefore, further investigation on this challenging issue is also needed in stranded cetaceans affected by central neuropathies.

  11. 间接免疫荧光法与线性免疫印迹法对自身抗体检测结果分析%Analysis of autoantibodies detection between indirect immunofluorescence method and linear western blot method

    Institute of Scientific and Technical Information of China (English)

    王梦涛; 赵婵媛; 刘小玲; 卢春利; 王锐

    2015-01-01

    目的:探讨自身特异性抗体筛查试验间接免疫荧光法(IIF)与确认试验线性免疫印迹法(LIA)检测结果的相关性。方法:用 IIF 法作为抗核抗体(ANA)的筛查试验,用 LIA 法作为抗核抗体谱(ANAs)特异性抗体的确认试验,对自身免疫性疾病组(AID)及非 AID 疾病对照组进行检测分析,并将检测结果分为四组:IIF-ANA+/LIA+组,IIF-ANA+/LIA-组,IIF-ANA-/LIA+组和 IIF-ANA-/LIA-组。结果:①在611例 AID 组中,IIF-ANA+/LIA+336例(55%),IIF-ANA+/LIA-116例(19%),IF-ANA-/LIA+55例(9%),IIF-ANA-/LIA-104例(17%)。②在100例非 AID 组中,IIF-ANA+/LIA+2例(2%),IIF-ANA+/LIA-7例(7%),IIF-ANA-/LIA+3例(3%),IIF-ANA-/LIA-88例(88%),总体符合率90%。AID 组与非 AID 组自身抗体的检测结果比较差异有统计学意义。③ IIF-ANA-/LIA+组中,以抗 SSA 抗体、抗 Ro-52抗体、抗 ds-DNA 抗体、抗组蛋白抗体、抗 Jo-1抗体为主要检测出的阳性自身抗体。AID 组 IIF-ANA+/LIA-中,ANA检测核型以均质型、斑点型为主,混合核型以均质斑点型为主,荧光检测滴度以1∶40~1∶320为主;非 AID 组 IIF-ANA+/LIA-中,核型以斑点型为主,荧光滴度以1∶40~1∶320为主。结论:AID 患者可能会出现 IIF-ANA 检测阳性而 LIA-ANAs 检测阴性或者 IIF-ANA 检测阴性而 LIA-ANAs 检测阳性,检查除了进行 IIF 筛查 ANA 外,还需进行自身抗体谱的特异性抗体确认实验。%Objective:To explore the correlation of the specific antibody results between screening test of IIF and validation test of LIA.Methods:IIF method as ANA screening test,using LIA method as the validation test of ANAs ,Analysis the group of AID and the disease control group of not AID,the test results can be divided into four groups:group IIF ANA + / LIA +,group IIF ANA + / LIA-,group IIF-ANA-/ LIA +,group IIF-ANA-/ LIA-.Results:①In 61 1 cases of AID groups:IIF ANA + / LIA + had 336 patients (55%),IIF ANA+ / LIA-had 1 1 6 patients (1 9%),IIF ANA-/ LIA

  12. Phenylglyoxal-Based Visualization of Citrullinated Proteins on Western Blots

    Directory of Open Access Journals (Sweden)

    Sanne M. M. Hensen

    2015-04-01

    Full Text Available Citrullination is the conversion of peptidylarginine to peptidylcitrulline, which is catalyzed by peptidylarginine deiminases. This conversion is involved in different physiological processes and is associated with several diseases, including cancer and rheumatoid arthritis. A common method to detect citrullinated proteins relies on anti-modified citrulline antibodies directed to a specific chemical modification of the citrulline side chain. Here, we describe a versatile, antibody-independent method for the detection of citrullinated proteins on a membrane, based on the selective reaction of phenylglyoxal with the ureido group of citrulline under highly acidic conditions. The method makes use of 4-azidophenylglyoxal, which, after reaction with citrullinated proteins, can be visualized with alkyne-conjugated probes. The sensitivity of this procedure, using an alkyne-biotin probe, appeared to be comparable to the antibody-based detection method and independent of the sequence surrounding the citrulline.

  13. Post-staining electroblotting for efficient and reliable peptide blotting.

    Science.gov (United States)

    Lee, Der-Yen; Chang, Geen-Dong

    2015-01-01

    Post-staining electroblotting has been previously described to transfer Coomassie blue-stained proteins from polyacrylamide gel onto polyvinylidene difluoride (PVDF) membranes. Actually, stained peptides can also be efficiently and reliably transferred. Because of selective staining procedures for peptides and increased retention of stained peptides on the membrane, even peptides with molecular masses less than 2 kDa such as bacitracin and granuliberin R are transferred with satisfactory results. For comparison, post-staining electroblotting is about 16-fold more sensitive than the conventional electroblotting for visualization of insulin on the membrane. Therefore, the peptide blots become practicable and more accessible to further applications, e.g., blot overlay detection or immunoblotting analysis. In addition, the efficiency of peptide transfer is favorable for N-terminal sequence analysis. With this method, peptide blotting can be normalized for further analysis such as blot overlay assay, immunoblotting, and N-terminal sequencing for identification of peptide in crude or partially purified samples.

  14. Changes in rRNA levels during stress invalidates results from mRNA blotting: Fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels

    DEFF Research Database (Denmark)

    Hansen, M.C.; Nielsen, A.K.; Molin, Søren

    2001-01-01

    experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates...... the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell...... in the hrcA-grpE-dnaK operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining...

  15. Western Red-tailed Skink Distribution in Southern Nevada: Pilot Study Results

    Energy Technology Data Exchange (ETDEWEB)

    Derek Hall, Paul Greger

    2008-11-12

    The western red-tailed skink (Eumeces gilberti rubricaudatus) is a sensitive species that is on the Nevada Natural Heritage Program’s “Animal and Plant At-Risk Tracking List.” Information about this species is lacking, especially for southern Nevada. A pilot project was initiated in 2006 on portions of the Nevada Test Site (NTS) to (1) develop techniques for determining western red-tailed skink distribution, (2) determine if skinks are still present at historic locations, (3) evaluate habitat use by trapping in a variety of habitats, and (4) collect tissue samples for genetic analysis. Skink capture success was compared in trap arrays with and without drift fences. A total of 9 western red-tailed skinks were captured in 6,092 trap days (0.1%, 1 skink/677 trap days). No skinks were captured in trap arrays with drift fences, which suggests that funnel traps set near rocks or vegetation without drift fences is a viable technique for capturing skinks. This greatly reduces the effort and cost to capture skinks. Skinks were captured at one of the three historic locations. Captures occurred in a variety of habitats including springs, ephemeral washes, and dry rocky areas. Genetic analysis revealed that NTS skinks are part of the Inyo clade, and are most closely related to skinks from slightly further north in Esmeralda County, Nevada, and west into the Panamint and Inyo/White Mountains in California. Results from this pilot study will be used to develop a western red-tailed skink distribution study for the entire NTS.

  16. Results of the influenza virus surveillance in wild birds in Western part of Mongolia

    Institute of Scientific and Technical Information of China (English)

    Marchenko V Yu; Otgonbaatar D; Shestopalov AM; Alekseev A Yu; Tserennorov D; Yurlov AK; Susloparov IM; Sharshov KA; Ilyinykh FA; Zolotykh SI; Abmed D

    2010-01-01

    Objective: To present results of virological study of wild birds inhabiting Western Mongolia. Methods: Over a period of 2003-2008, we isolated 13 influenza A viruses: H1N1, H3N6, H13N8 and H4N6 subtypes. We did not isolate any H5N1 subtype, that still cause epizooty in wild birds and poultry.Results: We revealed taxonomic and ecological heterogeneity of the birds involved in maintenance of circulation of influenza viruses in the given territory. Influenza viruses were isolated from birds of 6 orders; among them there are species preferring water and semi-aquatic biotopes, one species preferring dry plain region, and also one species which can inhabit both dry and water biotopes.Conclusions: Representatives of all main orders of Western Mongolia avifauna are involved in support of influenza A virus circulation, highly pathogenic H5N1 influenza viruses were registered in Mongolia thus it's necessary to continue permanent influenza virus surveillance in wild birds' populations.

  17. TLC-Blot (Far-Eastern Blot) and Its Application to Functional Lipidomics.

    Science.gov (United States)

    Taki, Takao

    2015-01-01

    A simple method for transfer of lipids-including phospholipids, glycolipids, and neutral lipids-from a high performance thin layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane, TLC-Blot (Far-Eastern Blot), and its biochemical applications are presented. This chapter presents the conventional procedures for separating lipid from tissue samples, cultured cells, and serum and the subsequent development of TLC. Individual lipids separated on an HPTLC plate can be transferred to the PVDF membrane quantitatively and also isolated from the lipid-blotted membrane by a one-step purification procedure. Immunodetection with monoclonal antibodies and treatment with lipid-metabolizing enzymes on the lipid-blotted membrane are possible. The method for identification of individual lipids transferred on the PVDF membrane using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (TLC-Blot/MALDI-TOF MS) is shown as a functional lipidomics application.

  18. First results of an ambient seismic noise analysis in western Corinth Gulf (Greece)

    Science.gov (United States)

    Giannopoulos, Dimitrios; Paraskevopoulos, Paraskevas; Sokos, Efthimios; Tselentis, G.-Akis

    2015-04-01

    We present the preliminary results of an ambient seismic noise analysis performed in the western Corinth Gulf, Greece. The Corinth Gulf is a continental rift which separates the central Greek mainland from Peloponnese. The rift is approximately 120 km long and 10-20 km wide, with a WNW-ESE orientation, extending from the Gulf of Patras in the west, to the Gulf of Alkionides in the east. It is considered as one of the most active extensional intra-continental rifts in the world, with the geodetically measured rates of extension varying from ~5 mm/yr at the eastern part, to ~15 mm/yr at the western part. We used data from three-component broad-band seismic stations operated under the framework of the Hellenic Unified Seismological Network (HUSN) and the Corinth Rift Laboratory (CRL). After the classical processing of continuous ambient seismic noise recordings, we used both auto-correlation and cross-correlation functions of single stations and station pairs, respectively, in order to retrieve empirical Green's functions (EGFs) of surface waves and estimate relative velocity changes. For estimating the relative velocity changes we used the moving-window cross spectrum analysis (MWCS) technique. This is the first attempt to characterize the ambient seismic noise properties in the area and study the possible relation between the detected relative velocity changes and the occurrence of moderate or strong earthquakes in the study area.

  19. First results from the SHIVA SONNE expedition to the tropical Western Pacific during November 2011

    Science.gov (United States)

    Krüger, K.; Quack, B.; Fuhlbrügge, S.; Tegtmeier, S.; Shiva Sonne Team

    2012-04-01

    The tropical oceans are a known source of reactive bromine and iodine to the atmosphere in the form of short-lived brominated and iodinated methanes as e.g. bromoform (CHBr3) and methyliodide (CH3I). These very short lived substances (VSLS) are expected to reach the stratosphere and deplete stratospheric ozone in the tropics especially if they have strong emissions over the tropical oceans, where high convective activity with fast efficient uplifts exists. Thus, the tropical Western Pacific is of special interest since the oceanic VSLS are largely uncharacterized and are projected to have hot spots for both their emissions and fast transport pathways to the stratosphere throughout the year. In this study, we present first results from the SHIVA Sonne expedition to the tropical Western Pacific during November 2011. The ship cruise was embedded within the frame work of the EU-project SHIVA (Stratospheric ozone: Halogen Impacts in a Varying Atmosphere). To investigate the large variability of VSLS sources in more detail the expedition cruised through Malaysian and Philippine coastal and open waters of various biogeochemical regimes. The ship expedition will be introduced to the audience, presenting the atmospheric and hydrographic setting. We will show highlights from the cruise including frequent meteorological profile soundings, characterizing the atmosphere from the marine boundary layer to the stratosphere in more detail, together with VSLS measurements both from the atmosphere and the ocean. First estimates of the VSLS transport into the stratosphere will be presented.

  20. Heterogeneity of PrP in Brains of Hamsters Infected with Scrapie 263K Strain and the Dynamic Detection by Western blot%羊瘙痒病263K毒株感染的仓鼠脑中PrP分子形式多样性的动态测定

    Institute of Scientific and Technical Information of China (English)

    高建梅; 高晨; 周晓勃; 张瑾; 韩俊; 张宝云; 董小平

    2003-01-01

    正常细胞的朊蛋白(PrPC)代谢和构象的改变是引发动物和人类可传播性海绵状脑病(transmissible spongiform encephalopathies,TSEs)的根本原因.将羊瘙痒病( scrapie)仓鼠适应株263K颅内接种仓鼠, 在接种后的第20、40、50、60、70、80天, 通过Western blot 动态检测仓鼠脑中PrP存在的形式.结果在接种后第40天, 在感染动物脑组织中即检测到 PrPSc分子,比临床症状出现的时间早(平均潜伏期为66.7±1.1天),且无糖基化形式的PrP分子所占百分比在接种后期增加明显.除了标准分子量大小(30kD~35kD)的 PrP分子外,在感染动物脑中存在着高分子量和低分子量形式的PrP分子 .定量分析显示,随着接种潜伏期的延长,不同形式PrP分子的含量也在增加,其中低分子量形式的PrP分子与临床症状的出现密切相关.蛋白去糖基化实验表明,在感染动物脑组织中,除了标准分子量大小的PrP蛋白外,还存在一条更小分子量的PrP条带,而正常动物脑组织仅存在标准大小的Pr P分子.低分子量形式的PrP分子具有与全长PrP分子相类似的糖基化模式.结果提示,scrapie 263K感染的仓鼠脑组织中存在不同分子形式的PrPSc,其PrP分子的代谢可能不同于正常动物.

  1. The Evaluation of Western Blotting Method for the Diagnosis of Helicobacter Pylori Infection in Children%免疫印迹试验在诊断儿童幽门螺旋杆菌感染中的应用评价

    Institute of Scientific and Technical Information of China (English)

    陈运生; 周少明; 刘彬; 孙玲玲

    2006-01-01

    目的:评价免疫印迹试验(Western Blotting)在诊断不同年龄组及不同病理表现儿童幽门螺旋杆菌(Helicobacter pylori,HP)感染中的应用价值.方法:对450例有消化道症状的患儿,进行胃镜检查并取病变处胃黏膜组织行病理学检查,以及13C-尿素呼气试验(13C-urea breath test,13C-UBT)、血清免疫印迹试验及胃液HP-DNA -PCR检查.结果:在4岁~6岁和6岁~12岁组患儿中,13C-UBT HP检出率明显高于免疫印迹试验(P<0.01);在12岁~18岁组患儿中,13C-UBT HP检出率与免疫印迹试验比较差异无显著性(P>0.05).细胞毒性基因相关蛋白(Cytotoxin-associated gene protein)阳性的HP感染在溃疡病组明显高于慢性胃炎组和正常组(P<0.05),慢性胃炎组CagA(Cytotoxin-associated gene protein,CagA) 阳性 HP显著高与正常组(P<0.05).结论:在12岁以上的少年患儿中,免疫印迹试验可作为快速诊断HP感染的可靠手段,并且能判断出是否为产毒菌株感染.在12岁以下的儿童中,免疫印迹试验最好与13C-UBT联合使用.

  2. Women Entrepreneurship in the Western Romania. Research Results and Policy Recommendations

    Directory of Open Access Journals (Sweden)

    Anca DODESCU

    2011-01-01

    Full Text Available Starting from the results of the empirical, quantitative, and qualitative research performed in the AntrES project, between 5.01.2009 - 4.01.2011, in the Western Romania, referring to the existence of obvious gender gaps regarding the start up of new businesses and the ownership of the businesses; the characteristics, the motivations, and the difficulties of the women entrepreneurs and potentially enterprising women; the successful women entrepreneurs' perceptions on the sex equality in the field of entrepreneurship - this paper presents arguments and recommendations of specific policies meant to support the potential women entrepreneurship in Romania, as an engine to overcome the economic crisis and to reconnect to the regional development mechanism facilitated by the European Union's regional policy.

  3. Electron spin resonance dating of teeth from Western Brazilian megafauna - preliminary results

    Energy Technology Data Exchange (ETDEWEB)

    Kinoshita, Angela, E-mail: angela.kinoshita@usc.br [Departamento de Fisica, FFCLRP, Universidade de Sao Paulo, 14040-901 Ribeirao Preto-SP (Brazil); Universidade Sagrado Coracao, Rua Irma Arminda 10-50, 17011-160 Bauru - Sao Paulo (Brazil); Jose, Flavio A. [Departamento de Fisica, FFCLRP, Universidade de Sao Paulo, 14040-901 Ribeirao Preto-SP (Brazil); Sundaram, Dharani; Paixao, Jesus da S.; Soares, Isabella R.M. [Universidade Federal de Mato Grosso, Departamento de Geologia Geral, 78090-000 Cuiaba-MT (Brazil); Figueiredo, Ana Maria [Instituto de Pesquisas Energeticas e Nucleares (IPEN), 05422-970 Sao Paulo-SP (Brazil); Baffa, Oswaldo [Departamento de Fisica, FFCLRP, Universidade de Sao Paulo, 14040-901 Ribeirao Preto-SP (Brazil)

    2011-09-15

    Electron Spin Resonance (ESR) was applied to determine ages of Haplomastodon teeth from Western Brazilian Megafauna. The Equivalent Doses (D{sub e}) of (1.3 {+-} 0.2)kGy, (800 {+-} 100)Gy and (140 {+-} 20)Gy were found and the software ROSY ESR dating was employed to convert D{sub e} in age, using isotope concentrations determined by neutron activation analysis (NAA) and other information, resulting in (500 {+-} 100)ka, (320 {+-} 50) and (90 {+-} 10)ka considering the Combination Uptake (CU) model for Uranium uptake, set as an Early Uptake (EU) for dentine and Linear Uptake (LU) for enamel. There are scarce reports about Pleistocene Megafauna in this area. This paper presents the first dating of megafauna tooth and this study could contribute to improve the knowledge about the paleoclimate and paleoenvironment of this region and prompt more investigations in this area.

  4. NEOLITHIC PLANT USE IN THE WESTERN MEDITERRANEAN REGION: PRELIMINARY RESULTS FROM THE AGRIWESTMED PROJECT

    Directory of Open Access Journals (Sweden)

    L. Peña-Chocarro

    2013-04-01

    Full Text Available This contribution focuses on the preliminary results of the AGRIWESTMED project which focuses on the archaeobotanical analyses of early Neolithic sites in the western Mediterranean region (both in Iberia and in northern Morocco. A large number of sites has been studied producing an interesting dataset of plant remains which places the earliest examples of domesticated plants in the second half of the 6th millennium cal BC. Plant diversity is high as it is shown by the large number of species represented: hulled and naked wheats, barley, peas, fava beans, vetches, lentils and grass peas. To more crops, poppy and flax, are also part of the first agricultural crops of the area. Although agriculture seems to occupy a first place in the production of food, gathering is well represented in the Moroccan sites where a large number of species has been identified. 

  5. CRITERIA OF POSITIVITY FOR Ig ANTIBODIES IN THE METHOD OF IMMUNE BLOTTING OF LYME DISEASE

    Directory of Open Access Journals (Sweden)

    V G Barskova

    2001-01-01

    Full Text Available There are currently no accepted criteria for positive Western blots in Russian patients with Lyme borreliosis. The purpose of the current study was to develop criteria for a positive IgG westem-blot to aid particularly in the diagnosis of patients with joint manifestation of the disorder. Patients: 97 with Lyme disease, 145 - control subjects. IgG antibody responses were determined to 3 species ofB.burgdorferi sensu lato by Western blotting, using blots prepared by manufacturer. The best discriminatory ability of test criteria was chained by requiring any 3 of 11 IgG bands, a definition that could be used with B. burgdorferi sensu stricto, B.garinii and B.afzelii strains. With these 3 antigen preparation, positive IgG blots were found in 0 to 18% of patients with localized erythema migrans of < 4 weeks duration, 23 to 39% of those with disseminated infection < 20 weeks duration, and in 39 to 46% of those with late arthritis/arthralgia of >6 months duration the specificity was 93 to 99%. Thus, IgG Western blotting may bring greater specificity to serologic testing in Lyme borreliosis, but the sensitivity is limited.

  6. Estimating mercury emissions resulting from wildfire in forests of the Western United States.

    Science.gov (United States)

    Webster, Jackson P; Kane, Tyler J; Obrist, Daniel; Ryan, Joseph N; Aiken, George R

    2016-10-15

    Understanding the emissions of mercury (Hg) from wildfires is important for quantifying the global atmospheric Hg sources. Emissions of Hg from soils resulting from wildfires in the Western United States was estimated for the 2000 to 2013 period, and the potential emission of Hg from forest soils was assessed as a function of forest type and soil-heating. Wildfire released an annual average of 3100±1900kg-Hgy(-1) for the years spanning 2000-2013 in the 11 states within the study area. This estimate is nearly 5-fold lower than previous estimates for the study region. Lower emission estimates are attributed to an inclusion of fire severity within burn perimeters. Within reported wildfire perimeters, the average distribution of low, moderate, and high severity burns was 52, 29, and 19% of the total area, respectively. Review of literature data suggests that that low severity burning does not result in soil heating, moderate severity fire results in shallow soil heating, and high severity fire results in relatively deep soil heating (wood>foliage>litter>branches. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. EXPERIMENTAL RESULTS ON THE POSSIBILITIES OF VEGETABLE GROWING IN THE AREA OF WESTERN CARPATHIAN MOUNTAINS FROM ROMANIA

    OpenAIRE

    Al. Silviu APAHIDEAN; Maria APAHIDEAN; Florin PACURAR

    2004-01-01

    In the area of Western Carpathian Mountains, the vegetable growing is less extended and the assortment of vegetables is limited to few species with smaller necessities regarding the pedoclimatic conditions. This research presents the results obtained after testing a number of 25 species and varieties of vegetables in the specific conditions of the area of Glacier - Western Carpathian Mountains, at an altitude of 1150 m. From these, the following vegetables reacted very well: onion, winter oni...

  8. Impacts on irrigated agriculture of changes in electricity costs resulting from Western Area Power Administration`s power marketing alternatives

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, B.K.; Flaim, S.J.; Howitt, R.E. [Argonne National Lab., IL (United States); Palmer, S.C. [Western Area Power Administration, Salt Lake City, UT (United States)

    1995-03-01

    Irrigation is a major factor in the growth of US agricultural productivity, especially in western states, which account for more than 85% of the nation`s irrigated acreage. In some of these states, almost all cropland is irrigated, and nearly 50% of the irrigation is done with electrically powered pumps. Therefore, even small increases in the cost of electricity could have a disproportionate impact on irrigated agriculture. This technical memorandum examines the impacts that could result from proposed changes in the power marketing programs of the Western Area Power Administration`s Salt Lake City Area Office. The changes could increase the cost of power to all Western customers, including rural municipalities and irrigation districts that rely on inexpensive federal power to pump water. The impacts are assessed by translating changes in Western`s wholesale power rate into changes in the cost of pumping water as an input for agricultural production. Farmers can adapt to higher electricity prices in many ways, such as (1) using different pumping fuels, (2) adding workers and increasing management to irrigate more efficiently, and (3) growing more drought-tolerant crops. This study projects several responses, including using less groundwater and planting fewer waterintensive crops. The study finds that when dependence on Western`s power is high, the cost of power can have a major effect on energy use, agricultural practices, and the distribution of planted acreage. The biggest percentage changes in farm income would occur (1) in Nevada and Utah (however, all projected changes are less than 2% of the baseline) and (2) under the marketing alternatives that represent the lowest capacity and energy offer considered in Western`s Electric Power Marketing Environmental Impact Statement. The aggregate impact on farm incomes and the value of total farm production would be much smaller than that suggested by the changes in water use and planted acreage.

  9. Holocene evolution and sedimentation rate of Alikes Lagoon, Zakynthos island, Western Greece – preliminary results

    Directory of Open Access Journals (Sweden)

    P. Avramidis

    2009-03-01

    Full Text Available In the present study we present preliminary results of Zakynthos Alikes lagoon, which is one of the most seismically active regions of Greece. In order to estimate – interpret the Holocene evolution of the area and to reconstruct the palaeonvironmental changes, we based on the data of a 21 m sediment core. Sediment types, structure, colour, as well as contact depths and bed characteristics, were recorded in the field. Standarised sedimentological analysis were carried out, on 46 samples including grain size analysis, calculation of moment measures, and micro- and molluscan fossils of 17 selected samples. Moreover, radiocarbon age determinations have been made on individual Cardium shells from two horizons and whole – core Magnetic Susceptibility (MS measurements were taken. The interpretation of depositional environments suggests a coastal environment (restricted-shallow with reduced salinity such as a lagoon margin and in a tidal flat and/or marsh particularly. The maximum age of the study sediments is about 8500 BP. The rate of sedimentation between 8280 BP and 5590 BP was 5.3 mm/yr and between 5590 BP and modern times 1.03 mm/yr. The rate of sedimentation was higher until mid-Holocene while decrease after to 1.03 mm/yr, results which are similar to other coastal areas of western Greece.

  10. Holocene evolution and sedimentation rate of Alikes Lagoon, Zakynthos island, Western Greece: preliminary results

    Science.gov (United States)

    Avramidis, P.; Kontopoulos, N.

    2009-07-01

    In the present study we present preliminary results from Alikes lagoon in Zakynthos island, an area that is one of the most seismically active regions of Greece. In order to estimate - interpret the Holocene evolution of the area and to reconstruct the palaeoenvironmental changes, we based on data derived from a 21 m sediment core. Sediment types, structure, colour, as well as contact depths and bed characteristics were recorded in the field. Standarised sedimentological analysis was carried out, on 46 samples including grain size analysis, calculation of moment measures, and micro- and molluscan fossils of 17 selected samples. Moreover, radiocarbon age determinations have been made on individual Cardium shells from two horizons and whole - core Magnetic Susceptibility (MS) measurements were taken. The interpretation of depositional environments suggests a coastal environment (restricted-shallow) with reduced salinity such as a lagoon margin and in a tidal flat and/or marsh particularly. The maximum age of the studied sediments is about 8500 BP. The rate of sedimentation between 8280 BP while 5590 BP was 5.3 mm/yr and between 5590 BP and modern times is on the order of 1.03 mm/yr. These sedimentation rates results are similar to other coastal areas of western Greece.

  11. Holocene evolution and sedimentation rate of Alikes Lagoon, Zakynthos island, Western Greece - preliminary results

    Science.gov (United States)

    Avramidis, P.; Kontopoulos, N.

    2009-03-01

    In the present study we present preliminary results of Zakynthos Alikes lagoon, which is one of the most seismically active regions of Greece. In order to estimate - interpret the Holocene evolution of the area and to reconstruct the palaeonvironmental changes, we based on the data of a 21 m sediment core. Sediment types, structure, colour, as well as contact depths and bed characteristics, were recorded in the field. Standarised sedimentological analysis were carried out, on 46 samples including grain size analysis, calculation of moment measures, and micro- and molluscan fossils of 17 selected samples. Moreover, radiocarbon age determinations have been made on individual Cardium shells from two horizons and whole - core Magnetic Susceptibility (MS) measurements were taken. The interpretation of depositional environments suggests a coastal environment (restricted-shallow) with reduced salinity such as a lagoon margin and in a tidal flat and/or marsh particularly. The maximum age of the study sediments is about 8500 BP. The rate of sedimentation between 8280 BP and 5590 BP was 5.3 mm/yr and between 5590 BP and modern times 1.03 mm/yr. The rate of sedimentation was higher until mid-Holocene while decrease after to 1.03 mm/yr, results which are similar to other coastal areas of western Greece.

  12. Resolving the Western Black Sea Fault Using Microtremor Measurements? -Preliminary Results-

    Science.gov (United States)

    Karabulut, Savas; Caglak, Faruk; Tun, Muammer; Cengiz Cinku, Mualla; Ustaomer, Timur; Ozel, Oguz; Parolai, Stefano; Tezel, Okan; Özçep, Ferhat

    2014-05-01

    çükçekmece Lakes. The first phase of the project collected 3- component single station microtremor measurements at 278 points in the study area between 18 and 24 December 2013. The grid space chosen was 600x600 meters. The duration of measurements at each point were between 20 minutes to 60 minutes using a Guralp CMG 6TD short period seismometer, with a sampling rate of 100 Hz. As a result of the evaluation of these observations, we obtained soil fundamental frequencies and amplifications, as well as bedrock depth from the peak frequency of the microtremors records. According to the resulting soil thickness maps, possible lineament structures were identified. Our results indicate two different frequency distribution in the studied area. The soil fundamental frequency is obtained below 1 Hz along the western and eastern section, while a gradual decrease is shown towards the west and then once again a increase. In the north however, soil transfer peaks of 1 Hz and above are remarkable. Three different types of basins are derived by mapping the thickness of the sediments. The evolution of these basins are still under discussion whether they arise from a tectonic origin or formed as a result of the alteration of the Eocene limestones which distributed in an wide area. In the next phase of the project, we will continue to collect other geophysical data such as gravity and electrical tomography etc. The results of this project will play an important role in the determination of the impact of the Western Black Sea Fault on an earthquake occurring in the Sea of Marmara and therefore serve as input to urban

  13. Regional economic impacts of changes in electricity rates resulting from Western Area Power Administration`s power marketing alternatives

    Energy Technology Data Exchange (ETDEWEB)

    Allison, T.; Griffes, P.; Edwards, B.K.

    1995-03-01

    This technical memorandum describes an analysis of regional economic impacts resulting from changes in retail electricity rates due to six power marketing programs proposed by Western Area Power Administration (Western). Regional economic impacts of changes in rates are estimated in terms of five key regional economic variables: population, gross regional product, disposable income, employment, and household income. The REMI (Regional Impact Models, Inc.) and IMPLAN (Impact Analysis for Planning) models simulate economic impacts in nine subregions in the area in which Western power is sold for the years 1993, 2000, and 2008. Estimates show that impacts on aggregate economic activity in any of the subregions or years would be minimal for three reasons. First, the utilities that buy power from Western sell only a relatively small proportion of the total electricity sold in any of the subregions. Second, reliance of Western customers on Western power is fairly low in each subregion. Finally, electricity is not a significant input cost for any industry or for households in any subregion.

  14. Preliminary result of 3-D attenuation tomography beneath Sunda Strait and western part of Java

    Science.gov (United States)

    Anshori, Muhajir; Nugraha, Andri Dian; Puspito, Nanang T.

    2017-07-01

    Sunda strait lies in the transition zone of two different subduction system that is almost perpendicular to the subduction in southern Java and oblique subduction in western Sumatra. The series of major disasters such as earthquakes and volcanic eruptions is a manifestation of its tectonic setting complexity. We used selected waveform data from724 earthquakes that occurred around the Sunda strait and western part of Java from 2009 - 2015 recorded by 21 MCGA network stations to construct three dimensional image of seismic attenuation. Frequency independent attenuation operators (t*) was determined using spectral fitting method for P and S wave arrivals, respectively. The inversion was performed using simul2000 algorithm to image the lateral and vertical variations of Qp and Qs value in the Sunda Strait and western part of Java. Some interesting features such as subducting slab, mantle wedge, magma chamber under volcanos and fault zones can be imaged well.

  15. Student Performance and Family Socioeconomic Status: Results from a Survey of Compulsory Education in Western China

    Science.gov (United States)

    Liu, Xiaofei; Lu, Ke

    2008-01-01

    This study used fifteen-year-old ninth-grade students from rural areas of five provinces in western China as samples to carry out research on the relationship between the socioeconomic status of Chinese families and student academic performance. Based on parents' educational background, occupation, family economic conditions, and other factors,…

  16. Patterns of Enterprise Flexibility: IAB Establishment Panel Results, Western Germany, 1993-95.

    Science.gov (United States)

    Bellmann, Lutz; Dull, Herbert; Kuhl, Jurgen; Lahner, Manfred; Lehmann, Udo

    1997-01-01

    Data on flexibility in German companies were compiled through interviews and followed up annually. Employment trends in western Germany indicated a historic peak of 29.5 million employed in 1992 followed by a steady decline of about 1 million to 28.5 million in 1995. Employment losses continued in 1996; 1997 showed some signs of stability.…

  17. Precipitation reconstruction using ring-width chronology of Himalayan cedar from western Himalaya: Preliminary results

    Indian Academy of Sciences (India)

    Ram R Yadav; Won-Kyu Park

    2000-09-01

    Himalayan cedar (Cedrus deodara (D. Don) G. Don) due to its long age and wide ecological amplitude in the Himalayan region has strong dendroclimatic potential. A well replicated ring-width chronology of it, derived from the ensemble of tree-ring samples of two adjacent homogeneous sites, has been used to reconstruct precipitation for the non-monsoon months (previous year October to concurrent May) back to AD 1171. This provides the first record of hydrological conditions for the western Himalayan region, India during the whole of the `Little Ice Age' and latter part of the `Medieval Warm Period'. The reconstruction revealed the wettest and the driest non-monsoon months during the fourteenth and the thirteenth centuries, respectively. The seventeenth century consistently recorded dry non-monsoon months in the western Himalayan region. Surplus precipitation, especially more pronounced since the 1950s, is recorded in the current century.

  18. Results of seismological observations in the western Kaliningrad region and in the Baltic Sea water area

    Science.gov (United States)

    Kovachev, S. A.

    2008-09-01

    In 2006 2007, researchers of the IO RAS conducted seismological observations in the Baltic Sea and western Kaliningrad region with the use of ocean-bottom and land-based autonomous seismic stations. According to maps of general seismic zoning of the territory of Russia, the Kaliningrad region is aseismic. However, a series of seismic phenomena with magnitudes of about 5 and sources located near the Bay of Gdansk coast occurred here in September 2004. The total duration of the IO RAS seismological observations in five areas of the region under investigation was more than 200 days. The analysis of seismic records of the IO RAS network located sources of two local weak earthquakes with magnitudes M L = 3.4 3.5, which indicates that the seismic process in the western part of the Kaliningrad region continues and the region is far from being seismically stable.

  19. Ozone over the Western Mediterranean Sea – results from two years of shipborne measurements

    Directory of Open Access Journals (Sweden)

    K. Velchev

    2011-01-01

    Full Text Available Ozone, along with other air pollutants, has been measured for two years from a monitoring station placed on a cruise ship that follows a regular track in the Western Mediterranean between April and October. Conditions favouring high ozone levels have been studied by analysis of weather maps and back trajectories. This analysis was focused on a transect over the open sea in the South Western Mediterranean between Tunis and Palma de Mallorca. High ozone levels were found in situations with an anticyclonic circulation over the Western Mediterranean when subsidence brings air masses down from altitudes between 1000 and 3500 m a.s.l. Analysis of composite meteorological maps suggests a relevant contribution of breeze circulation to subsidence during events with high surface ozone concentrations; this points to an important contribution from local ozone formation. A detailed back trajectory analysis of the origin of air masses with high ozone concentrations was carried out for two "hot spots" for ozone pollution, in the Gulf of Genoa and between Naples and Palermo, respectively. The main cause of high ozone levels in the Gulf of Genoa was found to be outflow from the Po Valley and the Genoa area while such episodes along the Naples-Palermo transect were most often associated with trajectories from the Rome or Naples areas. Analysis of the relationship between measured concentrations of Black Carbon and ozone allowed to evaluate the degree of photochemical "ageing" of the air masses encountered along the route of the cruise ship.

  20. Ozone over the Western Mediterranean Sea – results from two years of shipborne measurements

    Directory of Open Access Journals (Sweden)

    K. Velchev

    2010-03-01

    Full Text Available Ozone, along with other air pollutants, has been measured for two years from a monitoring station placed on a cruise ship that follows a regular track in the Western Mediterranean between April and October. Conditions favoring high ozone levels have been studied by analysis of weather maps and back trajectories. This analysis was focused on a transect over the open sea in the South Western Mediterranean between Tunis and Palma de Mallorca. High ozone levels were found in situations with an anticyclonic circulation over the Western Mediterranean when subsidence brings air masses down from altitudes between 1000 and 3500 m a.s.l. Analysis of composite meteorological maps suggest a relevant contribution of breeze circulation to subsidence during events with high surface ozone concentrations; this points to an important contribution from local ozone formation. A detailed back trajectory analysis of the origin of air masses with high ozone concentrations was carried out for two "hot spots" for ozone pollution, found along the coast south of Genova and between Napoli and Palermo, respectively. While it was found that the influence of plumes from areas with high pollutant levels might explain most episodes in the Northwestern transect, such "local" influences appeared to be of minor importance within the Napoli-Palermo transect.

  1. Geological Results from Drilling in the Poihipi (Western) Sector of the Wairakei Geothermal Field, NZ

    Energy Technology Data Exchange (ETDEWEB)

    Bogie, I.; Lawless, J.V.; MacKenzie, K.M.

    1995-01-01

    Four wells drilled into the Poihipi Sector on the Western margin of the Wairakei geothermal field have found a similar lithostratigraphy to that encountered in wells previously drilled in the general area. Young pumice breccias overly the Huka Falls Formation, with the latter containing intercalations of the Rautehuia Breccia. This in turn overlies ignimbrites and tuffaceous sediments of the Waiora Formation, which contains flows of Haparangi Rhyolite. This sequence is cut by steeply dipping normal faults which strike to the northeast and for the most part dip towards the northwest. Hydrothermal alteration is virtually limited to the Waiora and Haparangi units where a sequence of interlayered illite-smectite and illite clays are found along with chlorite, quartz, pyrite and calcite. There is a minor occurrence of zeolites. Despite large changes in the area's hydrology in response to exploitation, changes in alteration are limited to a comparatively deep occurrence of kaolinite and minor overprinting of epidote by illitic clay.

  2. Biogeochemistry of Lakes in Western Papua, Indonesia - First Results of a Pilot Study.

    Science.gov (United States)

    Kallmeyer, J.; Nomosatryo, S.; Henny, C.; Kopalit, H.

    2016-12-01

    Despite years of exploration for mineral and hydrocarbon resources, the lakes of Western Papua have received very little attention from a limnogeologic perspective. In some cases not even the maximum water depth of the lakes is published. The only research carried out so far focused on the fish and invertebrate fauna of the lakes, because the macrofauna of Papuan Lakes is significantly different from other islands of western Indonesia. Most lakes harbor numerous endemic species. We carried out a first limnogeologic pilot campaign in spring 2016 to measure water column profiles and take short (max 80 cm long) sediment cores.Lake Sentani is seated in Mesozoic mafic bedrock and consists of four separate basins with maximum water depths of 30 to 40 m. Three basins are connected by shallow sills and one by a natural canal. Although all four basins share almost identical surface water chemistry and exhibit sub- to anoxic bottom waters, each basin has its distinct water column stratification and sediment geochemistry. Despite its coastal location and minimal elevation we could not identify an influx of seawater into the lake. Lake Ayamaru is located further inland on a densely forested karstified carbonate platform. The lake level has dropped significantly in recent years due to water loss into the karst, further reduction of open water surface is caused by massive growth of Pistia. Currently the lake has a maximum depth of around 2 m. Its sediment is mainly composed of carbonate minerals and methane saturated. Due to the carbonate bedrock the lake is highly alkaline (up to 20 meq/L) despite its very low salinity. The initial analyses show that these lakes offer unique biogeochemical conditions that require further in-depth studies.Our research will expand to lakes Anggi Giji and Anggi Gida, which are at almost 2000 m elevation. They have maximum depths of around 200 m and much colder surface waters (12-20°C) compared to the other two lakes that have about 30

  3. Education in naturopathy and western herbal medicine in Australia: results of a survey of education providers.

    Science.gov (United States)

    McCabe, Pauline

    2008-08-01

    Questions about the risks and regulatory requirements of naturopathy and western herbal medicine (WHM) in Australia prompted research by the Department of Human Services Victoria. This article offers findings from a survey of education providers, which was one of several studies carried out for the report. (The full report can be found at http://www.health.vic.gov.au/pracreg/naturopathy.htm.) Questionnaires were sent to 43 Australian providers of naturopathy and WHM education. Unsustainable variation was found in award types, contact hours, clinical education, length of courses, and course content. Naturopaths and WHM practitioners are primary contact health professionals but educational standards vary widely, with some practitioners not likely to be adequately prepared. The degree of risk in their practice, and the need for better integration of complementary care with mainstream healthcare, require education at least to the level of a bachelor degree. Courses should be subject to independent external accreditation. However, attempts to determine appropriate minimum educational standards are unlikely to succeed without the support of a regulatory system that can mandate those minimum requirements.

  4. Results of a pilot programme of mammographic breast cancer screening in the Western Cape.

    Science.gov (United States)

    Apffelstaedt, J P; Hattingh, R; Baatjes, K; Wessels, N

    2014-04-01

    Mammographic screening programmes are now established in developing countries. We present an analysis of the first screening programme in sub-Saharan Africa. Women aged > or = 40 years were identified at three primary healthcare centres in the Western Cape Province, South Africa, and after giving informed consent underwent mammography at a mobile unit. After a single reading, patients with American College of Radiology Breast Imaging Reporting and Data System (BIRADS) 3 - 5 lesions were referred to a tertiary centre for further management. Between 1 February 2011 and 31 August 2012, 2 712 screening mammograms were performed. A total of 261 screening mammograms were reported as BIRADS 3 - 5 (recall rate 9.6%). Upon review of the 250 available screening mammograms, 58 (23%) were rated benign or no abnormalities (BIRADS 1 and 2) and no further action was taken. In 32 women, tissue was acquired (biopsy rate for the series 1.2%); 10 cancers were diagnosed (biopsy malignancy rate 31%). For the entire series of 2 712 screening mammograms, the cancer diagnosis rate was 3.7/1 000 examinations. Of 10 cancers diagnosed at screening, 5 were TNM clinical stage 0, 2 stage I and 3 stage II. The low cancer detection rate achieved, and the technical and multiple administrative problems experienced do not justify installation of a screening programme using the model utilised in this series.

  5. Pre-bomb marine reservoir ages in the western north Pacific: Preliminary result on Kyoto University collection

    Energy Technology Data Exchange (ETDEWEB)

    Yoneda, Minoru E-mail: myoneda@nies.go.jp; Kitagawa, Hiroyuki; Plicht, Johannes van der; Uchida, Masao; Tanaka, Atsushi; Uehiro, Takashi; Shibata, Yasuyuki; Morita, Masatoshi; Ohno, Terufumi

    2000-10-01

    The calibration of radiocarbon dates on marine materials involves a global marine calibration with regional corrections. The marine reservoir ages in the Western North Pacific have not been discussed, while it is quite important to determine the timing of palaeo-environmental changes as well as archaeological interpretation around this region. The lack of adequate collection of the pre-bomb shell from western north Pacific was the biggest problem. Recently we had a chance to examine specimens from an old shell collection stored in Kyoto University, including shell specimens from Japan, Korea, Taiwan and the Micronesia of 1920s and 1930s. We explored the possibility for usage of specimen without clear evidence of live collection by measuring 30 apparent radiocarbon ages of pre-bomb mollusk shells from 18 sites in Western North Pacific. The preliminary results showed several discrepancies with previously reported results and with each other. We have to carefully select the shell specimen that has biological signs such as articulating fulcrum. In order to exploit this big resource of pre-bomb shell collection, the new technique to distinguish fossils from live collected samples should be developed by using chemical and physical methods.

  6. Palaeoenvironmental evolution at Plio-Pleistocene Hominin sites in Western Kenya: Preliminary results

    Science.gov (United States)

    Vincent, Thomas; Whitfield, Elizabeth; Bishop, Laura; Plummer, Thomas; Ditchfield, Peter; Blumenthal, Scott; Finestone, Emma; Kiriakoulakis, Kostas

    2017-04-01

    This research uses a multiproxy approach to create detailed reconstructions of Plio-Pleistocene hominin landscapes and environments on the Homa Peninsula, western Kenya. Sedimentary sequences associated with this time period, containing hominin archaeological traces and faunal remains, are studied using analyses of particle size, phytoliths and lipid biomarkers. Reconstructing the palaeoenvironmental substrate here is critical in developing an understanding of the activity and behaviour of previous hominin inhabitants. Three archaeological sites exist in the region that form the bases of this research; these include Kanjera South, Nyayanga and Sare River. Research at Kanjera began in the early 20th century, but systematic investigation of the artefact and faunal assemblages of the southern exposures (Kanjera South), dating to ca. 2 Ma, did not begin until 1995. Field investigations and isotopic analyses of palaeosol carbonate nodules and fauna suggested that Kanjera South sediments were deposited in alluvial and lake marginal environments, on a grassy plain between wooded slopes and a permanent water body. Nyayanga and Sare River are new sites which have been little studied until present. Sediments at Nyayanga are thought to be ca. 2.6 Ma, whilst those at Sare River are ca. 1.77 Ma in age. This research refines previous reconstructions of the depositional environment at Kanjera South using analyses of particle size and end member mixing. These suggest that deposition occurred mainly through low-energy, ephemeral flows in a marginal lacustrine environment, with high energy, hyper-concentrated and viscous flows occurring intermittently. In contrast, these same techniques and field investigations at Nyayanga suggest there was a more consistent energy regime here than at Kanjera South, with sediments being deposited under low-energy conditions. Sediment samples at Sare River are undergoing similar analyses. Through detailed analyses of sediments at three archaeological

  7. Determining temperature changes in Western Iceland over the last millennium: issues and results

    Science.gov (United States)

    Caseldine, Chris; Langdon, Peter; Holmes, Naomi; Thompson, Gareth; Wastegard, Stefan; Larsen, Gudrun; Davies, Siwan; Leng, Melanie; Croudace, Ian

    2010-05-01

    High resolution multi-proxy studies of a core from Baulárvallavatn, western Iceland provide a record of climatic changes since the deposition of the Landnám tephra deposited in AD870±2. Derivation of the climate record has encountered a series of issues relating both to chronology and to proxy interpretation; but a millennial sequence of chironomid-based temperature change has been produced. Chronologically problems relate both to radiocarbon dating and tephrochronology. The lake is surrounded by loessial soils that have suffered from erosion, especially following settlement (the sedimentation rate in the lake has increased from 0.014mm yr-1 pre-settlement to 0.05mm yr-1 post-settlement). This has involved redeposition of both old carbon and tephra shards from earlier eruptions into the sediment. Thus post-Landnám radiocarbon dates are all relatively uniform between 2405-2155±30 BP. The high background levels of tephra made determination of specific post-Landnám horizons extremely difficult. Following over 550 individual shard analyses, and coupled with 137Cs dating of the uppermost sediments an age depth model was produced for the last millennium to provide a chronology for the climate proxies. Chironomid analyses at a resolution of c.10 yrs per sample showed a climatic response that is seen in the first axis of the PCA, rather than the Icelandic chironomid-July air temperature transfer function. The PCA axis correlates with winter/spring and autumn temperatures, possibly reflecting a response to summer season length rather than the expected response to July temperature. However, this has yet to be calibrated in more specific quantitative terms. The core was also subjected to ITRAX scanning; which identified key tephra horizons and reflected the relative consistency of sediment deposition over the last millennium. Oxygen isotope analyses of a limited number of diatom samples have also been undertaken and are the subject of continued research, as is diatom

  8. Hydrothermal Petroleum in Active Continental Rift: Lake Chapala, Western Mexico, Initial Results.

    Science.gov (United States)

    Zarate-del Valle, P. F.; Simoneit, B. R.; Ramirez-Sanchez, H. U.

    2003-12-01

    Lake Chapala in western Mexico is located partially in the Citala Rift, which belongs to the well-known neotectonic Jalisco continental triple junction. The region is characterized by active volcanism (Ceboruco, Volcan de Fuego), tectonic (1995 earthquake, M=8, 40-50 mm to SW) and hydrothermal (San Juan Cosala & Villa Corona spas and La Calera sinter deposit) activities. Hydrothermal petroleum has been described in active continental rift (East African Rift) and marine spreading zones (Guaymas Basin, Gulf of California). In 1868 the Mexican local press reported that manifestations of bitumen were appearing in front of the Columba Cap on the mid south shore of Lake Chapala. This bitumen is linked to the lake bottom and when the water level decreases sufficiently it is possible to access these tar bodies as islands. Because of these manifestations the Mexican oil company (PEMEX) drilled an exploration well (2,348m) at Tizapan El Alto without success. Hydrothermal activity is evident in the tar island zone as three in-shore thermal springs (26.8 m depth, 48.5° C, pH 7.8 and oriented N-S). The preliminary analyses by GC-MS of the tar from these islands indicate hydrothermal petroleum derived from lake sedimentary organic matter, generated at low temperatures (150° -200° C). The tars contain no n-alkanes, no PAH or other aromatics, but a major UCM of branched and cyclic hydrocarbons and mature biomarkers derived from lacustrine biota. The biomarkers consist of mainly 17α (H),21β (H)-hopanes ranging from C27 to C34 (no C28), gammacerane, tricyclic terpanes (C20-C26), carotane and its cracking products, and drimanes (C14-C16). The biomarker composition indicates an organic matter source from bacteria and algae, typical of lacustrine ecosystems. 14C dating of samples from two tar islands yielded ages exceeding 40 kyrs, i.e., old carbon from hydrothermal/tectonic remobilization of bitumen from deeper horizons to the surface. The occurrence of hydrothermal petroleum in

  9. THE FIRST RESULTS OF STUDIES OF TEMPORARY VARIATIONS IN SOILRADON ACTIVITY OF FAULTS IN WESTERN PRIBAIKALIE

    Directory of Open Access Journals (Sweden)

    К. Zh. Seminsky

    2015-09-01

    Full Text Available Radon concentrations in soil air are variable depending on factors that are considered external (planetary and internal (geodynamic relative to the Earth. In active fault zones, variations of gas emanations are most intense. A permanent monitoring station was established near Tyrgan settlement in Western Pribaikalie to study temporal variations of soil radon concentration, Q, in the faults of the Baikal rift, East Siberia. This station is located in the zone of the Primorsky normal fault that is the largest in the region. The station is equipped with radon radiometer PPA01M03 that records Q values every 85 minutes and also monitors a number of meteorological parameters, including atmospheric pressure, humidity, and air temperature.We analysed records of two measurement sessions (148 and 66 days covering a part of the year during which field measurement of Q are possible in the cold climate conditions of the area under study. According to the available monitoring data, variations of radon concentrations in the Primorsky fault zone may vary by more than one order of magnitude through a springsummerautumn period, and such variations are oscillatory. Significant changes of permeability in time occur due to intensive changes in the state of stresses of the rock massives under the impacts of the planetary and geodynamic factors. The influence of the first group of factors, i.e. planetary ones, is manifested by synchronous oscillations of radon concentrations and atmospheric pressure, which phases of occurrence are opposed. Domination of daily and fourday periods gives evidence that the state of stresses of the rock massives is impacted by the lunar tides and cyclonic phenomena associated with the interaction between the Earth and the Sun. The influence of the second group of factors, i.e. geodynamic ones, is suggested by an evident relation between radon emanations and seismic events, including the catastrophic earthquake in Japan (March 11, 2011, M=9

  10. Fast and Simple micro-RNA Northern Blots

    Directory of Open Access Journals (Sweden)

    Nham Tran

    2009-01-01

    Full Text Available RNA northern blots provide robust measurements of gene expression. The simple northern blot technique described in this report has been optimised to provide rapid, reproducible detection and analysis of mature and precursor forms of microRNAs. This protocol economises on the use of commercially available components and secondly reduces the hybridisation step to 2 hours.

  11. BLOTS AND ALL: A HISTORY OF THE RORSCHACH INK BLOT TEST IN BRITAIN.

    Science.gov (United States)

    Hubbard, Katherine; Hegarty, Peter

    2016-01-01

    Despite the easily recognizable nature of the Rorschach ink blot test very little is known about the history of the test in Britain. We attend to the oft-ignored history of the Rorschach test in Britain and compare it to its history in the US. Prior to the Second World War, Rorschach testing in Britain had attracted advocates and critiques. Afterward, the British Rorschach Forum, a network with a high proportion of women, developed around the Tavistock Institute in London and The Rorschach Newsletter. In 1968, the International Rorschach Congress was held in London but soon after the group became less exclusive, and fell into decline. A comparative account of the Rorschach in Britain demonstrates how different national institutions invested in the 'projective hypothesis' according to the influence of psychoanalysis, the adoption of a nationalized health system, and the social positioning of 'others' throughout the twentieth century. In comparing and contrasting the history of the Rorschach in Britain and the US, we decentralize and particularize the history of North American Psychology.

  12. Results of paleoflood investigations for Spring, Rapid, Boxedler, and Elk Creeks, Black Hills, western South Dakota

    Science.gov (United States)

    Driscoll, Daniel G.; O'Connor, James E.; Harden, Tessa M.

    2012-01-01

    Flood-frequency analyses for the Black Hills area are especially important because of severe flooding of June 9–10, 1972, that was caused by a large mesoscale convective system and resulted in at least 238 deaths. This paper summarizes results of paleoflood investigations for six study reaches in the central Black Hills. Stratigraphic records and resulting long-term flood chronologies, locally extending more than 2,000 years, were combined with observed and historical flood information to derive flood-frequency estimates. Results indicate that floods as large as and even substantially larger than 1972 have affected most of the study reaches. Results of the paleoflood investigations provide better physically based information on low-probability floods than has been previously available, substantially improving estimates of the magnitude and frequency of large floods in the central Black Hills and reducing associated uncertainties. Collectively, the results provide insights regarding regional flood-generation processes and their spatial controls, enable approaches for extrapolation of results for hazard assessment beyond specific study reaches, and provide a millennial-scale perspective on the 1972 flooding.

  13. Preliminary result of earthquake hypocenter determination using hypoellipse around western Java region

    Science.gov (United States)

    Supendi, Pepen; Nugraha, Andri Dian

    2016-05-01

    West Java region is located in high seismicity and active tectonic setting region influenced by subducting Indo-Australian plate beneath Eurasia plate. There are several on land active faults namely Cimandiri fault, Lembang fault, and Baribis fault in the region. MCGA earthquake data catalog from 2009 to 2014 shows the earthquakes were located not only around active faults but also relatively far away from the active faults in West Java region. In this study, we determined the earthquake location through re-picking of P-and S-wave arrival times recording by MCGA network. Earthquake location was determined by using Hypoellipse code that employs a single event determination method. We then relocated the events using hypocenter double-difference method. We also have been conducting focal mechanism analysis to estimate the type of fault slip. Our preliminary results show generally the epicenter location were distributed around the on land active fault which have the focus depth less than 30 km. For ongoing and future works, we have been determining focal mechanism of selected event related to on land active fault for this region..

  14. Possible future lakes resulting from continued glacier shrinkage in the Aosta Valley Region (Western Alps, Italy)

    Science.gov (United States)

    Viani, Cristina; Machguth, Horst; Huggel, Christian; Godio, Alberto; Perotti, Luigi; Giardino, Marco

    2017-04-01

    Aosta Valley (NW-Alps, Italy) is the region with the largest glaciarized area of Italy (133.73 km2). Like the other alpine regions it has shown a significant glacier retreat starting from the end of the Little Ice Age (LIA, ca. 1850 AD), by losing about 60% of its glaciarized area. As a direct consequence of glacier shrinkage, within glacially-sculpted landscapes, glacier-bed overdeepenings become exposed, offering suitable conditions for glacier lakes formation. In the Aosta Valley region, about 200 glacier lakes have been recognized in different time periods within LIA maximum extent boundaries, mainly dammed by bedrock landforms. With respect to human activities, glacier lakes represent both opportunities (e.g. Miage lake for tourism) and risks (e.g. outburst flood of the Gran Croux lake above Cogne in August 2016) in such a densely populated and developed region. The objective of this contribution is to assess locations of possible future glacier lakes in the Aosta Valley by using the GlabTop2 model (Glacier Bed Topography model version 2). Understanding where future lakes will appear is of fundamental importance for the identification of potential hazards and the interpretation of conditioning factors and dynamics. We first assessed ice thickness and consequently glacier bed topography over large glaciated areas of the region, by using both glaciers outlines related to 1999 (provided by the GlaRiskAlp project) and the regional DEM of 1990 (provided by the Aosta Valley Region) as input data. We performed several runs by varying different input parameters (e,g.: pixel size and basal shear stress). Then we compared modelled results on selected test glaciers (Rutor and Grand Etrèt) with available GPR data. As a validation, we also carried out a GPR survey during summer 2016 on the central area of Indren Glacier (Monte Rosa massif) where GlabTop2 shows the presence of a possible subglacial overdeepening morphology. We found that ice thickness and consequently the

  15. Results from stable isotope investigations of river waters in Western Croatia

    Science.gov (United States)

    Häusler, H.; Frančišković-Bilinski, S.; Rank, D.; Stadler, P.; Bilinski, H.

    2012-04-01

    During a campaign lasting from 27 October to 21 November 2010, sixty-one water samples were taken from the Kupa River, the catchment of which is about ten thousand square kilometres in size. Due to the fact that the upper tributaries of e.g. the Čabranka-, Dobra-, Korana-, Mrežnica- and Petrinjčica River comprise karstified Mesozoic carbonate formations, the hydrogeologic catchment of Kupa River extends the hydrologic one by far. The upper Kupa River is mainly charged by springs from big karst reservoirs in the Gorski Kotar mountain range, where a mean groundwater residence time of up to one year has to be considered. The rapid increase of discharge of these tributary rivers results from the rapid increase of discharge of karst wells after melting in springtime as well as from storm events. In general, the minimum mean discharge for all hydrographs in July reveals a dry summer season, with the maximum discharge in August/September resulting from an increase in precipitation. We interpret the d O-18 values of the Čabranka River (of about -8, 07‰) as signals from maritime precipitation in this karstified catchment area. The d O-18 value of upper Kupa River diminishes along its course from -8,09‰ near Osilnica to -9,06‰ west of Karlovac. After the inflow of tributaries south of Karlovac, the oxygen isotope ratio of Kupa River water reveals a significant change because the d O-18 values of the Dobra-, Korana- and Mrežnica River range from -10,45‰ to -9,58‰ . Due to the fact that the catchment of Dobra- and Korana River rises between 400 and 880 metres, we interpret the lower d O-18 values of river waters from recharge areas at those low mean altitudes as not caused by an altitude effect, but instead by precipitation out of more continental air masses. Our interpretation of stable isotope ratios in river waters is based on the relation between the weighted mean d O-18 and the altitude obtained from stations of the Global Network of Isotopes in

  16. Recent Opportunity Microscopic Imager Results from the Western Rim of Endeavour Crater

    Science.gov (United States)

    Herkenhoff, K. E.; Arvidson, R. E.; Jolliff, B. L.; Mittlefehldt, D. W.; Sullivan, R. J., Jr.; Gellert, R.; Sims, M. H.

    2016-12-01

    (dubbed "Joseph Collin") shed from the top of the rover's solar panels indicate that they were emplaced by active saltation of wind-driven, well-sorted fine sand grains in the current surface environment. The latest Opportunity MI results will be presented at the conference.

  17. Lipid-binding analysis using a fat blot assay.

    NARCIS (Netherlands)

    Munnik, T.; Wierzchowiecka, M.

    2013-01-01

    Protein-lipid interactions play an important role in lipid metabolism, membrane trafficking and cell -signaling by regulating protein localization, activation, and function. The Fat Blot assay is a relatively simple and inexpensive method to examine these interactions using nitrocellulose

  18. Mixed lubrication after rewetting of blotted pleural mesothelium.

    Science.gov (United States)

    Bodega, Francesca; Sironi, Chiara; Porta, Cristina; Pecchiari, Matteo; Zocchi, Luciano; Agostoni, Emilio

    2013-01-15

    Coefficient of kinetic friction (μ) of pleural mesothelium blotted with filter paper, and rewetted with Ringer solution markedly increases; this increase is removed if a sufficient amount of sialomucin or hyaluronan is added to Ringer (Bodega et al., 2012. Respiratory Physiology and Neurobiology 180, 34-39). In this research we found that μ of pleural mesothelium blotted, rewetted, and sliding at physiological velocities and loads, decreased with increase of velocity, mainly at low velocities. Despite this decrease, μ at highest velocity was still double that before blotting. With small concentration of sialomucin or hyaluronan μ was markedly smaller at each velocity, decreased less with increase of velocity, and at highest velocity approached preblotting value. These findings indicate a regime of mixed lubrication in post-blotting Ringer, at variance with boundary lubrication occurring before blotting or postblotting with sufficient macromolecule addition. Greater roughness of mesothelial surface, caused by blotting, likely induces zones of elastohydrodynamic lubrication, which increase with velocity, while contact area decreases.

  19. Accuracy of Reverse Dot-Blot PCR in Detection of Different β-Globin Gene Mutations.

    Science.gov (United States)

    El-Fadaly, N; Abd-Elhameed, A; Abd-Elbar, E; El-Shanshory, M

    2016-06-01

    Prevention programs for β-thalassemia based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation screening methods. The aim of this study was to compare between direct DNA sequencing, and reverse dot-blot PCR in detection of different β-globin gene mutations in Egyptian children with β-thalassemia. Forty children with β-thalassemia were subjected to mutation analysis, performed by both direct DNA sequencing and β-globin Strip Assay MED™ (based on reverse dot-blot PCR). The most frequent mutant alleles detected by reverse dot-blot PCR were; IVSI-110 G>A (31.25 %), IVS I-6 T > C (21.25 %), and IVS I-1 G>A (20 %). Relatively less frequent mutant alleles detected by reverse dot-blot PCR were "IVSII-1 G>A (5 %), IVSII-745 C>G (5 %), IVSII-848 C>A (2.5 %), IVSI-5 G>C (2.5 %), -87 C>G(2.5 %), and cd39 C>T (2.5 %)", While the genotypes of three patients (6 alleles 7.5 %) were not detected by reverse dot-blot PCR. Mutant alleles detected by direct DNA sequencing were the same as reverse dot-blot PCR method except it revealed the genotypes of 3 undetected patients (one patient was homozygous IVSI-110 G>A, and two patients were homozygous IVS I-1 G>A. Sensitivity of the reverse dot-blot PCR was 92.5 % when compared to direct DNA sequencing for detecting β-thalassemia mutations. Our results therefore suggest that, direct DNA sequencing may be preferred over reverse dot-blot PCR in critical diagnostic situations like genetic counseling for prenatal diagnosis.

  20. Large-Scale Forest Fires and Resulting Alterations to the Hydrologic Cycle in the Western U.S

    Science.gov (United States)

    Carr, J.; White, A. B.; Thomson, B.

    2012-12-01

    Recent changes in climate have resulted in a decrease in precipitation and snowpack amounts and increased temperatures in the western United States. Drier and warmer conditions coupled with forest management issues have led to an increase in the frequency and size of forest fires. The 2000 Cerro Grande fire in Los Alamos, New Mexico burned over 43,000 acres and 200 structures. Eleven years later, the Las Conchas fire burned over 156,000 acres and 100 structures, including areas previously burned in 2000, and was considered the largest fire in New Mexico's history. Both fires burned ponderosa, juniper, piñon and mixed conifer forests, resulting in dramatic decreases in vegetation, changes to surface soils, and alterations to the hydrologic cycle (decreased evapotranspiration, decreased infiltration, increased runoff volume and peak discharge, and decreased time to peak discharge) in surrounding watersheds. Burned Area Emergency Response (BAER) teams need to determine the flash-flood danger quickly in order to protect residents, fire-fighters, BAER-team field personnel, and property at risk. The USGS developed an analytical method for predicting post-fire peak discharges using data collected from eight different fires throughout the western United States. We use this method to predict peak discharge in Los Alamos watersheds post-Cerro Grande and post-Las Conchas, then compare predicted to measured peak discharge. We will evaluate the effectiveness of the three methodology levels presented by the USGS, which include varying levels of data input and processing. We expect the peak discharges to be similar in magnitude; however, we will also investigate different influential factors such as burn severity, soil type, vegetation type and density, ecological connectivity, topography, pre- and post-fire weather conditions, etc., as they relate to the fires and the results seen from the measured versus the analytical method. Determining the relative influence of these

  1. Report: Results of Technical Network Vulnerability Assessment: EPA’s National Health & Environment Effects Research Laboratory, Western Ecology Division

    Science.gov (United States)

    Report #11-P-0429, August 3, 2011. Vulnerability testing of EPA’s NHEERL Western Ecology Division network conducted in March 2011 identified Internet Protocol addresses with numerous high-risk and medium-risk vulnerabilities.

  2. TSE strain differentiation in mice by immunohistochemical PrPSc profiles and triplex Western blot

    NARCIS (Netherlands)

    Keulen, van L.J.M.; Langeveld, J.P.M.; Dolstra, C.H.; Jacobs, J.G.; Bossers, A.; Zijderveld, van F.G.

    2015-01-01

    TSE strains are routinely identified by their incubation period and vacuolation profile in the brain after intracerebral inoculation and serial passaging in inbred mouse lines. There are some major drawbacks to this method that are related to the variation in vacuolation that exists in the brains of

  3. Western Blot Analysis of the Exotoxin Components From Bacillus anthracis Separated by Isoelectric Focusing Gel Electrophoresis

    Science.gov (United States)

    2004-02-14

    adding hydroxylapatite (0.5% w/v; Calbiochem fast flow; Calbiochem, San Diego , CA). The suspensions were shaken on ice for 1–2 h, then incubated...Pellizzari, C. Recchi, G. Napolitani , M. Mock, C. Montecucco, Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine

  4. Indeterminate HIV-1 Western Blots: Etiology, Natural History, and Psychological Reactions

    Science.gov (United States)

    1992-09-16

    antibodies to retroviral proteins in parIents with primary Sjogren’s syndrome. Arthritis and Rheumatism 1990; 33:774-781. 51. Kunhl P, Seidl S, Holzberger...erythematosus, rheumatoid arthritis , juvenile onset diabetes, thyroiditis, and Crohn’s disease), were less likely to be tested electively for HIV, and a lower...association if both the IWB and autoantibodies were caused by another factor. Parity could reflect alloimmunization during pregnancy , producing

  5. Lithic technology and behavioural modernity: new results from the Still Bay site, Hollow Rock Shelter, Western Cape Province, South Africa.

    Science.gov (United States)

    Högberg, Anders; Larsson, Lars

    2011-08-01

    The Hollow Rock Shelter site in Western Cape Province, South Africa, was excavated in 1993 and 2008. This study presents new results from a technological analysis of Still Bay points and bifacial flakes from the site. The results show that Still Bay points from the site are standardized tools. The points in the assemblage consist of a complex mixture of whole and fragmented points in all phases of production. The fragmentation degree is high; approximately 80% of the points are broken. A high proportion of bending fractures shows that several of the points were discarded due to production failures, and points with impact damage or hafting traces show that used points were left in the cave. This illustrates that the production of points as well as replacement of used points took place at the site. The result also shows that worked but not finished preforms and points were left at the site, suggestive of future preparation. The points were produced within the framework of three different chaînes opératoires, all ending up in a typologically uniform tool. This shows that the manufacture of Still Bay points should be regarded as a special bifacial technology, only partly comparable with other bifacial technologies. A raw material analysis shows that locally available quartz and quartzite were used in the production, and that points made of silcrete were brought to the site. Based on the technological analysis, a discussion of behavioural modernity, focusing on hypotheses about social interaction, experimentation, different strategies for learning to knap, and landscape memories, results in an interpretation that behavioural modernity was established at Hollow Rock Shelter in the Still Bay phase of the southern African Middle Stone Age. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. GTP-blot analysis of small GTP-binding proteins. The C-terminus is involved in renaturation of blotted proteins.

    Science.gov (United States)

    Klinz, F J

    1994-10-01

    Recombinant c-Ha-ras, ralA and rap2, but not rap1A or rap1B proteins retained their ability to bind [alpha-32P]GTP after SDS/PAGE and transfer to nitrocellulose. Recombinant c-Has-ras missing the C-terminal 23 amino acid residues failed to bind [alpha-32P]GTP after the blot, and the ability of recombinant ralA missing the C-terminal 28 amino acid residues to bind [alpha-32P]GTP was decreased many-fold. The presence of nonionic detergents of the polyoxyethylene type such as Tween 20, Triton X-100, Nonidet P40 or Lubrol PX in the incubation buffer was necessary to induce renaturation of blotted recombinant c-Ha-ras protein, whereas other types of detergents were ineffective. We propose that detergents of the polyoxyethylene type induce the refolding of some types of blotted small GTP-binding proteins and that the C-terminus is involved in the refolding process. Membranes from NIH3T3 fibroblasts overexpressing c-Ha-ras protein showed much weaker binding of [alpha-32P]GTP as expected from the level of ras immunoreactivity. Treatment of fibroblasts with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, caused the accumulation of the unfarnesylated form of c-Ha-ras in the cytosol. Examination of [alpha-32P]GTP-binding and immunoreactivity for cytosolic and membrane-bound c-Ha-ras revealed that binding of [alpha-32P]GTP to unprocessed c-Ha-ras was increased about threefold compared to the same amount of processed c-Ha-ras. Our results demonstrate that detection and quantification of small GTP-binding proteins in eukaryotic cells by GTP-blot analysis is hampered by the fact that these proteins differ strongly in their ability to renature after blotting to nitrocellulose.

  7. Late Quaternary environments on the western Lomonosov Ridge (Arctic Ocean) - first results from RV Polarstern expedition PS87 (2014)

    Science.gov (United States)

    Spielhagen, Robert F.; Stein, Rüdiger; Mackensen, Andreas; PS87 Shipboard Scientific Party

    2016-04-01

    The interior Arctic Ocean is still one of the least known parts of the earth's surface. In particular this holds true for the deep-sea area north of Greenland which has been reached by research ships only within the last decade. The region is of special interest for climate researchers because numerical climate models predict that under future global warming the shrinking summer sea ice cover will finde a place of refuge here until it totally disappears. In summer 2014 several short and long undisturbed large-volume sediment cores were obtained from the western Lomonosov Ridge between 86.5°N and the North Pole. Here we present first results from site PS87/030 situated at 88°40'N. The combined sedimentary record of a box core and a kasten core analyzed so far is interpreted to represent the environmental variability in the last ca. 200,000 years and can be correlated to comparable records from the eastern Lomonosov Ridge and the Morris Jesup Rise. The well-defined coarse layers with abundant ice-rafted detritus reflect the history of circum-Arctic ice sheets. Planktic foraminifers with a distinct dominance of the polar species were found in most of the analyzed samples and allow to reconstruct the water mass history for this part of the Arctic Ocean. Planktic oxygen and carbon isotope records allow to identify several freshwater events which can be correlated to the decay of ice sheets surrounding the Arctic Ocean. The results presented are, however, preliminary and will be refined by future work including an improved temporal resolution of the records and the addition of further proxy records.

  8. Recovery potential of a western lowland gorilla population following a major Ebola outbreak: results from a ten year study.

    Directory of Open Access Journals (Sweden)

    Céline Genton

    Full Text Available Investigating the recovery capacity of wildlife populations following demographic crashes is of great interest to ecologists and conservationists. Opportunities to study these aspects are rare due to the difficulty of monitoring populations both before and after a demographic crash. Ebola outbreaks in central Africa have killed up to 95% of the individuals in affected western lowland gorilla (Gorilla gorilla gorilla populations. Assessing whether and how fast affected populations recover is essential for the conservation of this critically endangered taxon. The gorilla population visiting Lokoué forest clearing, Odzala-Kokoua National Park, Republic of the Congo, has been monitored before, two years after and six years after Ebola affected it in 2004. This allowed us to describe Ebola's short-term and long-term impacts on the structure of the population. The size of the population, which included around 380 gorillas before the Ebola outbreak, dropped to less than 40 individuals after the outbreak. It then remained stable for six years after the outbreak. However, the demographic structure of this small population has significantly changed. Although several solitary males have disappeared, the immigration of adult females, the formation of new breeding groups, and several birth events suggest that the population is showing potential to recover. During the outbreak, surviving adult and subadult females joined old solitary silverbacks. Those females were subsequently observed joining young silverbacks, forming new breeding groups where they later gave birth. Interestingly, some females were observed joining silverbacks that were unlikely to have sired their infant, but no infanticide was observed. The consequences of the Ebola outbreak on the population structure were different two years and six years after the outbreak. Therefore, our results could be used as demographic indicators to detect and date outbreaks that have happened in other, non

  9. Labor productivity losses over western Turkey in the twenty-first century as a result of alteration in WBGT

    Science.gov (United States)

    Altinsoy, Hamza; Yildirim, Haci Ahmet

    2015-04-01

    Occupational fatalities and work-related injuries are more common in Turkey than in most developing and developed countries. Several precautions have been taken concerning the matter, and The Occupational Health and Safety Act (OHSL 2012) has been passed. These efforts, however, have failed to incorporate in their framework the role of global warming. New legislation is underway for the prevention of occupational diseases, injuries, and fatalities. This is particularly worrisome given that Turkey is in the forefront of countries projected to be affected seriously by climate change. Consequently, a study on the direct and indirect impact of climate change on workers' health and labour productivity is paramount. The main purpose of this research is to present diminishing labour productivity as a consequence of decreased working hours via an estimate of rest hours of workers in manual labour. The climatic outputs of Regional Climate Model (RegCM3) obtained from the ENSEMBLES Project are used to calculate the wet bulb globe temperature (WBGT) over western Turkey. The study covers the span of years between 1971 and 2100. Moreover, spatial distributions of observed domain are estimated by means of a seasonal analysis, preliminary to a more detailed research. Critical regions, more adversely influenced than others, are identified. The total number of loss days for these critical regions are presented for various time periods. As a result, labour productivity particularly in agriculture and construction is expected to diminish seriously over Central Anatolia, Cyprus, and parts of the Aegean and eastern Mediterranean coastal areas. Between 2071 and 2100, deficiency in labour productivity may reach up to 52 % during the summer across some of these critical regions. Though it will be seen that the present study has focused primarily on manual labour and outdoor work in particular, it foreshadows nevertheless the dangerous impact of climate change on occupational health and

  10. Labor productivity losses over western Turkey in the twenty-first century as a result of alteration in WBGT.

    Science.gov (United States)

    Altinsoy, Hamza; Yildirim, Haci Ahmet

    2015-04-01

    Occupational fatalities and work-related injuries are more common in Turkey than in most developing and developed countries. Several precautions have been taken concerning the matter, and The Occupational Health and Safety Act (OHSL 2012) has been passed. These efforts, however, have failed to incorporate in their framework the role of global warming. New legislation is underway for the prevention of occupational diseases, injuries, and fatalities. This is particularly worrisome given that Turkey is in the forefront of countries projected to be affected seriously by climate change. Consequently, a study on the direct and indirect impact of climate change on workers' health and labour productivity is paramount. The main purpose of this research is to present diminishing labour productivity as a consequence of decreased working hours via an estimate of rest hours of workers in manual labour. The climatic outputs of Regional Climate Model (RegCM3) obtained from the ENSEMBLES Project are used to calculate the wet bulb globe temperature (WBGT) over western Turkey. The study covers the span of years between 1971 and 2100. Moreover, spatial distributions of observed domain are estimated by means of a seasonal analysis, preliminary to a more detailed research. Critical regions, more adversely influenced than others, are identified. The total number of loss days for these critical regions are presented for various time periods. As a result, labour productivity particularly in agriculture and construction is expected to diminish seriously over Central Anatolia, Cyprus, and parts of the Aegean and eastern Mediterranean coastal areas. Between 2071 and 2100, deficiency in labour productivity may reach up to 52% during the summer across some of these critical regions. Though it will be seen that the present study has focused primarily on manual labour and outdoor work in particular, it foreshadows nevertheless the dangerous impact of climate change on occupational health and

  11. Pre-bomb marine reservoir ages in the western north Pacific : Preliminary result on Kyoto University collection

    NARCIS (Netherlands)

    Yoneda, M; Kitagawa, H; van der Plicht, J; Uchida, M; Tanaka, A; Uehiro, T; Shibata, Y; Morita, M; Ohno, T

    2000-01-01

    The calibration of radiocarbon dates on marine materials involves a global marine calibration with regional corrections. The marine reservoir ages in the Western North Pacific have not been discussed, while it is quite important to determine the timing of palaeo-environmental changes as well as arch

  12. Echo-waveform classification using model and model free techniques: Experimental study results from central western continental shelf of India

    Digital Repository Service at National Institute of Oceanography (India)

    Chakraborty, B.; Navelkar, G.S.; Desai, R.G.P.; Janakiraman, G.; Mahale, V.; Fernandes, W.A.; Rao, N.

    Seafloor classifications and characterization using acoustic backscatter data from the central western continental shelf of India is presented in this work. Single beam sea-floor echo data using two frequencies: 33 kHz and 210 kHz along...

  13. Pre-bomb marine reservoir ages in the western north Pacific : Preliminary result on Kyoto University collection

    NARCIS (Netherlands)

    Yoneda, M; Kitagawa, H; van der Plicht, J; Uchida, M; Tanaka, A; Uehiro, T; Shibata, Y; Morita, M; Ohno, T

    2000-01-01

    The calibration of radiocarbon dates on marine materials involves a global marine calibration with regional corrections. The marine reservoir ages in the Western North Pacific have not been discussed, while it is quite important to determine the timing of palaeo-environmental changes as well as

  14. Pre-bomb marine reservoir ages in the western north Pacific : Preliminary result on Kyoto University collection

    NARCIS (Netherlands)

    Yoneda, M; Kitagawa, H; van der Plicht, J; Uchida, M; Tanaka, A; Uehiro, T; Shibata, Y; Morita, M; Ohno, T

    2000-01-01

    The calibration of radiocarbon dates on marine materials involves a global marine calibration with regional corrections. The marine reservoir ages in the Western North Pacific have not been discussed, while it is quite important to determine the timing of palaeo-environmental changes as well as arch

  15. Fingerprinting of Natural Product by Eastern Blotting Using Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Hiroyuki Tanaka

    2012-01-01

    Full Text Available We succeeded in developing the fingerprint of natural product by eastern blotting using monoclonal antibodies. After developing and separating them on a TLC plate, solasodine glycosides are oxidized by NaIO4 and reacted with a protein to give conjugates which are recognized with anti-solamargine monoclonal antibody (MAb. Anti-solamargine MAb having wide cross-reactivity can stain and detect all solasodine glycosides by fingerprint. Different sensitivity between solamargine and solasonine was observed. The detection limit was 1.6 ng of solasonine. The hydrolysed products of solamargine were determined by fingerprint of eastern blotting compared to their Rf values depending on the sugar number. Fingerprint by eastern blotting using anti-ginsenoside Rb1 MAb distinguished the formula containing ginseng prescribed in traditional Chinese medicine. By double-staining of ginsenosides it is possible to suggest that the staining color shows the pharmacological activity, such as the purple bands indicate ginsenosides having stimulation activity, and the blue color indicated compound like ginsenosides possessed the depression affect for the central nervous system (CNS, respectively.

  16. HOUSE DUST MITE ALLERGEN (Derp1 AND Blot5) LEVELS IN ASTHMATICS' HOME IN HONGKONG

    Institute of Scientific and Technical Information of China (English)

    Bao-qing Sun; Adrian Wu; Albert Chan; Stanley Chik; Dorothy Wong; Nan-shan Zhong

    2004-01-01

    Objective To measure Derpl and Blot5 allergen levels in asthmatics' homes in Hongkong.Methods Seventy houses were enrolled for a mite indoor environment study. Dust samples were obtained from two sites of each patients' house: bed and floor. Derpl and Blot5 levels were quantified by a two-site monoclonal antibody-based ELISA technique.Results The levels of Derpl allergens found in bed (geometric mean (GM) 3.43 μg/g of dust; 95%CI, 1.89-4.96 μg/g)and on the floor (GM 1.12 μg/g of dust; 95%CI, 0.71-1.53 μg/g) indicated significant differences (P=0.005). However, the levels of Blot5 allergens found in bed (GM 19.00 μg/g of dust; 95%CI, 0.89-38.90 μg/g) and on the floor (GM 6.14 μg/g of dust; 95%CI, 0.40-11.90 μg/g) showed no statistically significant difference. In addition, in regards to the exposure index for Derpl and Blot5 allergens found in bed and on the floor, 17.6% in bed and 8.6% on the floor had levels of Blot5 ≥ 10 μg/g of dust, higher than those obtained for Derp1 (7.2% and 0% in bed and on the floor respectively, P< 0.05); higher percentages in bed and on the floor (25.0% and 35.7%) were observed for levels of Blot5 =0 μg/g of dust as compared with Derpl in bed and on the floor (4.3% and 14.5% respectively, P< 0.05).Conclusions Derpl and Blot5 are the major allergens found in this regional study, Blot5 is a more potent allergen in Hongkong, probably reflecting the high level of exposure to Blomia tropicalis (Bt). Bt and Dermatophagoides pteronyssinus (Dp) allergens should be included for precise diagnosis and effective immuno-therapeutic treatment of mite allergy in Hongkong.

  17. Miniaturized fluorescent RNA dot blot method for rapid quantitation of gene expression

    Directory of Open Access Journals (Sweden)

    Yadetie Fekadu

    2004-06-01

    Full Text Available Abstract Background RNA dot blot hybridization is a commonly used technique for gene expression assays. However, membrane based RNA dot/slot blot hybridization is time consuming, requires large amounts of RNA, and is less suited for parallel assays of more than one gene at a time. Here, we describe a glass-slide based miniaturized RNA dot blot (RNA array procedure for rapid and parallel gene expression analysis using fluorescently labeled probes. Results RNA arrays were prepared by simple manual spotting of RNA onto amino-silane coated microarray glass slides, and used for two-color fluorescent hybridization with specific probes labeled with Cy3 and 18S ribosomal RNA house-keeping gene probe labeled with Cy5 fluorescent dyes. After hybridization, arrays were scanned on a fluorescent microarray scanner and images analyzed using microarray image analysis software. We demonstrate that this method gives comparable results to Northern blot analysis, and enables high throughput quantification of transcripts from nanogram quantities of total RNA in hundreds of samples. Conclusion RNA array on glass slide and detection by fluorescently labeled probes can be used for rapid and parallel gene expression analysis. The method is particularly well suited for gene expression assays that involve quantitation of many transcripts in large numbers of samples.

  18. Groundwater-quality data in the Western San Joaquin Valley study unit, 2010 - Results from the California GAMA Program

    Science.gov (United States)

    Mathany, Timothy M.; Landon, Matthew K.; Shelton, Jennifer L.; Belitz, Kenneth

    2013-01-01

    Groundwater quality in the approximately 2,170-square-mile Western San Joaquin Valley (WSJV) study unit was investigated by the U.S. Geological Survey (USGS) from March to July 2010, as part of the California State Water Resources Control Board (SWRCB) Groundwater Ambient Monitoring and Assessment (GAMA) Program's Priority Basin Project (PBP). The GAMA-PBP was developed in response to the California Groundwater Quality Monitoring Act of 2001 and is being conducted in collaboration with the SWRCB and Lawrence Livermore National Laboratory (LLNL). The WSJV study unit was the twenty-ninth study unit to be sampled as part of the GAMA-PBP. The GAMA Western San Joaquin Valley study was designed to provide a spatially unbiased assessment of untreated-groundwater quality in the primary aquifer system, and to facilitate statistically consistent comparisons of untreated groundwater quality throughout California. The primary aquifer system is defined as parts of aquifers corresponding to the perforation intervals of wells listed in the California Department of Public Health (CDPH) database for the WSJV study unit. Groundwater quality in the primary aquifer system may differ from the quality in the shallower or deeper water-bearing zones; shallow groundwater may be more vulnerable to surficial contamination. In the WSJV study unit, groundwater samples were collected from 58 wells in 2 study areas (Delta-Mendota subbasin and Westside subbasin) in Stanislaus, Merced, Madera, Fresno, and Kings Counties. Thirty-nine of the wells were selected by using a spatially distributed, randomized grid-based method to provide statistical representation of the study unit (grid wells), and 19 wells were selected to aid in the understanding of aquifer-system flow and related groundwater-quality issues (understanding wells). The groundwater samples were analyzed for organic constituents (volatile organic compounds [VOCs], low-level fumigants, and pesticides and pesticide degradates

  19. Acid Rain Effects on Adirondack Streams - Results from the 2003-05 Western Adirondack Stream Survey (the WASS Project)

    Science.gov (United States)

    Lawrence, Gregory B.; Roy, Karen M.; Baldigo, Barry P.; Simonin, Howard A.; Passy, Sophia I.; Bode, Robert W.; Capone, Susan B.

    2009-01-01

    Traditionally lakes have been the focus of acid rain assessments in the Adirondack region of New York. However, there is a growing recognition of the importance of streams as environmental indicators. Streams, like lakes, also provide important aquatic habitat, but streams more closely reflect acid rain effects on soils and forests and are more prone to acidification than lakes. Therefore, a large-scale assessment of streams was undertaken in the drainage basins of the Oswegatchie and Black Rivers; an area of 4,585 km2 in the western Adirondack region where acid rain levels tend to be highest in New York State.

  20. Use of a Combined Duplex PCR/Dot Blot Assay for more sensitive genetic characterization

    Directory of Open Access Journals (Sweden)

    Erin Curry

    2008-01-01

    Full Text Available A reliable and sensitive method of genetic analysis is necessary to detect multiple specific nucleic acid sequences from samples containing limited template. The most widely utilized method of specific gene detection, polymerase chain reaction (PCR, imparts inconsistent results when assessing samples with restricted template, especially in a multiplex reaction when copies of target genes are unequal. This study aimed to compare two methods of PCR product analysis, fluorescent detection following agarose gel electrophoresis or dot blot hybridization with chemiluminescent evaluation, in the detection of a single copy gene (SRY and a multicopy gene (β-actin. Bovine embryo sex determination was employed to exploit the limited DNA template available and the target genes of unequal copies. Primers were used either independently or together in a duplex reaction with purified bovine genomic DNA or DNA isolated from embryos. When used independently, SRY and β-actin products were detected on a gel at the equivalent of 4-cell or 1-cell of DNA, respectively; however, the duplex reaction produced visible SRY bands at the 256 cell DNA equivalent and β-actin products at the 64 cell DNA equivalent. Upon blotting and hybridization of the duplex PCR reaction, product was visible at the 1–4 cell DNA equivalent. Duplex PCR was also conducted on 186 bovine embryos and product was subjected to gel electrophoresis or dot-blot hybridization in duplicate. Using PCR alone, sex determination was not possible for 22.6% of the samples. Using PCR combined with dot blot hybridization, 100.0% of the samples exhibited either both the male specific and β-actin products or the β-actin signal alone, indicating that the reaction worked in all samples. This study demonstrated that PCR amplification followed by dot blot hybridization provided more conclusive results in the evaluation of samples with low DNA concentrations and target genes of unequal copies.

  1. Lithosphere-Asthenosphere boundary from a petrological perspective: Results form the Basin and Range, Western USA (Invited)

    Science.gov (United States)

    Gazel, E.; Plank, T.; Rau, C. J.; Forsyth, D. W.

    2010-12-01

    The lithosphere is the strong lid at the surface of the earth that defines the different tectonic plates and consists of the crust and rigid uppermost-mantle that moves on top of the viscous asthenospheric mantle. Magmas carry in their chemical codes the conditions of their mantle origin, and can be powerful tools for constraining the location of the LAB. Isotopic and trace element data are the traditional means by which magmas are ascribed to sub-continental lithospheric vs. asthenospheric sources, despite the fact that these tools provide no information on the depths or temperatures of melting. The causes of mantle melting and widespread mafic volcanism across the Basin and Range (B&R), Western USA, over the past 10 Ma is still debated. Lithospheric extension, mantle hydration, and local convective upwellings are different possible mechanisms. Constraining mantle temperature, compositional structure, and the relationship to the continental lithosphere are key to understanding the conditions of melt generation. Quantitative information is now becoming available due to improved mantle-melt thermobarometers, and our new data on the water content and oxidation state (fO2) of magmas in the B&R. Our work focuses on modeled primary magmas calibrated with data from undegassed melt inclusions trapped in olivine from young cinders. Preliminary work reveals large variations in melting conditions across the region, from cool (< 1300°C) and shallow (40-50 km) melting beneath in the west volcanic fields (e.g., Big Pine, CA) to higher temperatures (~1450°C) and deeper melting (80-100km) in the east volcanic fields (e.g., Hurricane, UT). These melting pressures are interpreted as the final melting pressures and coincide with a boundary at the top of the low velocity region in recent surface wave models using EarthScope data. Recent work on Big Pine lavas suggests that the depth of melting relates to the trace element composition of the magmas (e.g., Ce/Pb). Deeper melts have

  2. Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting.

    Science.gov (United States)

    Cheng, Tsai-Mu; Huang, Pei-Chen; Pan, Ju-Pin; Lin, Kuan-Yu; Mao, Simon J T

    2007-04-15

    Polyphenol oxidase (PPO) or tyrosinase is an important and ubiquitous enzyme responsible for browning in plants and melanization in animals. The molecular size of the plant PPO is varied among the species and its activity can be enhanced by a variety of anionic detergents. In the present study, we developed a simple method for the first-step identification of PPO in fruit and vegetable extracts. First, 3mm chromatographic paper was immersed in 0.5% (w/v) catechol solution as an immobilized PPO substrate. After running the extract with 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), one side of the glass plate was removed. The plate was immediately laid on top of the dried catechol-paper. A dark-brown band corresponding to PPO was visualized within 1 min and was further confirmed by a conventional Western blot using an antibody prepared against mushroom PPO. It also reveals that some vegetation (such as tomato, radish, and oriental melon) with low or no detectable activity in a conventional enzyme assay actually possessed marked levels of PPO activity when assessed by PAGE-blot. We propose that an inhibitor is associated with PPO in some plants; the inhibitor, however, is dissociated during the electrophoresis. Therefore, in addition to identify the molecular form of PPO, the present technique may explore the existence of PPO inhibitor(s) in plants. The detail of the method with respect to its relevance for searching a natural PPO inhibitor is described and discussed.

  3. Quantitative dot blot analysis (QDB), a versatile high throughput immunoblot method.

    Science.gov (United States)

    Tian, Geng; Tang, Fangrong; Yang, Chunhua; Zhang, Wenfeng; Bergquist, Jonas; Wang, Bin; Mi, Jia; Zhang, Jiandi

    2017-08-29

    Lacking access to an affordable method of high throughput immunoblot analysis for daily use remains a big challenge for scientists worldwide. We proposed here Quantitative Dot Blot analysis (QDB) to meet this demand. With the defined linear range, QDB analysis fundamentally transforms traditional immunoblot method into a true quantitative assay. Its convenience in analyzing large number of samples also enables bench scientists to examine protein expression levels from multiple parameters. In addition, the small amount of sample lysates needed for analysis means significant saving in research sources and efforts. This method was evaluated at both cellular and tissue levels with unexpected observations otherwise would be hard to achieve using conventional immunoblot methods like Western blot analysis. Using QDB technique, we were able to observed an age-dependent significant alteration of CAPG protein expression level in TRAMP mice. We believe that the adoption of QDB analysis would have immediate impact on biological and biomedical research to provide much needed high-throughput information at protein level in this "Big Data" era.

  4. Variations in the post-weaning human gut metagenome profile as result of Bifidobacterium acquisition in the Western microbiome

    Directory of Open Access Journals (Sweden)

    Matteo Soverini

    2016-07-01

    Full Text Available Studies of the gut microbiome variation among human populations revealed the existence of robust compositional and functional layouts matching the three subsistence strategies that describe a trajectory of changes across our recent evolutionary history: hunting and gathering, rural agriculture, and urban post-industrialized agriculture. In particular, beside the overall reduction of ecosystem diversity, the gut microbiome of Western industrial populations is typically characterized by the loss of Treponema and the acquisition of Bifidobacterium as an abundant inhabitant of the post-weaning gut microbial ecosystem. In order to advance the hypothesis about the possible adaptive nature of this exchange, here we explore specific functional attributes that correspond to the mutually exclusive presence of Treponema and Bifidobacterium using publically available gut metagenomic data from Hadza hunter-gatherers and urban industrial Italians. According to our findings, Bifidobacterium provides the enteric ecosystem with a diverse panel of saccharolytic functions, well suited to the array of gluco- and galacto-based saccharides that abound in the Western diet. On the other hand, the metagenomic functions assigned to Treponema are more predictive of a capacity to incorporate complex polysaccharides, such as those found in unrefined plant foods, which are consistently incorporated in the Hadza diet. Finally, unlike Treponema, the Bifidobacterium metagenome functions include genes that permit the establishment of microbe-host immunological cross-talk, suggesting recent co-evolutionary events between the human immune system and Bifidobacterium that are adaptive in the context of agricultural subsistence and sedentary societies.

  5. Biofilm detection by wound blotting can predict slough development in pressure ulcers: A prospective observational study.

    Science.gov (United States)

    Nakagami, Gojiro; Schultz, Gregory; Gibson, Daniel J; Phillips, Priscilla; Kitamura, Aya; Minematsu, Takeo; Miyagaki, Tomomitsu; Hayashi, Akitatsu; Sasaki, Sanae; Sugama, Junko; Sanada, Hiromi

    2016-12-26

    Bacteria have been found to form multicellular aggregates which have collectively been termed "biofilms." It is hypothesized that biofilm formation is a means to protect bacterial cells including protection form the immune response of humans. This protective mechanism is believed to explain persistent chronic wound infections. At times, the biofilms are abundant enough to see, and remove by simple wiping. However, recent evidence has shown that the removal of these visible portions are not sufficient, and that biofilms can continue to form even with daily wiping. In this work, we tested an approach to detect the biofilms which are present after clinically wiping or sharp wound debridement. Our method is based on a variation of impression cytology in which a nitrocellulose membrane was used to collect surface biofilm components, which were then differentially stained. In this prospective study, members of an interdisciplinary pressure ulcer team at a university hospital tested our method's ability to predict the generation of wound slough in the week that followed each blotting. A total of 70 blots collected from 23 pressure ulcers produced 27 wounds negative for staining and 43 positive. In the negative blots 55.6% were found to have decreased wound slough, while 81.4% with positive staining had either increase or unchanged wound slough generation. These results lead to an odds ratio of positive blotting cases of 9.37 (95% confidence intervals: 2.47-35.5, p = 0.001) for slough formation; suggesting that the changes in wound slough formation can be predicted clinically using a non-invasive wound blotting method.

  6. Multiculturalism of Tatar-Christians in Western Siberia as a result of confessional policy in the Russian Empire

    Directory of Open Access Journals (Sweden)

    Olga N. Naumenko

    2016-09-01

    Full Text Available One of the main tasks of the Russian state and the European countries is a formation of the uniform multinational people which are capable to resist to internal and external threats. Multiculturalism solves this problem, but migratory crisis in Europe has forced politicians to recognize impossibility of his realization in conditions when Christian and Arab-Muslim cultures are forced to coexist nearby. The article considers opposite experience – combination of orthodox and Islamic components in culture of the Siberian Christian Tatars in XVIII – the beginning of the 20th centuries is considered. Interpenetration of cultures has happened without loss of national identity of the Siberian Tatars; their culture has gained new unique lines. Authors consider that this combination has created a certain ethnic and national psychology of the Siberian baptized Tatars which allows them to coexist harmoniously with other people – Christian and Muslim. In article is considered multiculturalism of the Siberian Tatars in life, legal customs and religious art. Authors analyze the reasons of this phenomenon and emphasize that his major prerequisite – readiness of the Siberian people for interaction. It was based on uniform system of moral values, was formed throughout long evolution of ethnic and confessional development under the influence of a state policy. Authors come to the conclusion that the idea of multiculturalism is real and feasible in a combination "Christianity/Islam", but other conditions, than those developed in Western Europe now.

  7. Late Quaternary sediment deposition of core MA01 in the Mendeleev Ridge, the western Arctic Ocean: Preliminary results

    Science.gov (United States)

    Park, Kwang-Kyu; Kim, Sunghan; Khim, Boo-Keun; Xiao, Wenshen; Wang, Rujian

    2014-05-01

    Late Quaternary deep marine sediments in the Arctic Ocean are characterized by brown layers intercalated with yellowish to olive gray layers (Poore et al., 1999; Polyak et al., 2004). Previous studies reported that the brown and gray layers were deposited during interglacial (or interstadial) and glacial (or stadial) periods, respectively. A 5.5-m long gravity core MA01 was obtained from the Mendeleev Ridge in the western Arctic Ocean by R/V Xue Long during scientific cruise CHINARE-V. Age (~450 ka) of core MA01 was tentatively estimated by correlation of brown layers with an adjacent core HLY0503-8JPC (Adler et al., 2009). A total of 22 brown layers characterized by low L* and b*, high Mn concentration, and abundant foraminifera were identified. Corresponding gray layers are characterized by high L* and b*, low Mn concentration, and few foraminiferal tests. Foraminifera abundance peaks are not well correlated to CaCO3 peaks which occurred with the coarse-grained (>0.063 mm) fractions (i.e., IRD) both in brown and gray layers. IRDs are transported presumably by sea ice for the deposition of brown layers and by iceberg for the deposition of gray layers (Polyak et al., 2004). A strong correlation coefficient (r2=0.89) between TOC content and C/N ratio indicates that the major source of organic matter is terrestrial. The good correlations of CaCO3 content to TOC (r2=0.56) and C/N ratio (r2=0.69) imply that IRDs contain detrital CaCO3 which mainly originated from the Canadian Arctic Archipelago. In addition, high kaolinite/chlorite (K/C) ratios mostly correspond to CaCO3 peaks, which suggests that the fine-grained particles in the Mendeleev Ridge are transported from the north coast Alaska and Canada where Mesozoic and Cenozoic strata are widely distributed. Thus, the Beaufort Gyre, the predominant surface current in the western Arctic Ocean, played an important role in the sediment delivery to the Mendeleev Ridge. It is worthy of note that the TOC and CaCO3 peaks are

  8. Breast cancer literacy among higher secondary students: results from a cross-sectional study in Western Nepal.

    Science.gov (United States)

    Bhandari, Parash Mani; Thapa, Kiran; Dhakal, Sarmila; Bhochhibhoya, Shristi; Deuja, Rashmi; Acharya, Pawan; Mishra, Shiva Raj

    2016-02-18

    Being the most common cancer among women worldwide, it is vital to be well-aware of breast cancer risk factors, symptoms and curability. However, few studies have reported breast cancer literacy in students using a validated instrument. A cross-sectional study was conducted among students of grades 11 and 12 from eleven higher secondary schools, one selected randomly from each ilaka of Parbat district. Questionnaire with modified Comprehensive Breast Cancer Knowledge Test was self-administered to 516 students. Knowledge score was categorized into two categories: 'good knowledge' and 'poor knowledge' taking median score as the cut-off. Chi-square test was used to determine difference in knowledge by socio-demographic factors, including gender. Only 4.8% of the students responded correctly to at least half of the items, and 1.4% did not respond correctly to any of the items on risk factors and curability. Physical exercise was identified as a protective factor of breast cancer by 62.4% of the students. Presence of noncancerous breast lumps (56.6%) and being overweight (36.4%) were recognized as the risk factors. Knowledge of lumpectomy and radiation therapy for treatment of breast cancer was reported by 42.8% of students, while only 39.0% were aware of the availability of treatment therapies other than mastectomy. Males were significantly better informed than females (χ(2) = 4.02, p = 0.045). Pain in the breast (23.3 %), change in the shape of the breast (20.0%) and discharge of pus (14.1%) were the three most commonly recognized symptoms. Nearly one in two (47.1%) students indicated that the school curriculum inadequately informed them on breast cancer. Our study demonstrates poor knowledge on breast cancer risk factors, symptoms and curability among higher secondary school students in Western Nepal. Still, several myths regarding breast cancer persist. Half of the students had the perception that school curriculum inadequately informed them on breast cancer

  9. Standardization of Licorice and TCM Formulations Using Eastern Blot Fingerprinting Analysis

    Directory of Open Access Journals (Sweden)

    Yukihiro Shoyama

    2013-01-01

    Full Text Available To prepare the antiglycyrrhizin (GC monoclonal antibody (MAb, GC was treated with NaIO4 resulting in aldehyde which can be combined with carrier protein. An antigen conjugate was performed by a matrix-assisted laser desorption/ionization TOF mass spectrometry to determine the hapten numbers in the conjugate. Anti-GC MAb was prepared from a hybridoma which was fixed from the spleen cells producing anti-GC MAb and the myeloma cells after immunization. The TCM and licorice extract were developed by TLC and blotted to a polyvinylidene difluoride (PVDF membrane. The membrane was treated by NaIO4 and protein, enzyme labeled secondary MAb, and finally substrate was added. Clear spot appeared on PVDF membrane identifying GC against a background containing large amount of impurities. In eastern blotting, the GC molecule was divided into two functions. The aglycone part is recognized as an epitope and the sugar moiety can be combined to membrane. The specific reactivity of sugar moiety in the GC molecule against anti-GC MAb might be modified by the NaIO4 treatment on the membrane because glycyrrhetic acid 3-O-glucuronide can be stained although the cross-reactivity is only 4.3%. Eastern blotting for GC can not only apply for the standardization of licorice and TCM, but also it can open for the other bioactive products.

  10. Precisions on the structure of the Basque Arc (western Pyrenees, Spain): preliminary results from magnetic fabrics from the Biscay Synclinorium

    Science.gov (United States)

    Vegas, Néstor; Aranguren, Aitor; Rodríguez-Méndez, Lidia; Cuevas, Julia; María Tubía, José; Julián Esteban, José

    2017-04-01

    The Mesozoic Basque-Cantabrian basin (western Pyrenees) was inverted during the Alpine Orogeny in late Cretaceous-Eocene times. The central sector of the basin, the Basque Arc is characterized by the existence of large folds (80 km long) that outline an arc. This study focuses on the interpretation of AMS fabrics in rocks from the Biscay Synclinorium, a major fold system of the Basque Arc that verges to the NE, with sub-horizontal, N110˚ E trending axes and axial planes striking to N110˚ E and dipping steeply to the SW (Calvo-Rathert et al., 2007). The aim of this contribution is characterize the deformation around the Biscay Synclinorium. For this reason we combined fieldwork with magnetic fabric analysis of 95 cores in Upper Cretaceous sedimentary rocks of the Biscay Synclinorium. 68 cores come from the Calcareous formation (marls, sandy limestones and limestones of Cenomanian to Campanian age) that makes part of the northeastern limb of the synclinorium and the remaining 27 cores from the Detrital-calcareous flysch (a multilayer sequence with sandy limestones and marls of Maastrichtian age) that crops out in the synclinorium core (Garrote et al., 1991). In the Upper Cretaceous Calcareous formation there is a penetrative cleavage that mainly strikes to N110˚ E dipping 50˚ to 60˚ to the SW (S1). In the Detrital-calcareous flysch of the core, the best-preserved planar structure is the bedding and only locally an axial planar cleavage is observed. Intersection lineations are sub-horizontal and N110˚ E-trending. The measurements of the magnetic susceptibility provide low k values ranging between 99x10-6 and 403x10-6 SI. The anisotropy of the magnetic susceptibility, P, reaches values of 1.213, pointing to the overprinting of tectonic deformation on primary magnetic fabrics of sedimentary origin. The magnetic foliation shows a fairly uniform arrangement that is nearly coincident with the dominant planar structures of the rocks, S0 or S1in the core and the limb

  11. Delinquent behavior among young people in the western world : First results of the international self-report delinquency study

    NARCIS (Netherlands)

    Junger-Tas, J.; Terlouw, G.-J.; Klein, M.W.

    1994-01-01

    This volume presents the first results of the self-report in 13 individual countries. A second volume will follow in 1995, which will present the results of in-depth comparative analysis. The outcomes of these studies are presented in individual chapters. Some conclusions on the basis of this

  12. Integrating systematic screening for gender-based violence into sexual and reproductive health services: results of a baseline study by the International Planned Parenthood Federation, Western Hemisphere Region.

    Science.gov (United States)

    Guedes, A; Bott, S; Cuca, Y

    2002-09-01

    Three Latin American affiliates of the International Planned Parenthood Federation, Western Hemisphere Region, Inc. (IPPF/WHR) have begun to integrate gender-based violence screening and services into sexual and reproductive health programs. This paper presents results of a baseline study conducted in the affiliates. Although most staff support integration and many had already begun to address violence in their work, additional sensitization and training, as well as institution-wide changes are needed to provide services effectively and to address needs of women experiencing violence.

  13. Spectrophotometric quantitation of DNA on blots after ethanol-solubilization of the MTT-formazan from anti-digoxigenin-based detection of nucleic acids.

    Science.gov (United States)

    Colgan, D J

    1993-01-01

    The tetrazolium salt, 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) has recently been established as a substitute for Nitro-blue tetrazolium (NBT) in stain mixtures using antibody-conjugated alkaline phosphatase for the location of proteins on Western blots (Heegaard, 1990). Experiments reported here show that MTT is as sensitive as NBT in digoxigenin-labeled probe localization (on nucleic acid blots) utilizing alkaline-phosphatase-labelled, anti-digoxigenin antibodies. Moreover, as the formazan from MTT is soluble in ethanol, it is shown that spectrophotometric quantitation can be used to estimate the amount of target DNA on dot and Southern blots. For dot blotting, pBR328 was used as the probe and pBR322 as target. For Southern blots, human rDNA was used as the probe and total genomic calf DNA as the target. Staining response was linear over at least six twofold DNA dilutions in both types of blot.

  14. Results of an Assessment to Identify Potential Barriers to Sustainable Agriculture on American Indian Reservations in the Western United States

    Science.gov (United States)

    Singletary, Loretta; Emm, Staci; Brummer, Fara Ann; Hill, George C.; Lewis, Steve; Hebb, Vicki

    2016-01-01

    Purpose: This paper reports the results of survey research conducted with tribal producers between 2011 and 2012 on 19 of the largest American Indian reservations in Idaho, Nevada, North Dakota, Oregon, South Dakota, and Washington. The purpose of the research was to identify potential barriers to sustainable agriculture on reservation lands. This…

  15. Results of an Assessment to Identify Potential Barriers to Sustainable Agriculture on American Indian Reservations in the Western United States

    Science.gov (United States)

    Singletary, Loretta; Emm, Staci; Brummer, Fara Ann; Hill, George C.; Lewis, Steve; Hebb, Vicki

    2016-01-01

    Purpose: This paper reports the results of survey research conducted with tribal producers between 2011 and 2012 on 19 of the largest American Indian reservations in Idaho, Nevada, North Dakota, Oregon, South Dakota, and Washington. The purpose of the research was to identify potential barriers to sustainable agriculture on reservation lands. This…

  16. Mineralogical and Spectroscopic Characterization of Some Products Resulting from the Weathering Process on the Tomb of Nakht-Djehuty (TT189, Western Thebes, Upper Egypt

    Directory of Open Access Journals (Sweden)

    Hussein Hassan M.H. Mahmoud

    2010-12-01

    Full Text Available The present study deals with the mineralogical and spectroscopic characterization of some weathering products formed on the  decorated surfaces of the tomb of Nakht-Djehuty (TT189, during the time of Ramesses II (the 19th Dynasty, c.1279-1213 BC, western Thebes, Upper Egypt. The wall paintings in the ancient tombs of western Thebes are subjected to salt weathering as a result of the geological structure of the Theban formation and the environmental conditions of the region which enhance the formation of several forms of damage. The weathering forms observed are mainly flakes, salt efflorescence, biofilms and hard crusts. Damaged layers have been collected and investigated using scanning electron microscopy equipped with energy dispersive X-ray analysis system (SEM-EDS, Powder X-ray diffraction analysis (PXRD and Fourier transform spectroscopy (FTIR. The results showed that the main dominant salts affecting the site are sodium chloride (halite, NaCl and phases of sulphates [gypsum, CaSO4 · 2H2O; bassanite, CaSO4 · 0.5H2O and anhydrite, CaSO4] were also detected. FTIR spectra collected on a KBr pellet of thin dark layers covering the decorated surfaces indicated the detection of calcium oxalate probably derived from biodegradation of the organic binders in tempera techniques or the organic coatings used in old restorations of the murals.

  17. Drainage evolution of the Heihe River in western Hexi Corridor, China, derived from sedimentary and magnetostratigraphic results

    Science.gov (United States)

    Pan, Baotian; Chen, Dianbao; Hu, Xiaofei; Cao, Xilin; Chen, Jinjun; Mao, Junwei

    2016-10-01

    The uplifting of the Tibetan Plateau has significantly changed the environment in surrounding areas by delivering abundant water and sediment. The Heihe River draining the Qilian Shan in the NE Tibetan Plateau acts as a dominant sediment routing system from the uplifted NE Tibetan Plateau to the Hexi Corridor as well as the Badain Jaran Desert. Reconstructing the evolution of the Heihe River could provide evidence for the birth of the Badain Jaran Desert and enhance the understanding of sedimentary basin fill and the relationship between tectonism, drainage evolution and environmental changes. With this aim, two parallel cores (DWJ and XKJD with depths of 140 and 68.2 m, respectively) were drilled in the floodplain of the Heihe River. The facies analysis of the sedimentary sequences from the drilling cores showed that the sedimentary environment changed from the lake system to a delta system and finally to a fluvial system at the depths of ∼133.3 and ∼68 m, respectively. The magnetostratigraphic results revealed ages of approximately 1.75 and 1.12 Ma for the DWJ and XKJD cores, respectively, and an age of approximately 1.1 Ma for the transition from delta to fluvial environment in both the cores. The change of the sedimentary environment at approximately 1.1 Ma was caused by the formation of the integrated Heihe River. The integrated Heihe River may have developed via mechanisms such as river capture and river diversion due to the uplifting of the North Qilian Shan and the Longshou Shan. The present study suggested that the formation of large inland rivers, such as the Heihe River and the Shiyang River in NW China, played an important role in the evolution of the deserts and the Loess Plateau.

  18. The Western Tauern Window (Eastern Alps): Timing and Interplay of Folds and Sinistral Shear Zones as Result of South-Alpine Indentation

    Science.gov (United States)

    Schneider, Susanne; Rosenberg, Claudio; Hammerschmidt, Konrad

    2010-05-01

    Zentralgneiss to the Upper Austroalpine units. Within the Upper Penninic nappes N-side up kinematic indicators occur, in addition to the sinistral ones. Newly formed biotites of Zentralgneiss rocks have been dated with the Rb/Sr technique (Kitzig et al. 2009), yielding 18-20Ma for their formation during sinistral deformation. Fine-grained phengites from the axial plane foliation of the upright folds were dated with the K/Ar method, yielding 14-17Ma. Ar/Ar in-situ LA analyses of sinistral mylonites (Ahorn, Olperer and Greiner) yield formation ages of syn-kinematic phengites between 24-12Ma. These grains are overgrown by post-kinematic phengites of 12-9Ma. Northeast of the western TW, sinistral shear is accommodated by the brittle sinistral SEMP Fault system, whose activity has been dated to 17Ma (Peresson & Decker 1997). Several sinistral shear zones (Ahorn, Greiner, Ahrntal) of the western TW may coalesce into the SEMP Fault (e.g., Linzer et al., 2002). In the west, the Ahorn Shear Zone terminates nearly 10km east of the Brenner Fault, into a NW-striking fold belt. The Ahrntal Fault continues into the Jaufen Fault, which merges with the brittle sinistral Giudicarie Fault. Motion along the Giudicarie Fault initiated in the Miocene (Stipp et al., 2004), or already in the Oligocene (Müller et al 2001). Based on these results, a temporal, kinematic and geometric continuity between sinistral shearing along the Giudicarie Fault, along the SEMP Fault, and throughout the western TW, can be assessed. The sinistral shear zones of the western TW are kinematically linked to upright folds, hence to crustal thickening. Upright folding and sinistral shearing were active since 24Ma and terminated at 12Ma. In summary, the sinistral displacements of the Giudicarie System appear to be partitioned into upright folds and sinistral, transpressive shear zones in the western Tauern Window, both of which contribute to its exhumation. The coalescence of the sinistral shear zones into the SEMP Fault

  19. Seroprevalence of toxocariasis in hypereosinophilic individuals in Ahwaz, south-western Iran.

    Science.gov (United States)

    Maraghi, S; Rafiei, A; Hajihossein, R; Sadjjadi, S M

    2012-06-01

    Eosinophilia in human peripheral blood is caused by different agents, including toxocariasis. The present study aimed to evaluate the prevalence of toxocariasis in hypereosinophilic individuals in the city of Ahwaz, located in south-western Iran, using enzyme-linked immunosorbent assay and Western blot techniques. Serum samples were examined from 100 individuals with peripheral blood eosinophilia and also from another 100 individuals without eosinophilia as the control group. In hypereosinophilic individuals seroprevalence antibodies against Toxocara were found in 19 (19%), of whom 12 (63.15%) were female and 7 (36.85%) were male. Positive sera were subsequently confirmed by Western blot. All of the observed bands ranged from 24 to 100 kDa. Antibodies against Toxocara were found in 1% of the control group, but were not confirmed by Western blot. The results showed significant differences between the frequency of infection within age and gender (P toxocariasis as a hygienic problem among hypereosinophilic individuals in this area. It is, therefore, necessary to examine these individuals for toxocariasis.

  20. Monoclonal Antibodies against Small Molecule Natural Products and Their Applications, Eastern Blotting and Knockout Extract

    Directory of Open Access Journals (Sweden)

    Yukihiro Shoyama

    2011-06-01

    Full Text Available To determine the hapten number in hapten-carrier protein conjugate matrix-assisted laser desorption/ionization (MALDI tof mass spectrometry was applied. Highly specific anti-ginsenoside Rb1 and Rg1 monoclonal antibodies (MAbs were prepared. Ginsenosides were developed on thin layer chromatography (TLC plates which were covered by a polyvinylidene difluoride (PVDF membrane resulting in blotting. The membrane was treated with NaIO4 solution to release the aldehyde group on the sugar moiety of the ginsenosides. By treatment of the membrane with a protein solution the ginsenoside-protein conjugation as a Schiff-base occurred, which can function to fix it to the PVDF membrane. A part of the ginsenoside aglycone was reacted with anti-ginsenoside Rb1 MAb, secondary MAb conjugated with enzyme and finally a substrate was added, resulting in a specific and highly sensitive staining that we named Eastern blotting. Furthermore, it makes one-step isolation of ginsenoside Rb1 possible using an immuno-affinity column conjugated with anti-ginsenoside Rb1 MAb. Furthermore, immunoaffinity concentration was carried out allowing high sensitivity analysis of lower concentrations of ginsenoside Rb1 so that several unknown bands could be structurally determined.

  1. Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

    Directory of Open Access Journals (Sweden)

    N.S. Sato

    2004-07-01

    Full Text Available Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples, from healthy blood donors (50 samples, individuals with sexually transmitted disease other than syphilis (3 samples, and from individuals with other spirochetal diseases such as Lyme disease (20 samples and leptospirosis (3 samples. The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7% and a specificity of 94.7% (95% CI, 87.0 to 98.7%; a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

  2. Western Sufism

    DEFF Research Database (Denmark)

    Sedgwick, Mark

    Western Sufism is sometimes dismissed as a relatively recent "new age" phenomenon, but in this book, Mark Sedgwick argues that it actually has very deep roots, both in the Muslim world and in the West. In fact, although the first significant Western Sufi organization was not established until 1915......, the first Western discussion of Sufism was printed in 1480, and Western interest in some of the ideas that are central to Sufi thought goes back to the thirteenth century. Sedgwick starts with the earliest origins of Western Sufism in late antique Neoplatonism and early Arab philosophy, and traces later...

  3. A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot.

    Science.gov (United States)

    Albers, Eliene; Sbroggiò, Mauro; Martin-Gonzalez, Javier; Avram, Alexandra; Munk, Stephanie; Lopez-Contreras, Andres J

    2017-01-19

    The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific genomic locus, but limits the detection of its correct genomic integration by standard PCR methods. Genomic Long Range PCR (LR-PCR), using primers adjacent to the homology arms, has been used as an alternative to radioactive-based Southern blot screenings. However, LR-PCRs are often difficult and render many false positive and false negative results. Here, we propose an alternative screening method based on the detection of a genetic modification at the mRNA level, which we successfully optimized in two mouse models. This screening method consists of a reverse-transcription PCR (RT-PCR) using primers that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting constructs which can also be useful to screen KO and KI mutant mice or cell lines including those generated by CRISPR/Cas9.

  4. Simultaneous preparation of RNA and nuclei for Northern blot and flow cytometric analysis

    Energy Technology Data Exchange (ETDEWEB)

    Tesfaigzi, J.; Jaramillo, R. [Inhalation Toxicology Research Inst., Albuquerque, NM (United States)

    1996-11-01

    Several methods have been developed to quantify RNA synthesis during the progression of the cell cycle. The rate of RNA synthesis can be detected during different stages of the cell cycle by staining cells with agents that intercalate with nucleic acids. For example, following staining of mammalian cells with acridin orange, the green and red fluorescence that correlates with DNA and RNA content, respectively, can be analyzed by flow cytometry. Increase in RNA content during the progression of cells through the cell cycle can be measured after staining with acridin orange. RNA synthesis resulting from the stimulation of quiescent cells with various growth factors has also been demonstrated by labeling cells with bromo-uridine and using the anti-bromo-deoxyuridine antibody. These methods allow measurement of the overall RNA content in cells; however, they do not allow the measurement of the levels of specific mRNAs throughout the cell cycle. Current methods to quantify specific mRNAs generally require the preparation of a large number of cells (5--10 {times} 10{sup 6} cells) to carry out flow cytometric analyses and to isolate RNA for Northern blot analysis or solution hybridization. In this report, the authors describe a method of simultaneously preparing RNA and nuclei for Northern blot and flow cytometric analyses, respectively. The minimum number of nuclei required to obtain flow cytometric data and the effect of conserving nuclei in methanol for several days are also presented.

  5. High-Resolution Late Holocene Climatic Records From Kucukcekmece Lagoon and Uludag Glacial, Yeniçaga, Bafa Lakes in Western Turkey: Some Preliminary Results

    Science.gov (United States)

    Akcer On, S.; Cagatay, M.; Sakinc, M.

    2010-12-01

    This study focuses on climatic and environmental sedimentary records of the last 4000 years in Kucukcekmece Lagoon (Istanbul), Uludag glacial (Bursa) , Yenicaga (Bolu) and Bafa (Mugla) Lakes in western Turkey. The water bodies are located on a N-S transect in western Turkey, and as such the’r records are important for assesment of Late Holocene eastern Mediterranean climatic changes. A total of 12 cores varying between 0.6 and 4.8 m are analyzed at 5mm resolution using Multi Sensor Core Logger (MSCL) having magnetic susceptibilty, P-Wave, density and resistivity sensors, XRF(X-Ray Fluoresance) core scanner multi element analysis at a 0.2 mm resolution. The cores are then sampled at 20, 50 and 100 mm intervals for different analyses. The samples are analyzed for total inorganic (TIC) and organic carbon (TOC). The ostracoda and benthic foraminifera shells in the sand size fraction of the sediment samples are identified under binocular microscope and suitable species are picked and analyzed for the stable oxygen and carbon isotope analysis. The cores are dated using AMS 14C analysis. The data from these lakes and lagoon are interpreted for environmental and climatic changes and compared with historical and other regional and global climate signals. The results indicate dry periods during 3300 -2500 ka BP and 867-1790 AD and1070-1690 AD with some brief intervening humid periods. Abrupt environmental change starting around1950 due to mainly human activities has also been clearly detected. These changes in the sedimentds are well correlated with the historical records.

  6. RNase protection assays and RNA gel blots: a direct comparison of sensitivity.

    Science.gov (United States)

    Higgs, D C; Colbert, J T

    1992-01-01

    RNase protection assays are commonly thought to be a more sensitive means of detecting and quantitating specific mRNAs than are RNA gel blots (Northern blots). We have directly compared the sensitivity of these two approaches by assaying for known amounts of in vitro synthesized beta-glucuronidase mRNA. With the probes and protocols employed here, the ability to detect a specific mRNA was similar whether RNase protection or RNA gel blot analyses were performed.

  7. RESULTS OF COMPREHENSIVE STUDIES OF THE UNDERGROUND HYDROSPHERE WITHIN THE WESTERN SHOULDER OF THE BAIKAL RIFT (AS EXEMPLIFIED BY THE BAYANDAI – KRESTOVSKY CAPE SITE

    Directory of Open Access Journals (Sweden)

    Konstantin Zh. Seminsky

    2015-09-01

    Full Text Available The subject of comprehensive studies is the underground hydrosphere of the upper crust of the western shoulder of the Baikal rift, being characterized by high tectonic activity in the recent stage of tectogenesis. The studies were focused on the Bayandai – Krestovsky Cape site, considering it as a benchmark for the territory of the Western Pribiakalie (Fig. 1. The available hydrogeological survey database is used to study underground waters circulating at depth of several kilometers. Analyses of deeper waters are conducted on the basis of geophysical data. According to results of initial geological and geophysical studies [Семинский и др., 2010], the crust at the junction of the Siberian crater and the SayanBaikal folded belt is characterized by a hierarchic zoneblock structure (Fig. 2. Regardless of the scale of studies, the territory under study can be divided into sections of two types, that alternate from NW to SE and represent wide highly destructed zones and relatively monolithic blocks of the crust. The Obruchev fault system is distinguished as the main interblock zone (the 2nd hierarchic level in the study area. It represents the 50 km long NW shoulder of the Baikal rift (the1st hierarchic level and includes the Morskaya, Primorskaya and Prikhrebtovaya interplate zones (the 3rd hierarchic level. These zones are traced from depth of dozens of kilometers; at the surface, they are represented by fault structures of the highest hierarchic levels.Specific features of the current zoneblock divisibility of the crust serve as the structural basis for interpreting the materials obtained by hydrogeological studies conducted on the Bayandai – Krestovsky Cape site to research the distribution, mineralization and macrocomponent compositions of waters which represent the subsurface part of the underground lithosphere in the study area. The research is based on analyses of the underground water samples from 46 observation points

  8. Immunological diagnosis of human hydatid cyst using Western immunoblotting technique

    Science.gov (United States)

    Hadipour, Mahboubeh; Nazari, Mohammad; Sanei, Behnam; Ghayour, Zahra; Sharafi, Seyedeh Maryam; Yazdani, Hajar; Darani, Hossein Yousofi

    2016-01-01

    Background: Echinococcosis is a parasitic disease with worldwide distribution which is caused by the tapeworms Echinococcus granulosus. Diagnosis of the disease relies on imaging techniques, but the techniques are not able to differentiate the cyst from benign or malignant tumors; hence, appropriate serologic methods are required for the differential diagnosis of the infection. Materials and Methods: In this investigation, different sheep hydatid cyst antigens probed with thirty sera of patients with hydatid cyst and also thirty human normal sera using Western immunoblotting technique. Considering results of surgery as gold standard, sensitivity and specificity of Western blotting was estimated. Results: Sera of 29, 26, and 16 patients with hydatid cyst reacted with specific bands of hydatid cyst fluid (HCF), protoscolex crude antigen, and cyst wall crude antigen, respectively. However, none of the normal human sera reacted with those specific bands. Conclusion: A 20 kDa band of sheep HCF is an appropriate antigen for serodiagnosis of hydatid cyst infection. PMID:28331516

  9. Immunological diagnosis of human hydatid cyst using Western immunoblotting technique

    Directory of Open Access Journals (Sweden)

    Mahboubeh Hadipour

    2016-01-01

    Full Text Available Background: Echinococcosis is a parasitic disease with worldwide distribution which is caused by the tapeworms Echinococcus granulosus. Diagnosis of the disease relies on imaging techniques, but the techniques are not able to differentiate the cyst from benign or malignant tumors; hence, appropriate serologic methods are required for the differential diagnosis of the infection. Materials and Methods: In this investigation, different sheep hydatid cyst antigens probed with thirty sera of patients with hydatid cyst and also thirty human normal sera using Western immunoblotting technique. Considering results of surgery as gold standard, sensitivity and specificity of Western blotting was estimated. Results: Sera of 29, 26, and 16 patients with hydatid cyst reacted with specific bands of hydatid cyst fluid (HCF, protoscolex crude antigen, and cyst wall crude antigen, respectively. However, none of the normal human sera reacted with those specific bands. Conclusion: A 20 kDa band of sheep HCF is an appropriate antigen for serodiagnosis of hydatid cyst infection.

  10. Cathepsin D Expression in Colorectal Cancer: From Proteomic Discovery through Validation Using Western Blotting, Immunohistochemistry, and Tissue Microarrays

    Directory of Open Access Journals (Sweden)

    Chandra Kirana

    2012-01-01

    Full Text Available Despite recent advances in surgical techniques and therapeutic treatments, survival from colorectal cancer (CRC remains disappointing with some 40–50% of newly diagnosed patients ultimately dying of metastatic disease. Current staging by light microscopy alone is not sufficiently predictive of prognosis and would benefit from additional support from biomarkers in order to stratify patients appropriately for adjuvant therapy. We have identified that cathepsin D expression was significantly greater in cells from invasive front (IF area and liver metastasis (LM than those from main tumour body (MTB. Cathepsin D expression was subsequently examined by immunohistochemistry in tissue microarrays from 119 patients with CRC. Strong expression in tumour cells at the IF did not correlate significantly with any clinico-pathological parameters examined or patient survival. However, cathepsin D expression in cells from the MTB was highly elevated in late stage CRC and showed significant correlation with subsequent distant metastasis and shorter cancer-specific survival. We also found that macrophages surrounding tumour cells stained strongly for cathepsin D but there was no significant correlation found between cathepsin D in macrophages at IF and MTB of CRC patient with the clinic-pathological parameters examined.

  11. Western-blot detection of PrP**sc in archived paraffin-embedded brainstem from scrapie-affected sheep

    Science.gov (United States)

    Scrapie is a naturally occurring fatal neurodegenerative disease of adult sheep and goats, one of a group of mammalian diseases known as transmissible spongiform encephalopathies or prion diseases. Immunoassays that identify disease-associated prion protein (PrP**Sc) are integral to the diagnosis o...

  12. Lectin staining and Western blot data showing differential sialylation of nutrient-deprived cancer cells to sialic acid supplementation

    OpenAIRE

    Badr, Haitham A.; Dina M. M. AlSadek; Mohit P. Mathew; Chen-Zhong Li; Djansugurova, Leyla B.; Yarema, Kevin J.; Hafiz Ahmed

    2015-01-01

    This report provides data that are specifically related to the differential sialylation of nutrient deprived breast cancer cells to sialic acid supplementation in support of the research article entitled, “Nutrient-deprived cancer cells preferentially use sialic acid to maintain cell surface glycosylation" [1]. Particularly, breast cancer cells, when supplemented with sialic acid under nutrient deprivation, display sialylated glycans at the cell surface, but non-malignant mammary cells show s...

  13. Characterization of Polyclonal Antibody Induced by Autoantibody TPO (Thyroidperoxidase) From Autoimmune Thyroid Disease (AITD) Serum with ELISA and Western Blotting

    OpenAIRE

    Maulidya Aulia Fiqriyana; Aulanni'am Aulanni'am; Anna Roosdiana

    2013-01-01

    Autoantibody TPO is a potential marker for early detection of autoimmune thyroid disease (AITD). Autoantibody TPO has a specifity and a sensitivity ranging from 82% to100% in comparison to other AITD serology markers. Concentration of autoantibody TPO in sera had a positive correlation with activities of chronic AITD. This research have been conducted to investigate the characteristic of polyclonal antibody TPO induced by autoantibody TPO from serum of AITD patients. The autoantibody TPO was ...

  14. Identification of hantavirus infection by Western blot assay and TaqMan PCR in patients hospitalized with acute kidney injury.

    Science.gov (United States)

    Oldal, Miklós; Németh, Viktória; Madai, Mónika; Kemenesi, Gábor; Dallos, Bianka; Péterfi, Zoltán; Sebők, Judit; Wittmann, István; Bányai, Krisztián; Jakab, Ferenc

    2014-06-01

    Hantaviruses, one of the causative agents of viral hemorrhagic fevers, represent a considerable healthcare threat. In Hungary, Dobrava-Belgrade virus (DOBV) and Puumala virus (PUUV) are the main circulating hantavirus species, responsible for the clinical picture known as hemorrhagic fever with renal syndrome, a disease that may be accompanied by acute kidney injury (AKI), requiring hospitalization with occasionally prolonged recovery phase. A total of 20 patient sera were collected over a 2-year period from persons hospitalized with AKI, displaying clinical signs and laboratory findings directly suggestive for hantavirus infection. Samples were tested using an immunoblot assay, based on complete viral nucleocapsid proteins to detect patients' IgM and IgG antibodies against DOBV and PUUV. In parallel, all specimens were also tested by 1-step real-time TaqMan reverse-transcriptase polymerase chain reaction to confirm infection and to determine the causative hantavirus genotype. We present here the first Hungarian clinical study spanning across 2 years and dedicated specifically to assess acute kidney injuries, in the context of hantavirus prevalence.

  15. Electrospun nitrocellulose and nylon: Design and fabrication of novel high performance platforms for protein blotting applications

    Directory of Open Access Journals (Sweden)

    Bowlin Gary L

    2007-10-01

    Full Text Available Abstract Background Electrospinning is a non-mechanical processing strategy that can be used to process a variety of native and synthetic polymers into highly porous materials composed of nano-scale to micron-scale diameter fibers. By nature, electrospun materials exhibit an extensive surface area and highly interconnected pore spaces. In this study we adopted a biological engineering approach to ask how the specific unique advantages of the electrospinning process might be exploited to produce a new class of research/diagnostic tools. Methods The electrospinning properties of nitrocellulose, charged nylon and blends of these materials are characterized. Results Nitrocellulose electrospun from a starting concentration of Conclusion The flexibility afforded by electrospinning process makes it possible to tailor blotting membranes to specific applications. Electrospinning has a variety of potential applications in the clinical diagnostic field of use.

  16. Application of a Dot Blot Hybridization Platform to Assess Streptococcus uberis Population Structure in Dairy Herds

    Science.gov (United States)

    Albuquerque, Pedro; Ribeiro, Niza; Almeida, Alexandre; Panschin, Irena; Porfirio, Afonso; Vales, Marta; Diniz, Francisca; Madeira, Helena; Tavares, Fernando

    2017-01-01

    Streptococcus uberis is considered one of the most important pathogens associated with bovine mastitis. While traditionally acknowledged as an environmental pathogen, S. uberis has been shown to adopt a contagious epidemiological pattern in several dairy herds. Since different control strategies are employed depending on the mode of transmission, in-depth studies of S. uberis populations are essential to determine the best practices to control this pathogen. In this work, we optimized and validated a dot blot platform, combined with automatic image analysis, to rapidly assess the population structure of infective S. uberis, and evaluated its efficiency when compared to multilocus sequence analysis (MLSA) genotyping. Two dairy herds with prevalent S. uberis infections were followed in a 6 month period, in order to collect and characterize isolates from cows with persistent infections. These herds, located in Portugal (Barcelos and Maia regions), had similar management practices, with the herd from Barcelos being smaller and having a better milking parlor management, since infected cow segregation was immediate. A total of 54 S. uberis isolates were obtained from 24 different cows from the two herds. To overcome operator-dependent analysis of the dot blots and increase the technique's consistency and reliability, the hybridization signals were converted into probability values, with average probabilities higher than 0.5 being considered positive results. These data allowed to confirm the isolates' identity as S. uberis using taxa-specific markers and to determine the presence of virulence- and antibiotic resistance-related genes. In addition, MLSA allowed to disclose the most prevalent S. uberis clonal lineages in both herds. Seven different clusters were identified, with Barcelos showing a high clonal diversity and Maia a dominant lineage infecting most cows, suggesting distinct epidemiological patterns, with S. uberis displaying an environmental or contagious

  17. Field observations and results of a 1-D boundary layer model for developing near-surface temperature maxima in the Western Arctic

    Directory of Open Access Journals (Sweden)

    Shawn G. Gallaher

    2017-03-01

    Full Text Available Summer sea ice extent in the Western Arctic has decreased significantly in recent years resulting in increased solar input into the upper ocean. Here, a comprehensive set of 'in situ' shipboard, on-ice, and autonomous ice-ocean measurements were made of the early stages of formation of the near-surface temperature maximum (NSTM in the Canada Basin. These observations along with the results from a 1-D turbulent boundary layer model indicate that heat storage associated with NSTM formation is largely due to the absorption of penetrating solar radiation just below a protective summer halocline. The depth of the summer halocline was found to be the most important factor for determining the amount of solar radiation absorbed in the NSTM layer, while halocline strength controlled the amount of heat removed from the NSTM by turbulent transport. Observations using the Naval Postgraduate School Turbulence Frame show that the NSTM was able to persist despite periods of intermittent turbulence because transport rates were too small to remove significant amounts of heat from the NSTM layer. The development of the early and late summer halocline and NSTM were found to be linked to summer season buoyancy and wind events. For the early summer NSTM, 1-D boundary layer model results show that melt pond drainage provides sufficient buoyancy to the summer halocline to prevent subsequent wind events from mixing out the NSTM. For the late summer NSTM, limited freshwater inputs reduce the strength of the summer halocline making the balance between interfacial stresses and buoyancy more tenuous. As a result, the late summer NSTM is an ephemeral feature dependent on local wind conditions, while the early summer NSTM is more persistent and able to store heat in the near-surface ocean beyond the summer season.

  18. Nuclear and mitochondrial DNA analyses of golden eagles (Aquila chrysaetos canadensis) from three areas in western North America; initial results and conservation implications

    Science.gov (United States)

    Craig, Erica H; Adams, Jennifer R.; Waits, Lisette P.; Fuller, Mark R.; Whittington, Diana M.

    2016-01-01

    Understanding the genetics of a population is a critical component of developing conservation strategies. We used archived tissue samples from golden eagles (Aquila chrysaetos canadensis) in three geographic regions of western North America to conduct a preliminary study of the genetics of the North American subspecies, and to provide data for United States Fish and Wildlife Service (USFWS) decision-making for golden eagle management. We used a combination of mitochondrial DNA (mtDNA) D-loop sequences and 16 nuclear DNA (nDNA) microsatellite loci to investigate the extent of gene flow among our sampling areas in Idaho, California and Alaska and to determine if we could distinguish birds from the different geographic regions based on their genetic profiles. Our results indicate high genetic diversity, low genetic structure and high connectivity. Nuclear DNA Fst values between Idaho and California were low but significantly different from zero (0.026). Bayesian clustering methods indicated a single population, and we were unable to distinguish summer breeding residents from different regions. Results of the mtDNA AMOVA showed that most of the haplotype variation (97%) was within the geographic populations while 3% variation was partitioned among them. One haplotype was common to all three areas. One region-specific haplotype was detected in California and one in Idaho, but additional sampling is required to determine if these haplotypes are unique to those geographic areas or a sampling artifact. We discuss potential sources of the high gene flow for this species including natal and breeding dispersal, floaters, and changes in migratory behavior as a result of environmental factors such as climate change and habitat alteration. Our preliminary findings can help inform the USFWS in development of golden eagle management strategies and provide a basis for additional research into the complex dynamics of the North American subspecies.

  19. Development of a monoclonal antibody-based colony blot immunoassay for detection of thermotolerant Campylobacter species.

    Science.gov (United States)

    Huang, Hongsheng; Phipps-Todd, Beverley; McMahon, Tanis; Elmgren, Catherine L; Lutze-Wallace, Cheryl; Todd, Zoe A; Garcia, Manuel M

    2016-11-01

    Campylobacter species, particularly thermotolerant Campylobacter spp., such as C. jejuni, are major human foodborne pathogens. Culture methods have been routinely used for the detection of this organism in various types of samples. An alternative, simple and rapid confirmation test(s) without further tedious biochemical tests would be useful. Meanwhile, Campylobacter-like colonies can be difficult to identify on agar plates overgrown with competitive bacteria, which can lead to false-negative results. This study was to develop a simple colony blot immunoassay using a new monoclonal antibody (Mab) produced in the present study for rapid screening, confirmation and quantification of campylobacters on culture agar plates. The procedure developed in this study was able to specifically detect thermotolerant Campylobacter spp., but not other non-thermotolerant Campylobacter and non-Campylobacter reference strains tested. This assay could detect 10(5) cells in a single dot. This assay showed 100% correlation with the culture method for the blotted membranes from 21 either chicken meat or vegetable samples experimentally inoculated with thermotolerant campylobacters. Among 101 natural samples of chicken meat (n=44), chicken feces (n=20) and vegetables (n=37), this assay also showed positive for 23 chicken meat and 14 fecal samples that were positive for thermotolerant campylobacters by culture method, and identified four additional suspects that were culture negative. Membranes stored at 4°C for at least 4years could also be used for this assay. The assay developed in this study can be used in quantitative study for immediate or archival usage, and for diagnostic test to preliminarily confirm the presence of thermotolerant Campylobacter on agar plates. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  20. A new scheme for joint surface wave and earthquake travel-time inversion and resulting 3-D velocity model for the western North Island, New Zealand

    Science.gov (United States)

    Eberhart-Phillips, Donna; Fry, Bill

    2017-08-01

    We have developed a joint inversion of surface wave group velocity (U) and local earthquake travel-time (LET) data and applied it to the North Island, New Zealand, to improve the existing New Zealand wide 3-D seismic velocity model. This approach takes full advantage of the differing sensitivities of surface and body waves. The data are complementary, particularly at shallow depths where LET tomography suffers from vertical smearing and surface wave tomography is susceptible to horizontal smearing. The employed U observations are 2-D models at discrete periods which were developed for Rayleigh wave dispersion curves measured from the 1744 interstation Green's Functions obtained by stacked cross-correlations of broadband ambient noise data. In the volume surrounding each U observation, we distribute numerous points for relating the U observation to the gridded 3-D tomography model, analogous to points along a raypath. The partial derivatives at the points are computed using the U sensitivity kernels for Vp and Vs, with Vs related to Vp and Vp/Vs perturbations. Thus, the U observations are included along with the travel-time observations in a joint inversion to best fit the data and the existing tomography model. The resulting model favors the U where there is little travel-time resolution. The combined inversion used 2949 U observations at 6-16 s period and LET from 1509 earthquakes that extend to 370 km depth, and improved the model fit by reducing the U residual data variance by 62% and the LET by 9%. The resulting model generally has better constrained depth of shallow anomalies, with decreased velocity in the upper 2 km in the western North Island, and slight focusing of crustal high velocity features at 8 km depth. Significantly, the increased resolution in the shallowest 5 km of the model improves the utility of the 3-D model for use in seismic hazard assessment, wave propagation studies, and studies comparing seismic velocities to geological mapping.

  1. Reactive Nitrogen in Asian Continental Outflow over the Western Pacific: Results from the NASA Transport and Chemical Evolution over the Pacific (TRACE-P)Airborne Mission

    Science.gov (United States)

    Talbot, R.; Dibb, J.; Scheuer, E.; Seid, G.; Russo, R.; Sandholm, S.; Tan, D.; Blake, D.; Blake, N.; Singh, H.

    2003-01-01

    We present here results for reactive nitrogen species measured aboard the NASA DC-8 aircraft during the Transport and Chemical Evolution over the Pacific TRACE-P) mission. The large-scale distributions total reactive nitrogen (NO(sub y,sum) = NO + NO2 + HNO3 + PAN + C(sub 1)-C(sub 5) alkyl nitrates) and O3 and CO were better defined in the boundary layer with significant degradation of the relationships as altitude increased. Typically, NO(sub y,sum) was enhanced over background levels of approx.260 pptv by 20-to-30-fold. The ratio C2H2/CO had values of 1-4 at altitudes up to 10 km and as far eastward as 150degE, implying significant vertical mixing of air parcels followed by rapid advection across the Pacific. Analysis air parcels originating from five principal Asian source regions showed that HNO3 and PAN dominated NO(sub y,sum). Correlations of NO(sub y,sum) with C2Cl4 (urban tracer) were not well defined in any of the source regions, and they were only slightly better with CH3Cl (biomass tracer). Air parcels over the western Pacific contained a complex mixture of emission sources that are not easily resolvable as shown by analysis of the Shanghai mega-city plume. It contained an intricate mixture of pollution emissions and exhibited the highest mixing ratios of NO(sub y,sum) species observed during TRACE-P. Comparison of tropospheric chemistry between the earlier PEM-West B mission and the recent TRACE-P data showed that in the boundary layer significant increases in the mixing ratios of NO(sub y,sum)species have occurred, but the middle and upper troposphere seems to have been affected minimally by increasing emissions on the Asian continent over the last 7 years.

  2. 108 WESTERN INFLUENCE ON CHINESE AND NIGERIAN ...

    African Journals Online (AJOL)

    Ike Odimegwu

    western cultures and globalization; and as a result of these, ... In Nigeria, westernization is seen as the effects of western invasion and colonization on some native societies of the world which had both positive and negative impacts. Nigerians.

  3. Shipboard determination of radiocesium in seawater after the Fukushima accident: results from the 2011-2012 Russian expeditions to the Sea of Japan and western North Pacific Ocean.

    Science.gov (United States)

    Ramzaev, V; Nikitin, A; Sevastyanov, A; Artemiev, G; Bruk, G; Ivanov, S

    2014-09-01

    A total of 88 seawater samples were collected during two Russian research expeditions (April-May 2011 and August-September 2012) to the Sea of Japan, the Oyashio Current region near Kuril Islands and the Kuroshio-Oyashio transition area in the western North Pacific Ocean. The observations were made aboard the R/V Pavel Gordienko and Akademik Shokalsky in order to study the impact of the Fukushima accident on radioactive contamination of the marine environment. On the board of a ship, the water samples were passed through filters to retain particles with the size of >1 micron. Cesium was extracted from the large volumes (100-3000 L) of the filtrated water using a selective fiber chemisorbent impregnated with copper ferrocyanide. Measurements of (134)Cs and (137)Cs activities in 83 samples of sorbents and 21 samples of filters were performed in the ship-based laboratory with a semiconductor HP-Ge detector. The quantified activity concentrations of dissolved radiocesium ranged from 1 Bq m(-3) to 34 Bq m(-3) for (137)Cs and from 0.2 Bq m(-3) to 29 Bq m(-3) for (134)Cs. Activity concentrations of (137)Cs and (134)Cs were strongly correlated with each other (r = 0.993, n = 59). The (137)Cs/(134)Cs activities ratio in the Fukushima-derived radiocesium inventory for the study areas was deduced to be 0.99 ± 0.03 (on 15 March 2011) and the pre-Fukushima background level of (137)Cs in seawater was estimated as 1.3 ± 0.3 Bq m(-3). The lowest activities of both isotopes were determined in the western part of the Sea of Japan near the Russian coast, while the maximal levels were observed in the open Pacific Ocean, some 500-800 km offshore the Fukushima Dai-ichi Nuclear Power Plant. Contamination with (134)Cs at a level of 0.3-2.6 Bq m(-3) was registered in seawater samples collected in 2011 near the Kuril Islands and Kamchatka in the Oyashio Current region. During the period from April-May 2011 to August-September 2012, activity concentrations of (137)Cs and (134)Cs in surface

  4. Peculiarities in atmospheric particle number and size-resolved speciation in an urban area in the western Mediterranean: Results from the DAURE campaign

    Science.gov (United States)

    Reche, C.; Viana, M.; Moreno, T.; Querol, X.; Alastuey, A.; Pey, J.; Pandolfi, M.; Prévôt, A.; Mohr, C.; Richard, A.; Artiñano, B.; Gomez-Moreno, F. J.; Cots, N.

    2011-09-01

    Air quality degradation problems in urban environments are mainly due to road traffic emissions. In northern and central European cities extensive investigations have been carried out on the variability of number of ultrafine particles and black carbon, whereas studies in southern-Europe have traditionally focused on chemical speciation, resulting in insufficient information to characterise urban aerosol processes. Accordingly, sources and processes controlling atmospheric pollutants were evaluated during the international DAURE campaign in Barcelona (Spain), a typical urban environment in the western Mediterranean, by means of continuous measurements of particle number, black carbon, PMx, particulate nitrate and particulate sulphate concentrations. Results provided evidence of the influence of three PM sources of interest: road traffic, construction-demolition works and shipping emissions. Road traffic governs the daily cycle of black carbon, a number of gaseous pollutants and the finer fraction of PM. Evaluation of the coarse aerosol (PM 2.5-10) daily cycle furnished evidence of the influence of construction-demolition works. These activities could increase hourly PM 2.5-10 levels by up to 8 μg m -3 on an annual hourly mean basis. Finally, shipping emissions were traced by means of SO 2 concentrations, which peak at a time different from that of other gaseous pollutants owing to the regular daytime onshore breeze bringing harbour emissions at midday. Moreover, a major local contribution of secondary aerosols was detected with elevated particle number levels at midday when nucleation processes are favoured by photochemistry and lower pollution levels. Particle number data were also analysed in search for the primary emission of vehicle exhaust (N1) and the secondary parts from gaseous precursors, primary parts from non-traffic sources, and/or particles inherited in the air mass (N2). N2 fraction accounted for 40% of number concentrations, ranging from 37% during

  5. PCR-Reverse Blot Hybridization Assay for Screening and Identification of Pathogens in Sepsis

    OpenAIRE

    Choi, Yeonim; Wang, Hye-young; Lee, Gyusang; Park, Soon-Deok; Jeon, Bo-Young; Uh, Young; Kim, Jong Bae; Lee, Hyeyoung

    2013-01-01

    Rapid and accurate identification of the pathogens involved in bloodstream infections is crucial for the prompt initiation of appropriate therapy, as this can decrease morbidity and mortality rates. A PCR-reverse blot hybridization assay for sepsis, the reverse blot hybridization assay (REBA) Sepsis-ID test, was developed; it uses pan-probes to distinguish Gram-positive and -negative bacteria and fungi. In addition, the assay was designed to identify bacteria and fungi using six genus-specifi...

  6. Alcohol, tobacco and illicit drug use amongst same-sex attracted women: results from the Western Australian Lesbian and Bisexual Women's Health and Well-Being Survey

    Directory of Open Access Journals (Sweden)

    McManus Alexandra

    2009-09-01

    Full Text Available Abstract Background The prevalence of alcohol, tobacco and illicit drug use has been reported to be higher amongst lesbian and bisexual women (LBW than their heterosexual counterparts. However, few studies have been conducted with this population in Australia and rates that have been reported vary considerably. Methods A self-completed questionnaire exploring a range of health issues was administered to 917 women aged 15-65 years (median 34 years living in Western Australia, who identified as lesbian or bisexual, or reported having sex with another woman. Participants were recruited from a range of settings, including Perth Pride Festival events (67.0%, n = 615, online (13.2%, n = 121, at gay bars and nightclubs (12.9%, n = 118, and through community groups (6.9%, n = 63. Results were compared against available state and national surveillance data. Results LBW reported consuming alcohol more frequently and in greater quantities than women in the general population. A quarter of LBW (25.7%, n = 236 exceeded national alcohol guidelines by consuming more than four standard drinks on a single occasion, once a week or more. However, only 6.8% (n = 62 described themselves as a heavy drinker, suggesting that exceeding national alcohol guidelines may be a normalised behaviour amongst LBW. Of the 876 women who provided data on tobacco use, 28.1% (n = 246 were smokers, nearly double the rate in the female population as a whole. One third of the sample (33.6%, n = 308 reported use of an illicit drug in the previous six months. The illicit drugs most commonly reported were cannabis (26.4%, n = 242, meth/amphetamine (18.6%, n = 171, and ecstasy (17.9%, n = 164. Injecting drug use was reported by 3.5% (n = 32 of participants. Conclusion LBW appear to use alcohol, tobacco and illicit drugs at higher rates than women generally, indicating that mainstream health promotion messages are not reaching this group or are not perceived as relevant. There is an urgent

  7. Evaluation of an enzyme-linked immunoelectrotransfer blot test for the confirmatory serodiagnosis of human toxocariasis

    Directory of Open Access Journals (Sweden)

    William H Roldán

    2009-05-01

    Full Text Available To improve the serodiagnosis of human toxocariasis, a sensitive and specific enzyme-linked immunoelectrotransfer blot (EITB-IgG test was developed and evaluated using Toxocara canislarvae excretory-secretory antigens for detecting anti-Toxocara IgG antibodies. The EITB-IgG profile of toxocariasis was characterized by comparing 27 sera from patients with toxocariasis, 110 sera from healthy subjects and 186 sera from patients with other helminth diseases (ascariasis, ancylostomiasis, trichuriasis, enterobiasis, strongyloidiasis, hymenolepiasis, diphyllobothriasis, taeniasis, cysticercosis, hydatidosis and fascioliasis. Antigenic bands of 24, 28, 30, 35, 56, 117, 136 and 152 kDa were predominantly recognized in sera from all patients with toxocariasis. However, only bands of 24-35 kDa were highly specific for Toxocara infection (98.3%, whereas other antigenic bands observed displayed cross-reactivity. Additionally, when the results of the EITB-IgG test were compared to those of the ELISA-IgG test, a 100% concordance was observed for positive results in human toxocariasis cases. The concordance for negative results between the two tests for healthy subjects and patients with other helminth diseases were 96.3% and 53.7%, respectively, showing that the EITB-IgG test has a higher specificity than ELISA. In conclusion, the EITB-IgG test is a very useful tool to confirm the serological diagnosis of human toxocariasis.

  8. Evaluation of an enzyme-linked immunoelectrotransfer blot test for the confirmatory serodiagnosis of human toxocariasis.

    Science.gov (United States)

    Roldán, William H; Espinoza, Yrma A

    2009-05-01

    To improve the serodiagnosis of human toxocariasis, a sensitive and specific enzyme-linked immunoelectrotransfer blot (EITB-IgG) test was developed and evaluated using Toxocara canislarvae excretory-secretory antigens for detecting anti-Toxocara IgG antibodies. The EITB-IgG profile of toxocariasis was characterized by comparing 27 sera from patients with toxocariasis, 110 sera from healthy subjects and 186 sera from patients with other helminth diseases (ascariasis, ancylostomiasis, trichuriasis, enterobiasis, strongyloidiasis, hymenolepiasis, diphyllobothriasis, taeniasis, cysticercosis, hydatidosis and fascioliasis). Antigenic bands of 24, 28, 30, 35, 56, 117, 136 and 152 kDa were predominantly recognized in sera from all patients with toxocariasis. However, only bands of 24-35 kDa were highly specific for Toxocara infection (98.3%), whereas other antigenic bands observed displayed cross-reactivity. Additionally, when the results of the EITB-IgG test were compared to those of the ELISA-IgG test, a 100% concordance was observed for positive results in human toxocariasis cases. The concordance for negative results between the two tests for healthy subjects and patients with other helminth diseases were 96.3% and 53.7%, respectively, showing that the EITB-IgG test has a higher specificity than ELISA. In conclusion, the EITB-IgG test is a very useful tool to confirm the serological diagnosis of human toxocariasis.

  9. Association of extraintestinal manifestations of inflammatory bowel disease in a province of western Hungary with disease phenotype: Results of a 25-year follow-up study

    Institute of Scientific and Technical Information of China (English)

    Tunde Pandur; Gyula David; Zsuzsanna Balogh; Pal Kuronya; Arpad Tollas; Peter Laszlo Lakatos

    2003-01-01

    IBD is a systemic disease associated with a large number of extraintestinal manifestations (EIMs). Our aim was to determine the prevalence of EIMs in a large IBD cohort in Veszprem Province in a 25-year follow-up study.METHODS: Eight hundred and seventy-three IBD patients were enrolled (ulcerative colitis/UC/: 619, m/f: 317/302,mean age at presentation: 38.3 years, average disease duration: 11.2 years; Crohn's disease/CD/: 254, m/f: 125/129,mean age at presentation: 32.5 years, average disease duration: 9.2 years). Intestinal, extraintestinal signs and laboratory tests were monitored regularly. Any alteration suggesting an EIMs was investigated by a specialist.RESULTS: A total of 21.3% of patients with IBD had EIM (UC: 15.0%, CD: 36.6%). Age at presentation did not affect the likelihood of EIM. Prevalence of EIMs was higher in women and in CD, ocular complications and primary sclerosing cholangitis (PSC) were more frequent in UC. In UC there was an increased tendency of EIM in patients with a more extensive disease.Joint complications were more frequent in CD (22.4% VS UC 10.2%, P<0.01). In UC positive family history increased the risk of joint complications (OR:3.63). In CD the frequency of type-1 peripheral arthritis was increased in patients with penetrating disease (P=0.028). PSC was present in 1.6% in UC and 0.8% in CD. Dermatological complications were present in 3.8% in UC and 10.2% in CD, the rate of ocular complications was around 3% in both diseases. Rare complications were glomerulonephritis, autoimmune hemolytic anaemia and celiac disease.CONCLUSION: Prevalence of EIM in Hungarian IBD patients is in concordance with data from Western countries. The high number of EIM supports a role for complex follow-up in these patients.ACKNOWLEDGEMENTDr. Zsuzsanna Erdelyi, Dr. Agnes Horvath, Dr Gabor Mester (Veszprem), Dr. Sandor Meszaros (Ajka), Dr. Csaba Molnar help in data collection and to Gabriella Demenyi for technical assistance.

  10. Rapid Detection of Rifampin-resistant Clinical Isolates of Mycobacterium tuberculosis by Reverse Dot Blot Hybridization

    Institute of Scientific and Technical Information of China (English)

    GUO Qian; WAN Kang Lin; YU Yan; ZHU Yan Ling; ZHAO Xiu Qin; LIU Zhi Guang; ZHANG Yuan Yuan; LI Gui Lian; WEI Jian Hao; WU Yi Mou

    2015-01-01

    Objective A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in‘hot mutation region’ of Mycobacterium tuberculosis (M. tuberculosis). Methods 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. Results The sensitivity and specificity of the RDBH assay were 91.2%(165/181) and 98.3%(117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7%(293/300), 98.2%(164/167), and 97.0%(129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. Conclusion Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.

  11. Rapid detection of intestinal pathogens in fecal samples by an improved reverse dot blot method

    Institute of Scientific and Technical Information of China (English)

    Jian-Ming Xing; Su Zhang; Ying Du; Dan Bi; Li-Hui Yao

    2009-01-01

    AIM:To develop a new, rapid and accurate reverse dot blot (RDB) method for the detection of intestinal pathogens in fecal samples.METHODS:The 12 intestinal pathogens tested were Salmonella spp., Brucella spp., Escherichia coli O157:H7,Clostridium botulinum, Bacillus cereus,Clostridium perfringens, Vibrio parahaemolyticus,Shigella spp., Yersinia enterocolitica, Vibrio cholerae,Listeria monocytogenes and Staphylococcus aureus.The two universal primers were designed to amplify two variable regions of bacterial 16S and 23S rDNA genes from all of the 12 bacterial species tested. Five hundred and forty fecal samples from the diarrhea patients were detected using the improved RDB assay.RESULTS:The methods could identify the 12 intestinal pathogens specifically, and the detection limit was as low as 103 CFUs. The consistent detection rate of the improved RDB assay compared with the traditional culture method was up to 88.75%.CONCLUSION:The hybridization results indicated that the improved RDB assay developed was a reliable method for the detection of intestinal pathogen in fecal samples.

  12. Determinants of Primary School Non-Enrollment and Absenteeism: Results from a Retrospective, Convergent Mixed Methods, Cohort Study in Rural Western Kenya.

    Science.gov (United States)

    King, Nia; Dewey, Cate; Borish, David

    2015-01-01

    Education is a key element in the socioeconomic development required to improve quality of life in Kenya. Despite the introduction of free primary education, primary school enrollment and attendance levels remain low. Drawing on qualitative and quantitative data, this study explores the determinants of non-enrollment and absenteeism in rural western Kenya and potential mitigation strategies to address these issues. The study was conducted in Bwaliro village in rural western Kenya. A random sample of 64 students was obtained by blocking the village primary school's student population according to grade level, gender, and orphan status. Qualitative and quantitative data were collected through interviews with parents, guardians, and key informants, and focus group discussions with students. Quantitative data were compared using chi-square tests, Student's T-test, and Poisson regressions. Qualitative data were analyzed using thematic content analysis. Malaria, menstruation, and lack of money were among the most notable determinants of primary school dropout and absenteeism, and these factors disproportionately impacted orphans and female students. Potential mitigation strategies suggested by the community included provision of malaria treatment or prevention, reduction in education costs, expansion of the established school-feeding program, and provision of sanitary pads. Despite free primary education, numerous factors continue to prevent children in rural western Kenya from attending primary school. The findings suggest that interventions should primarily target orphaned and female students. Prior to implementation, suggested mitigation strategies should be assessed for cost-effectiveness.

  13. Determinants of Primary School Non-Enrollment and Absenteeism: Results from a Retrospective, Convergent Mixed Methods, Cohort Study in Rural Western Kenya.

    Directory of Open Access Journals (Sweden)

    Nia King

    Full Text Available Education is a key element in the socioeconomic development required to improve quality of life in Kenya. Despite the introduction of free primary education, primary school enrollment and attendance levels remain low. Drawing on qualitative and quantitative data, this study explores the determinants of non-enrollment and absenteeism in rural western Kenya and potential mitigation strategies to address these issues.The study was conducted in Bwaliro village in rural western Kenya. A random sample of 64 students was obtained by blocking the village primary school's student population according to grade level, gender, and orphan status. Qualitative and quantitative data were collected through interviews with parents, guardians, and key informants, and focus group discussions with students. Quantitative data were compared using chi-square tests, Student's T-test, and Poisson regressions. Qualitative data were analyzed using thematic content analysis.Malaria, menstruation, and lack of money were among the most notable determinants of primary school dropout and absenteeism, and these factors disproportionately impacted orphans and female students. Potential mitigation strategies suggested by the community included provision of malaria treatment or prevention, reduction in education costs, expansion of the established school-feeding program, and provision of sanitary pads.Despite free primary education, numerous factors continue to prevent children in rural western Kenya from attending primary school. The findings suggest that interventions should primarily target orphaned and female students. Prior to implementation, suggested mitigation strategies should be assessed for cost-effectiveness.

  14. Comparison of Diagnostic Value of Antigen B and Protoscoleces Antigen in Diagnosis of Hydatid Cyst by Blotting Method

    Directory of Open Access Journals (Sweden)

    F. Oreizi

    2006-01-01

    Full Text Available Introduction & Objective : Hydatidosis, a disease caused by the cestod helminth echinococcus granulosus, is one of the most important parasitic zoonosis in man and a variety of animals. Sensitive and reliable serologic methods are necessary to confirm the diagnosis. In this study, Ag B and Psc Ag were purified as two specific parasitic antigens and evaluated by Dot blotting used on the serum of hydatidosis patients and control group in order to identify the most sensitive and specific subunits.Materials and Methods: In an analytic and comparative study, serum samples collected from 22 patients under operation of hydatid cyst. As a control group, 4 patients with acute toxoplasmosis, 4 patients with leishmaniasis, 4 patients infected by non-hydatid cestods(Tenia saginata and H.nana and 4 normal subjects were included in this investigation. Infected sheep’s liver and lung were used for the preparation of antigen. Cyst fluid containing protoscoleces was extracted and then partially purified with a protein A column. AgB and Psc Ags were interacted with hydatid and control sera, with Dot blot method and sensitivity and specificity of these antigens were evaluated. Results: Sensitivity and specificity were estimated 95.9% and 81% respectively, for AgB and 100% and 63% respectively, for Psc Ag in Dot blot Method. Conclusion: Evaluation of sensitivity and specificity of AgB and Psc Ag using Dot blotting revealed that AgB has high value for diagnosis of hydatidosis. and presumably can help physicians to diagnose hydatid cyst easier than other routine tests.

  15. Development of PCR/dot blot assay for specific detection and differentiation of taeniid cestode eggs in canids.

    Science.gov (United States)

    Armua-Fernandez, Maria Teresa; Nonaka, Nariaki; Sakurai, Tatsuya; Nakamura, Seita; Gottstein, Bruno; Deplazes, Peter; Phiri, Isaac G K; Katakura, Ken; Oku, Yuzaburo

    2011-01-01

    We report the development of a colourimetric PCR/dot blot assay targeting the mitochondrial gene NADH dehydrogenase subunit 1 (nad1) for differential diagnosis of taeniid eggs. Partial sequences of the cestode nad1 gene were aligned and new primers were designed based on conserved regions. Species-specific oligonucleotide probes (S-SONP) for canine taeniid cestodes were then designed manually based on the variable region between the conserved primers. Specifically, S-SONP were designed for the Taenia crassiceps, T. hydatigena, T. multiceps, T. ovis, T. taeniaeformis, Echinococcus granulosus (genotype 1), E. multilocularis and E. vogeli. Each probe showed high specificity as no cross-hybridisation with any amplified nad1 fragment was observed. We evaluated the assay using 49 taeniid egg-positive samples collected from dogs in Zambia. DNA from 5 to 10 eggs was extracted in each sample. Using the PCR/dot blot assay, the probes successfully detected PCR products from T. hydatigena in 42 samples, T. multiceps in 3 samples, and both species (mixed infection) in the remaining 4 samples. The results indicate that the PCR/dot blot assay is a reliable alternative for differential diagnosis of taeniid eggs in faecal samples.

  16. Renaturation of blotted allergens increases the sensitivity of specific IgE detection.

    Science.gov (United States)

    Muro, M D; Fernández, C; Moneo, I

    1996-01-01

    Several authors have demonstrated that renaturation is an essential step for the appropriate recognition of blotted proteins. The use of nonionic detergents has been described as a useful alternative to enhance the antigenicity in immunoblotting, although elution from proteins by detergents has been observed. To measure the influence of different factors on the sensitivity of specific IgE by immunoblotting, we used twenty human sera from atopic patients who were allergic or nonallergic to a common, reliable allergen (grass pollen mixture). The use of Nonidet-P40 was found to be a useful alternative for the renaturation of the allergens. No elution from the membrane was found when employing this detergent, even at high concentrations (3%), and its use gave better sensitivity than methanol. On the other hand, we detected that methanol possessed renaturing properties. A transfer method using diffusion instead of electric transfer gave the best results and two membranes could be obtained from each gel. Using this method, we found that after NP-40 incubation of the membrane, the use of bovine albumin could be omitted as blocking agent and that its use had even deleterious effects.

  17. NORTHERN BLOT ANALYSIS OF nm23 GENE EXPRESSION IN HUMAN LUNG CANCER

    Institute of Scientific and Technical Information of China (English)

    LIU Lun-xu; ZHOU Qing-hua; SHI Ying-kang; QIN Yang; SUN Zhi-lin; SUN Ze-fang

    1999-01-01

    Objective: To investigate the role of nm23 gene expression in human lung cancer. Methods: Forty human lung cancer tissues and 19 non-cancer pulmonary tissues were studied for their nm23-H1 and nm23-H2 mRNA expression with non-radioactive Northern blot hybridization. The correlation of nm23 mRNA expression with clinical features of lung cancer was analyzed. Results: The mRNA expression of nm23-H2 gene in poorly differentiated squamous cell carcinoma was significantly decreased compared to that in moderate-high differentiated squamous cell carcinoma. The mRNA expression of nm23-H1 and nm23-H2 gene in small cell lung cancer was significantly decreased compared to that in squamous cell carcinoma. No significant difference in nm23 mRNA expression was observed between lung cancer with and without lymph node metastasis, nor was there significant difference between tumor stage. Conclusion: The mRNA expression of nm23 gene is correlated with the degree of differentiation of lung cancer, but there is no evidence of metastasis suppression effect by nm23 gene.

  18. Utility of slot-blot-ELISA as a new, fast, and sensitive immunoassay for detection of carcinoembryonic antigen in the urine samples of patients with various gastrointestinal malignancies.

    Science.gov (United States)

    El-Masry, Samir; El-Sayed, Ibrahim H; Lotfy, Mahmoud; Mahmoud, Lamiaa; El-Naggar, Mohamed

    2007-01-01

    Carcinoembryonic antigen (CEA) is the most widely used clinical tumor marker. CEA immunoassay has found acceptance as a diagnostic adjunct in clinical diagnosis of gastrointestinal tumors (GIT). Several immunoassays have been established for detection of CEA in plasma, serum, tissue, feces, and urine of cancer patients using polyclonal or monoclonal antibodies raised against CEA. Some of these assays display both high sensitivity and specificity for the detection of CEA. However, these assays require special and highly expensive equipment and the procedures require long periods for their completion. In the present study, we established a Slot-Blot Enzyme Linked Immunosorbent Assay (SB-ELISA), based on anti-CEA monoclonal antibody (CEA-mAb), as a new, simple, fast, cheap, and non-invasive immunodiagnostic technique for detection of CEA in the urine of GIT patients. Urine and serum samples were collected from 248 GIT patients (58 with pancreatic cancer, 20 with hepatoma, 23 with ampullary carcinoma, 15 with hilar cholangiocarcinoma, 28 with gastric cancer, 14 with esophageal cancer, and 90 with colorectal cancer). Moreover, urine and serum samples were collected from 50 healthy individuals to serve as negative controls. The traditional ELISA technique was used for determination of CEA in the sera of GIT patients using anti-CEA monoclonal antibody. A comparison between the results of both techniques (ELISA and SB-ELISA) was carried out. The traditional ELISA detected CEA in the sera of 154 out of 248 GIT patients with a sensitivity of 59.8%, 51.7% positive predictive value (PPV) and 75.37% negative predictive value (NPV). In addition, it identified 15 false positive cases out of 50 healthy individuals with a specificity of 70%. The urinary CEA was identified by a Western blotting technique and CEA-mAb at a molecular mass of 180 Kda. The developed SB-ELISA showed higher sensitivity, specificity, PPV, and NPV (70.1%, 78%, 62.4%, and 82.13%, respectively) for detection

  19. The timberline as result of the interactions among forest, abiotic environment and human activity in the Babia Gora massif, Western Carpathians

    Science.gov (United States)

    Lajczak, Adam

    2016-04-01

    The timberline is one of the clearest and most easily discernible boundaries in nature (Troll 1973). Among the existing conceptual models clarifying the complex nature of the relationship between the timberline and the environment and human impact, the fullest seems to be the one presented by K. Holtmeier (2009). The quoted author comprehensively characterizes the timberline, taking into account its course, appearance and ecological characteristics. The aim of the work is to quantitatively assess the factors influencing the course of timberline and its changes over the last ca. 400 years in the Babia Góra massif (1725 m a.s.l.), the highest flysch ridge in the Western Carpathians, which is formed as an asymmetric ridge of kuesta type. Forests mainly in the upper montane zone have preserved their natural character in many areas. Old spruce tree growths are a particular advantage of the Babia Góra Mt., being there on the predominant section of the altitude zone of the timberline. In the rest of this zone, spruce forests have been under a direct or indirect influence of changes resulting from grazing with its 400-year history and forestry taking place about 100 years ago. The 60 m difference between the average altitude of the timberline on the northern (1335 m a.s.l.) and the southern slope (1395 m a.s.l.) on the Babia Góra Mt. can be explained with climatic conditions of these areas. The average gradient of the slope within the timberline ecotone on the southern slope of the massif is by 100 smaller, and the amount of solar energy delivered during the growing season is 40% higher (960 kWh/m2) than on the northern slope. The higher position of the timberline on the windward southern slope is favored by the dominant winds from S-W sector, thinner snow cover and earlier melting due to the lower precipitation on the slope and also due to the winnowing of snow onto the steep northern slope of the ridge. On the southern slope of the ridge there are no larger snow

  20. Geochemistry of Coesite-Bearing Pyrope Quartzites and Related Rocks From the Dora Maira Massif, Western Alps: New Results and the Enigma of the Jadeite-Rocks

    Science.gov (United States)

    Schertl, H.

    2008-12-01

    demonstrate that the whiteschist-type pyrope quartzites can be clearly distinguished chemically from metasedimentary whiteschists of former evaporite environments. Field relations of trails of pyrope quartzites, now forming lensoid inclusions within a former granite intrusion, would also make a metasedimentary origin rather unlikely. On the other hand, consistent genetical relationships between pyrope quartzites and their adjacent granitic country rocks were obtained, which virtually match those of Mg-metasomatic leucophyllites from the Eastern Alps occurring along shear zones within granitic gneisses. Similar Mg-rich rocks to be found within the Tauern Window and the Monte Rosa and Gran Paradiso Massivs of the Western Alps indicate that throughout the entire range of the Alps local processes of Mg-metasomatism occurred. References Chopin, C. (1984): Coesite and pure pyrope in high-grade blueschists of the Western Alps: a first record and some consequences. Contrib. Mineral. Petrol., 86, 107-118. Compagnoni, R. and Hirajima, T. (2001): Superzoned garnets in the coesite-bearing Brossasco-Isasca Unit, Dora-Maira massif, Western Alps, and the origin of the whiteschists. Lithos, 57, 219- 236. Schertl, H.-P. and Schreyer, W. (2008): Geochemistry of coesite-bearing "pyrope quartzites" and related rocks from the Dora-Maira Massif, Western Alps. Eur. J. Mineral., in press.

  1. PET-blot analysis contributes to BSE strain recognition in C57Bl/6 mice.

    Science.gov (United States)

    Lezmi, Stéphane; Bencsik, Anna; Baron, Thierry

    2006-10-01

    Identification of the strain of agent responsible for bovine spongiform encephalopathy (BSE) can be made histologically through the analysis of both distribution and intensity of brain vacuolar lesions after BSE transmission to mouse. Another useful way to distinguish the BSE agent from other prion strains is the study of the distribution of the abnormal prion protein (PrP(res)). For that purpose, paraffin-embedded tissue blot (PET-blot) method was applied on brains from C57Bl/6 mice infected with cattle BSE, experimental sheep BSE, or feline spongiform encephalopathy (FSE) from a cheetah. PrP(res) distribution was comparable, whichever of the three BSE agent sources was considered and was distinct from the PrP(res) distribution in C57Bl/6 mice inoculated with a French scrapie isolate or with a mouse-adapted scrapie strain (C506M3). These data confirm a common origin of infectious agent responsible for the British and French cattle BSE. They also indicate that PET-blot method appears as a precise complementary tool in prion strain studies because it offers easy and quick assessment of the PrP(res) mapping. Advantages and limits of the PET-blot method are discussed and compared with other established and validated methods of strain typing.

  2. Northern and Southern blot analysis of human RNA and DNA in autopsy material

    DEFF Research Database (Denmark)

    Larsen, S; Rygaard, K; Asnaes, S

    1992-01-01

    Fresh biopsy material for molecular biological investigations is not obtainable from all relevant normal human tissues. We studied the feasibility of using RNA and DNA from autopsies for Northern and Southern blot analysis. Tissue samples from seven organs were obtained from 10 autopsies performed...

  3. Eyelash Epilation in the Absence of Trichiasis: Results of a Population-Based Prevalence Survey in the Western Division of Fiji.

    Directory of Open Access Journals (Sweden)

    Colin Macleod

    2017-01-01

    Full Text Available The WHO definition of trachomatous trichiasis (TT is "at least one eyelash touching the globe, or evidence of recent epilation of in-turned eyelashes", reflecting the fact that epilation is used as a self-management tool for TT. In Fiji's Western Division, a high TT prevalence (8.7% in those aged ≥15 years was reported in a 2012 survey, yet a 2013 survey found no TT and Fijian ophthalmologists rarely see TT cases. Local anecdote suggests that eyelash epilation is a common behaviour, even in the absence of trichiasis. Epilators may have been identified as TT cases in previous surveys.We used a preliminary focus group to design an interview questionnaire, and subsequently conducted a population-based prevalence survey to estimate the prevalence of epilation in the absence of trichiasis, and factors associated with this behaviour, in the Western Division of Fiji.We sampled 695 individuals aged ≥15 years from a total of 457 households in 23 villages. 125 participants (18% reported epilating their eyelashes at least once within the past year. Photographs were obtained of the eyes of 121/125 (97% individuals who epilated, and subsequent analysis by an experienced trachoma grader found no cases of trachomatous conjunctival scarring or trichiasis. The age- and sex- adjusted prevalence of epilation in those aged ≥15 years was 8.6% (95% CI 5.7-11.3%. iTaukei ethnicity, female gender, and a higher frequency of drinking kava root were independently associated with epilation.Epilation occurs in this population in the absence of trichiasis, with sufficient frequency to have markedly inflated previous estimates of local TT prevalence. Individuals with epilated eyelashes should be confirmed as having epilated in-turned eyelashes in an eye with scarring of the conjunctiva before being counted as cases of TT.

  4. Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis.

    Science.gov (United States)

    Aravalli, Rajagopal N; Park, Chang W; Steer, Clifford J

    2016-08-26

    The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.

  5. Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis

    Energy Technology Data Exchange (ETDEWEB)

    Aravalli, Rajagopal N., E-mail: aravalli@umn.edu [Department of Radiology, University of Minnesota Medical School, MMC 292, 420 Delaware Street SE, Minneapolis, MN 55455 (United States); Park, Chang W. [Department of Medicine, University of Minnesota Medical School, MMC 36, 420 Delaware Street SE, Minneapolis, MN 55455 (United States); Steer, Clifford J., E-mail: steer001@umn.edu [Department of Medicine, University of Minnesota Medical School, MMC 36, 420 Delaware Street SE, Minneapolis, MN 55455 (United States); Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455 (United States)

    2016-08-26

    The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.

  6. First results from Acoustic Doppler Current Profiler measurements of meltwater flux in a large supraglacial river in western Greenland compared with downstream proglacial river outflow

    Science.gov (United States)

    Pitcher, L. H.; Smith, L. C.; Overstreet, B. T.; Chu, V. W.; Rennermalm, A. K.; Cooper, M. G.; Gleason, C. J.; Yang, K.

    2015-12-01

    A vast network of seasonally evolving, thermally eroding supraglacial rivers on the southwestern Greenland Ice Sheet (GrIS) is the preeminent transporter of meltwater across this area of the ablation zone. Supraglacial rivers are important for estimating surface water storage and transport into moulins and into the en-, sub-, and proglacial environments. Yet, little is known about their role in the GrIS cryo-hydrologic system. To that end, supraglacial river discharge in a large river, the "Rio Behar" (67.05°, -49.02°; ~75 km from the Kangerlussuaq International Airport), was measured in situ over 300 times: approximately four times per hour over three consecutive days from July 19 - 22, 2015. The Rio Behar drains a ~ 70 km2 ice catchment and enters a large moulin in the Watson River land-ice watershed in western Greenland. River discharge was measured using a Sontek M9 Acoustic Doppler Current Profiler. Each profile records water temperature, depth-integrated velocity, channel width and channel bathymetry. This novel dataset can be used to assess diurnal variations in river discharge, slope, velocity, stream power, and channel incision in order to enhance process-level understanding of GrIS meltwater routing, storage and transport. Future work will compare supraglacial river discharge in the Rio Behar with in situ estimates of proglacial river outflow upstream of the Watson River bridge in Kangerlussuaq, Greenland.

  7. How are countries in the Western Pacific Region tracking the HIV epidemic? Results from a 2011 survey of ministries of health

    Science.gov (United States)

    Wi, Teodora; Calleja, Jesus Garcia

    2012-01-01

    In 2011, as part of the World Health Organization global reporting tool to collect data on the progress of improving the health sector response to HIV/AIDS towards universal access, a questionnaire was sent to ministries of health of Western Pacific Region Member States on the scope and functioning of their HIV surveillance systems. Of the 17 countries that responded, 13 were low- to middle-income countries and four were high-income countries. Regular serosurveillance surveys are conducted with female sex workers in all lower- and middle-income countries that responded to the survey but less so with people who inject drugs and men who have sex with men. Furthermore, there are no surveillance activities of the key populations in most of the Pacific island countries. It is recommended that estimations of high-risk populations be conducted in priority Pacific island countries and tailored surveillance systems be designed. Efforts should also be made to gather and accumulate data from sufficient geographic coverage to allow the HIV epidemic to continue to be monitored. PMID:23971003

  8. DISKRIMINASI KELAMIN PADA IKAN TUNA SIRIP KUNING, Yellowfin tuna MENGGUNAKAN ANALISIS DOT BLOT DAN ELISA

    Directory of Open Access Journals (Sweden)

    Gusti Ngurah Permana

    2014-08-01

    Full Text Available Pemahaman tentang penentuan jenis kelamin dalam populasi induk merupakan hal yang sangat penting bagi keberhasilan program pembenihan. Pengukuran reaksi antibodi dan aktivitas hormon testosterone, serta estradiol adalah metode dengan potensi yang secara akurat dapat menentukan jenis kelamin ikan tanpa mematikan ikan. Tujuan penelitian ini adalah untuk mengetahui akurasi metode dot blot dan ELISA dengan 11-ketotestorsterone (11-KT yang tersedia secara komersial EIA-kit untuk membedakan jenis kelamin ikan tuna sirip kuning. Hasil analisis menunjukkan bahwa metode dot blot menghasilkan ekspresi vitelogenin tampak jelas pada individu betina dan efek plasma terlihat transparan, jika dibandingkan dengan individu jantan. Interpretasi dari metode ini memerlukan pengalaman dan keahlian dalam akurasi pembacaan hasil. Aktivitas hormon 11-KT dengan sampel klip sirip dan plasma memberikan hasil yang baik dengan aktivitas hormon terlihat jelas.

  9. Changes in Health-Seeking Behavior Did Not Result in Increased All-Cause Mortality During the Ebola Outbreak in Western Area, Sierra Leone.

    Science.gov (United States)

    Vygen, Sabine; Tiffany, Amanda; Rull, Monica; Ventura, Alexandre; Wolz, Anja; Jambai, Amara; Porten, Klaudia

    2016-10-05

    Little is known about the residual effects of the west African Ebola virus disease (Ebola) epidemic on non-Ebola mortality and health-seeking behavior in Sierra Leone. We conducted a retrospective household survey to estimate mortality and describe health-seeking behavior in Western Area, Sierra Leone, between May 25, 2014, and February 16, 2015. We used two-stage cluster sampling, selected 30 geographical sectors with probability proportional to population size, and sampled 30 households per sector. Survey teams conducted face-to-face interviews and collected information on mortality and health-seeking behavior. We calculated all-cause and Ebola-specific mortality rates and compared health-seeking behavior before and during the Ebola epidemic using χ(2) and Fisher's exact tests. Ninety-six deaths, 39 due to Ebola, were reported in 898 households. All-cause and Ebola-specific mortality rates were 0.52 (95% confidence interval [CI] = 0.29-0.76) and 0.19 (95% CI = 0.01-0.38) per 10,000 inhabitants per day, respectively. Of those households that reported a sick family member during the month before the survey, 86% (73/85) sought care at a health facility before the epidemic, compared with 58% (50/86) in February 2015 (P = 0.013). Reported self-medication increased from 4% (3/85) before the epidemic to 23% (20/86) during the epidemic (P = 0.013). Underutilization of health services and increased self-medication did not show a demonstrable effect on non-Ebola-related mortality. Nevertheless, the residual effects of outbreaks need to be taken into account for the future. Recovery efforts should focus on rebuilding both the formalized health system and the population's trust in it. © The American Society of Tropical Medicine and Hygiene.

  10. Associations of intakes of magnesium and calcium and survival among women with breast cancer: results from Western New York Exposures and Breast Cancer (WEB) Study.

    Science.gov (United States)

    Tao, Meng-Hua; Dai, Qi; Millen, Amy E; Nie, Jing; Edge, Stephen B; Trevisan, Maurizio; Shields, Peter G; Freudenheim, Jo L

    2016-01-01

    Magnesium (Mg) and calcium (Ca) antagonizes each other in (re) absorption, cell cycle regulation, inflammation, and many other physiologic activities. However, few studies have investigated the association between magnesium and calcium intakes and breast cancer survival, and the interaction between calcium and magnesium intake. In a cohort of 1,170 women with primary, incident, and histologically confirmed breast cancer from Western New York State, we examined the relationship between intakes of these two minerals and survival. Cox regression models were used to estimate hazard ratios (HR) and 95% confidence intervals (95% CI). Mean follow-up time was 87.4 months after breast cancer diagnosis; there were 170 deaths identified. After adjustment for known prognostic factors, and intakes of energy, total vitamin D and total calcium, higher dietary intake of magnesium was inversely associated with risk of all-cause mortality (HR = 0.50, 95% CI, 0.28-0.90 for highest vs. lowest tertile; p trend = 0.02). Likewise, a marginal association was found for total Magnesium intake from foods and supplements combined (HR = 0.58, 95% CI, 0.31-1.08; p trend = 0.09). The inverse association of higher total magnesium intake with all-cause mortality was primarily presented among postmenopausal women and was stronger among women who had a high Ca:Mg intake ratio (>2.59). There were no clear associations for prognosis with intake of calcium. We found that magnesium intake alone may improve overall survival following breast cancer, and the association may be stronger among those with high Ca:Mg intake ratio.

  11. DIFFERENTIATION OF PSEUDOCONDYLOMA OF VULVA AND CONDYLOMA ACUMINATA BY DOT BLOT HYBRIDIZATION AND POLYMERASE CHAIN REACTION

    Institute of Scientific and Technical Information of China (English)

    刘跃华; 王家璧; 司静懿

    1996-01-01

    This study differentiated pseudocondyloma of vulva from condyloma acunainata using dot blot hybridization and polymerase chain reaction (PCR). A total of 27 cases o{ pseudocondyloma of vulva and 65 cases of condyloma acuminata were selected for the sttldy. The genital lesions were examined clinically and were biopsled. Each biopsy v-as subjected to histological examination and HPV DNA analysis by dot blot hybridization and PCR. Dot blot analysis detected HPV DNA in 19(82.6%) out of 23 cases of condyloma acuminata and 2(25%) out of 8 cases pseudocondyloma of vulvae(P<0. 05). PCR detected HPV DNA in 51(92.7%) our of 55 cases of eondyloma acuminata, compared with none in 23 cases of pseudocondylorna(P<0. 001). HPV DNA was present in the majority of condyloma acuminata specimens, HPV 6 and 11 were the predominant types. Peudocondyloma is probably not associated with HPV. PCR was the most sensitive and useful techntque for HPV DNA detection.

  12. A slot blot immunoassay for quantitative detection of Plasmodium falciparum circumsporozoite protein in mosquito midgut oocyst.

    Directory of Open Access Journals (Sweden)

    Sanjai Kumar

    Full Text Available There is still a need for sensitive and reproducible immunoassays for quantitative detection of malarial antigens in preclinical and clinical phases of vaccine development and in epidemiology and surveillance studies, particularly in the vector host. Here we report the results of sensitivity and reproducibility studies for a research-grade, quantitative enhanced chemiluminescent-based slot blot assay (ECL-SB for detection of both recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP and native PfCSP from Oocysts (Pf Oocyst developing in the midguts of Anopheles stephensi mosquitoes. The ECL-SB detects as little as 1.25 pg of rPfCSP (linear range of quantitation 2.5-20 pg; R2 = 0.9505. We also find the earliest detectable expression of native PfCSP in Pf Oocyst by ECL-SB occurs on day 7 post feeding with infected blood meal. The ECL-SB was able to detect approximately as few as 0.5 day 8 Pf Oocysts (linear quantitation range 1-4, R2 = 0.9795 and determined that one Pf Oocyst expressed approximately 2.0 pg (0.5-3 pg of native PfCSP, suggesting a similar range of detection for recombinant and native forms of Pf CSP. The ECL-SB is highly reproducible; the Coefficient of Variation (CV for inter-assay variability for rPf CSP and native PfCSP were 1.74% and 1.32%, respectively. The CVs for intra-assay variability performed on three days for rPf CSP were 2.41%, 0.82% and 2% and for native Pf CSP 1.52%, 0.57%, and 1.86%, respectively. In addition, the ECL-SB was comparable to microscopy in determining the P. falciparum prevalence in mosquito populations that distinctly contained either high and low midgut Pf Oocyst burden. In whole mosquito samples, estimations of positivity for P. falciparum in the high and low burden groups were 83.3% and 23.3% by ECL-SB and 85.7% and 27.6% by microscopy. Based on its performance characteristics, ECL-SB could be valuable in vaccine development and to measure the parasite prevalence in mosquitoes

  13. Dot-Blot Hybridization for Detection of Five Cucurbit Viruses by Digoxigenin-Labelled cDNA Probes

    Institute of Scientific and Technical Information of China (English)

    MENG Juan; GU Qin-sheng; LIN Shi-ming; PENG Bin; LIU Li-feng; TIAN Yan-ping; LI Li

    2007-01-01

    Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops,Zuccini yellow mosaic virus(ZYMV),Watermelon mosaic virus(WMV),Cucumber mosaic virus(CMV),Papaya ringspot virus watermelon strain(PRSV-W)and Squash mosaic virus(SqMV),as a good alternative assay in seed health test and epidemiological and transgenic research.Digoxigenin-labelled cDNA probes of the five viruses were synthesized by PCR with the specific primers and applied in dot-blot hybridization to detect five viruses in crude extraction of the infected leaves.And three SqMV probes of different lengths(0.55,1.6,and 2.7 kb,respectively)were designed to investigate the effect of hybridization.The results showed that the sensitivity for detecting the crude extraction of infected leaves by ZYMV,WMV,CMV,PRSV-W,and SqMV was down to 1:160,1:160,1:320,1:160,and 1:320,respectively.Three SqMV probes of different length showed no differences on the sensitivity and specificity.The digoxigenin-labelled probes prepared by PCR could be used for accurate and rapid identification of 5 viruses infecting cucurbitaceous crops with good stabilities,sensitivities,specificity,and reproducibilities.

  14. Fluorescent detection of Southern blots and PCR-based genetic typing tests

    Energy Technology Data Exchange (ETDEWEB)

    Mansfield, E.S.; Worley, J.M. [Molecular Dynamics, Inc., Sunnyvale, CA (United States); Zimmerman, P.A. [Laboratory of Parasitic Diseases, Bethesda, MD (United States)] [and others

    1994-09-01

    The Southern blot is used to study gene organization, to identify disease-causing genomic rearrangements, or for typing RFLP markers in forensic, paternity, or prenatal diagnostic testing. Fluorescence offers a much greater dynamic range and a more linear response than film used in radioactive or chemiluminescent detection of RFLPs. We therefore investigated using the Fluorimager{trademark} 575 (Molecular Dynamics, Inc.) for analyzing Southern blots. Using a single-locus probe to D2S44 (YNH24) (Promega Corp.), we detect as little as 100 ng (0.05 attomole) genomic DNA. The alkaline phosphatase-labeled probe is detected using AttoPhos (JBL Scientific), and the developed membrane is scanned with the Fluorimager. Biotinylated hybridization probes can also be developed using a streptavidin-alkaline phosphatase conjugate and AttoPhos. The instrument scan parameters can be adjusted to prevent overexposure and accompanying loss of resolution in images of blots, gels, or 96-well microplates. We have used these other sample formats in PCR-based genetic typing assays. We use FluorKit DQS (Molecular Dynamics) to accurately quantify PCR template DNA (1-500 ng) in 96-well microplates scanned using the same instrument. Mutation detection assays run include heteroduplex gels (5% polyacrylamide, 2.7 M urea), short tandem repeat (STR) markers, amplified fragment length polymorphisms (AmpFLP), competitive priming PCR, and allele-specific oligotyping. These assays are run using either 1- or 2-color labeling. We detect unlabeled PCR products, such as the AmpFLP marker D1S80 (Perkin-Elmer) by post-staining gels for 10 minutes with SYBR Green 1 (Molecular Probes) and scanning the wet gel. The Fluorimager scans a 20 x 25 cm sample within three minutes, allowing rapid optimization of fluorescent protocols and high sample throughput.

  15. Autoantibody profiling of patients with primary biliary cirrhosis using a multiplexed line-blot assay.

    Science.gov (United States)

    Villalta, Danilo; Sorrentino, Maria Concetta; Girolami, Elia; Tampoia, Marilina; Alessio, Maria Grazia; Brusca, Ignazio; Daves, Massimo; Porcelli, Brunetta; Barberio, Giuseppina; Bizzaro, Nicola

    2015-01-01

    To evaluate the autoantibody profile in patients with primary biliary cirrhosis (PBC) using a new multiplexed line-blot assay specifically designed for the diagnosis of autoimmune liver diseases. Sera of 58 consecutive PBC patients and 191 disease controls (144 with autoimmune liver diseases other than PBC, and 67 with non-autoimmune chronic liver diseases) were tested by both the multiplexed line-blot Autoimmune Liver Disease Profile 2 (ALD2) and by IIF on HEp-2 cells and on rat kidney/liver/stomach tissues. ALD2 contains the following PBC-associated antigens: AMA-M2, natively purified from bovine heart; M2-E3, a recombinant fusion protein including the E2 subunits of PDC, BCOADC and OGDC; sp100, PML and gp210 recombinant proteins. With the ALD2 assay, a positive reaction to AMA-M2, M2-E3, sp100, PML and gp210 in PBC patients was observed in 77.6%, 84.5%, 34.5%, 15.1% and 18.9%, respectively, of the PBC sera. The overall sensitivity and specificity for PBC were 98.3% and 93.7%. Using IIF, positivity rates to AMA, and to antinuclear autoantibodies with membranous/rim-like and multiple nuclear dot patterns were 86.2%, 8.6% and 22.4%, respectively. The overall sensitivity and specificity for PBC of the IIF method were 86.2% and 97.9%, respectively. The ALD2 line-blot showed a good diagnostic accuracy for PBC and a higher sensitivity than the IIF method to detect sp100 and gp210 autoantibodies. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose

    OpenAIRE

    Rozier, Claude; Mache, Régis

    1984-01-01

    Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20° C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloropl...

  17. A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot

    DEFF Research Database (Denmark)

    Albers, Eliene; Sbroggiò, Mauro; Martin Gonzalez, Javier

    2017-01-01

    The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific...... that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting...

  18. Molecular evidence for the occurrence of beet western yellows virus on chickpea in Morocco.

    NARCIS (Netherlands)

    Fortass, M.; Wilk, van der F.; Heuvel, van de J.F.J.M.; Goldbach, R.W.

    1997-01-01

    A luteovirus isolate infecting chickpea in Morocco was experimentally transmitted by Myzus persicae to Physalis floridana, on which it produced mild symptoms. When tested in western blots against antisera to known legume luteoviruses, this isolate reacted strongly to beet western yellows virus (BWYV

  19. Molecular evidence for the occurrence of beet western yellows virus on chickpea in Morocco.

    NARCIS (Netherlands)

    Fortass, M.; Wilk, van der F.; Heuvel, van de J.F.J.M.; Goldbach, R.W.

    1997-01-01

    A luteovirus isolate infecting chickpea in Morocco was experimentally transmitted by Myzus persicae to Physalis floridana, on which it produced mild symptoms. When tested in western blots against antisera to known legume luteoviruses, this isolate reacted strongly to beet western yellows virus

  20. A sensitive non-radioactive northern blot method to detect small RNAs.

    Science.gov (United States)

    Kim, Sang Woo; Li, Zhihua; Moore, Patrick S; Monaghan, A Paula; Chang, Yuan; Nichols, Mark; John, Bino

    2010-04-01

    The continuing discoveries of potentially active small RNAs at an unprecedented rate using high-throughput sequencing have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Currently, northern blot is the most widely used method for validating small RNAs that are identified by methods such as high-throughput sequencing. We describe a new northern blot-based protocol (LED) for small RNA (approximately 15-40 bases) detection using digoxigenin (DIG)-labeled oligonucleotide probes containing locked nucleic acids (LNA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for cross-linking the RNA to the membrane. LED generates clearly visible signals for RNA amounts as low as 0.05 fmol. This method requires as little as a few seconds of membrane exposure to outperform the signal intensity using overnight exposure of isotope-based methods, corresponding to approximately 1000-fold improvement in exposure-time. In contrast to commonly used radioisotope-based methods, which require freshly prepared and hazardous probes, LED probes can be stored for at least 6 months, facilitate faster and more cost-effective experiments, and are more environmentally friendly. A detailed protocol of LED is provided in the Supplementary Data.

  1. Production of hybrids between western gray wolves and western coyotes.

    Directory of Open Access Journals (Sweden)

    L David Mech

    Full Text Available Using artificial insemination we attempted to produce hybrids between captive, male, western, gray wolves (Canis lupus and female, western coyotes (Canis latrans to determine whether their gametes would be compatible and the coyotes could produce and nurture offspring. The results contribute new information to an ongoing controversy over whether the eastern wolf (Canis lycaon is a valid unique species that could be subject to the U. S. Endangered Species Act. Attempts with transcervically deposited wolf semen into nine coyotes over two breeding seasons yielded three coyote pregnancies. One coyote ate her pups, another produced a resorbed fetus and a dead fetus by C-section, and the third produced seven hybrids, six of which survived. These results show that, although it might be unlikely for male western wolves to successfully produce offspring with female western coyotes under natural conditions, western-gray-wolf sperm are compatible with western-coyote ova and that at least one coyote could produce and nurture hybrid offspring. This finding in turn demonstrates that gamete incompatibility would not have prevented western, gray wolves from inseminating western coyotes and thus producing hybrids with coyote mtDNA, a claim that counters the view that the eastern wolf is a separate species. However, some of the difficulties experienced by the other inseminated coyotes tend to temper that finding and suggest that more experimentation is needed, including determining the behavioral and physical compatibility of western gray wolves copulating with western coyotes. Thus although our study adds new information to the controversy, it does not settle it. Further study is needed to determine whether the putative Canis lycaon is indeed a unique species.

  2. Production of hybrids between western gray wolves and western coyotes.

    Science.gov (United States)

    Mech, L David; Christensen, Bruce W; Asa, Cheryl S; Callahan, Margaret; Young, Julie K

    2014-01-01

    Using artificial insemination we attempted to produce hybrids between captive, male, western, gray wolves (Canis lupus) and female, western coyotes (Canis latrans) to determine whether their gametes would be compatible and the coyotes could produce and nurture offspring. The results contribute new information to an ongoing controversy over whether the eastern wolf (Canis lycaon) is a valid unique species that could be subject to the U. S. Endangered Species Act. Attempts with transcervically deposited wolf semen into nine coyotes over two breeding seasons yielded three coyote pregnancies. One coyote ate her pups, another produced a resorbed fetus and a dead fetus by C-section, and the third produced seven hybrids, six of which survived. These results show that, although it might be unlikely for male western wolves to successfully produce offspring with female western coyotes under natural conditions, western-gray-wolf sperm are compatible with western-coyote ova and that at least one coyote could produce and nurture hybrid offspring. This finding in turn demonstrates that gamete incompatibility would not have prevented western, gray wolves from inseminating western coyotes and thus producing hybrids with coyote mtDNA, a claim that counters the view that the eastern wolf is a separate species. However, some of the difficulties experienced by the other inseminated coyotes tend to temper that finding and suggest that more experimentation is needed, including determining the behavioral and physical compatibility of western gray wolves copulating with western coyotes. Thus although our study adds new information to the controversy, it does not settle it. Further study is needed to determine whether the putative Canis lycaon is indeed a unique species.

  3. Production of hybrids between western gray wolves and western coyotes

    Science.gov (United States)

    Mech, L. David; Christensen, Bruce W.; Asa, Cheryl S.; Callahan, Magaret; Young, Julie K.

    2014-01-01

    Using artificial insemination we attempted to produce hybrids between captive, male, western, gray wolves (Canis lupus) and female, western coyotes (Canis latrans) to determine whether their gametes would be compatible and the coyotes could produce and nurture offspring. The results contribute new information to an ongoing controversy over whether the eastern wolf (Canis lycaon) is a valid unique species that could be subject to the U. S. Endangered Species Act. Attempts with transcervically deposited wolf semen into nine coyotes over two breeding seasons yielded three coyote pregnancies. One coyote ate her pups, another produced a resorbed fetus and a dead fetus by C-section, and the third produced seven hybrids, six of which survived. These results show that, although it might be unlikely for male western wolves to successfully produce offspring with female western coyotes under natural conditions, western-gray-wolf sperm are compatible with western-coyote ova and that at least one coyote could produce and nurture hybrid offspring. This finding in turn demonstrates that gamete incompatibility would not have prevented western, gray wolves from inseminating western coyotes and thus producing hybrids with coyote mtDNA, a claim that counters the view that the eastern wolf is a separate species. However, some of the difficulties experienced by the other inseminated coyotes tend to temper that finding and suggest that more experimentation is needed, including determining the behavioral and physical compatibility of western gray wolves copulating with western coyotes. Thus although our study adds new information to the controversy, it does not settle it. Further study is needed to determine whether the putative Canis lycaon is indeed a unique species.

  4. 用DNA探针检测沙眼衣原体%Detection of Chlamydia Trachomatis by DNA Probe Blot

    Institute of Scientific and Technical Information of China (English)

    王卫萍; 陈亚利; 武建国

    2001-01-01

    目的: 建立一种敏感而特异的沙眼衣原体分子生物学检测方法. 方法: 用PCR扩增517bp的沙眼衣原体种特异性基因片段并标记成探针,建立DNA探针杂交检测沙眼衣原体的方法.结果: 探针只与沙眼衣原体L2、TE55株DNA呈阳性杂交斑点,与其他两种衣原体、解脲支原体、淋病奈瑟菌、大肠埃希菌、金黄色葡萄球菌、流感嗜血杆菌及白色念珠菌DNA斑点膜无阳性杂交信号.从100例慢性宫颈炎和前列腺炎病人生殖道分泌物中检出阳性22例,阳性率22%. 结论: 建立的DNA探针检测沙眼衣原体方法具有较高的敏感性和特异性,可用于批量临床标本的检测.%Objectives: To establish a sensitive and specific molecular biologicalmethod for detecting Chlamydia trachomatis of genital tract infection patients. Methods: A DNA blot assay was developed by coating DNA of Chlamydia trachomatisand/or extract of clinical samples on nitrocellulose (NC) membrane, blotting witha DNA probe labeled with DIG. Results: There was no positive blotting in Chlamydiapneumoniae, Chlamydia psittaci, Ureaplasma urealyticum, Neisseria gonorrhoeae,Esherichia coli, Staphylococcus aureus, Haemophilus infuenzae and Candida albicansexcept Chlamydia trachomatis. The sensitivity could be improved to 1pg. The positivepercentage was 22% (22/100) in detection of swabs collected from 100 chroniccervicitis and prostatitis patients. Conclusions: This method was not only sensitive, rapid and specific but also could be applied to detect batch samples. Natl J Androl,2001,7(2):102~104

  5. The influence of variability of calculation grids on the results of numerical modeling of geothermal doublets - an example from the Choszczno area, north-western Poland

    Science.gov (United States)

    Wachowicz-Pyzik, A.; Sowiżdżał, A.; Pająk, L.

    2016-09-01

    The numerical modeling enables us to reduce the risk related to the selection of best localization of wells. Moreover, at the stage of production, modeling is a suitable tool for optimization of well operational parameters, which guarantees the long life of doublets. The thorough selection of software together with relevant methodology applied to generation of numerical models significantly improve the quality of obtained results. In the following paper, we discuss the impact of density of calculation grid on the results of geothermal doublet simulation with the TOUGH2 code, which applies the finite-difference method. The study area is located between the Szczecin Trough and the Fore-sudetic Monocline, where the Choszczno IG-1 well has been completed. Our research was divided into the two stages. At the first stage, we examined the changes of density of polygon calculation grids used in computations of operational parameters of geothermal doublets. At the second stage, we analyzed the influence of distance between the production and the injection wells on variability in time of operational parameters. The results demonstrated that in both studied cases, the largest differences occurred in pressures measured in production and injection wells whereas the differences in temperatures were less pronounced.

  6. Patch Test Results in Patients with Allergic Contact Dermatitis in the Western Black Sea Region: A Five-Year Retrospective Study

    Directory of Open Access Journals (Sweden)

    Rafet

    2011-12-01

    Full Text Available Background and Design: In this retrospective study, patch test results of 304 patients with allergic contact dermatitis (ACD, who were tested with the European Standard Series, were evaluated. The aim of the study was to determine the frequency of positive reactions to different sensitizers in patients with ACD and to identify sex and age differences.Material and Method: We evaluated a total of 304 patients (195 (64.1% female and 109 (35.9% male who applied to our clinic between the period of January 2003 and December 2008 and who were tested with patch test with the consideration of ACD. Patch test results were assessed according to the International Contact Dermatitis Research Group at 48 and 72 hours after initial application.Results: Lesions of ACD were found to be most frequent on the hands (65.5%. A total of 95 (31.3% patients had one or more positive patch test reactions. The most common allergens were nickel sulfate (14.8% followed by cobalt chloride (9.2%, potassium dichromate (6.6%, balsam of Peru (3.6%, fragrance mix (3.3%, mercaptobenzothiazole (3% and colophonium (2.3%. Positive reactions to nickel sulfate were more common among women, especially those in younger group. Frequency of contact allergy to lanolin, mercapto mix, epoxy resin, paraben mix and quaternium-15 were relatively low, while no positive reaction was obtained to formaldehyde in any case. In 51.6% of patients with positive reaction, the responsible allergen was detected by testing with the European Standard Series.Conclusion: Although this study shows that sensitization rate to metals and fragrance are still high in our region, as well as in our country, and are more frequent in females than in males, future prospective multicenter studies with large number of patients would point out the adequacy of the European Standard Series in our country. (Turk­derm 2011; 45: 198-202

  7. Climate-sensitive carbon cycling on the western Antarctic continental shelf: results from the Amundsen Sea Polynya International Research Expedition (ASPIRE)

    Science.gov (United States)

    Yager, P. L.; Sherrell, R. M.; Stammerjohn, S. E.

    2016-02-01

    The Amundsen Sea hosts the most productive polynya in all of coastal Antarctica, with its vibrant green waters exceeding 20 µg Chl a per liter. It is also one of the global regions most vulnerable to climate change, experiencing rapid losses in both sea ice cover and nearby ice sheets. During the Amundsen Sea Polynya International Research Expedition (ASPIRE) in austral summer 2010-11, we aimed to determine mechanisms driving the production and fate of this extraordinary algal bloom, with an eye towards predicting how this system will respond to further change. Here we summarize and synthesize results from the expedition, highlight results now being published in a special ASPIRE feature in the journal Elementa, and present an effort to balance the carbon budget for the region. We collected water column profiles for total dissolved inorganic carbon (DIC) and nutrients, particulate and dissolved organic matter, chlorophyll a, macrozooplankton, and microbial biomass. We also measured primary and secondary production, community respiration rates, vertical particle flux and fecal pellet production and grazing. With observations arranged along a gradient of increasing integrated nitrate depletion, changes in DIC in the upper water column (ranging from 0.2 to 4.7 mol C m-2) and gas exchange are compared to nutrient and organic matter inventories to estimate export. Comparisons to short-term (days) drifting traps and a year-long moored sediment trap capturing the downward flux confirmed that a high fraction (up to 60%) of the net community production was exported to sub-euphotic depths during the early part of the bloom in this productive region. The ultimate fate of this carbon is also estimated. We discuss the importance of this carbon sequestration, particularly in light of changing climate conditions in this region, informed by ongoing data synthesis and modeling efforts (INSPIRE).

  8. Ground-water flow near two radioactive-waste-disposal areas at the Western New York Nuclear Service Center, Cattaraugus County, New York; results of flow simulation

    Science.gov (United States)

    Bergeron, M.P.; Bugliosi, E.F.

    1988-01-01

    Two adjacent burial areas were excavated in a clay-rich till at a radioactive waste disposal site near West Valley in Cattaraugus County, N.Y.: (1) which contains mainly low-level radioactive wastes generated onsite by a nuclear fuel reprocessing plant, has been in operation since 1966; and (2) which contains commercial low-level radioactive wastes, was operated during 1963-75. Groundwater below the upper 3 meters of till generally moves downward through a 20- to 30-meter thick sequence of tills underlain by lacustrine and kame-delta deposits of fine sand and silt. Groundwater in the weathered, upper 3 meters of till can move laterally for several meters before either moving downward into the kame-delta deposits or discharging to the land surface. A two-dimensional finite-element model that simulates two vertical sections was used to evaluate hydrologic factors that control groundwater flow in the till. Conditions observed during March 1983 were reproduced accurately in steady-state simulations that used four isotropic units of differing hydraulic conductivity to represent two fractured and weathered till units near land surfaces, an intermediate group of isolated till zones that contain significant amounts of fine sand and silt, and a sequence of till units at depths that have been consolidated by overburden pressure. Recharge rates used in the best-fit simulation ranged from 1.4 cm/yr along smooth, sloping or compacted surfaces to 3.8 cm/yr near swampy areas. Values of hydraulic conductivity and infiltration used in the calibrated best-fit model were nearly identical to values used in a previous model analysis of the nearby commercial-waste burial area. Results of the model simulations of a burial pit assumed to be filled with water indicate that water near the bottom of the burial pit would migrate laterally in the shallow, weathered till for 5 to 6 meters before moving downward into the unweathered till, and water near the top of the pit would move laterally

  9. Placer Gold Composition and Provenance Studies in the Kuznetskiy Alatau and Western Sayan, South-East Siberia: Results of Field Trip, Summer 2014

    Directory of Open Access Journals (Sweden)

    G. J.G. Paxman

    2015-03-01

    Full Text Available This article presents the results of a study of gold samples obtained by students during a practical field trip in the summer of 2014. Placer gold samples retrieved from four rivers in southeast Siberia (Khakassia and Tyva territories by panning and sluicing were described and analyzed compositionally by Scanning Electron Microscopy. There is evidence from grain flattening and morphology for significant variations in gold transport distance, both within and between sample locations. The composition and texture of gold is compared to similar studies in the Yukon, and it is inferred that most of the placer gold in the region originated from orogenic lode sources. This orogenic gold is of Devonian to Carboniferous age. There is also evidence for a contribution from igneous intrusion-related bedrock gold, which is supported by the presence of granite, granodiorite and sienite intrusions of Devonian age. There is scope for further study, since relatively few grains were analyzed here. In addition, if compositional data of the prospective primary gold deposits can be obtained, there would be potential for more precise determination of provenance.

  10. Source regions of ragweed pollen arriving in south-western Poland and the influence of meteorological data on the HYSPLIT model results.

    Science.gov (United States)

    Bilińska, Daria; Skjøth, Carsten Ambelas; Werner, Małgorzata; Kryza, Maciej; Malkiewicz, Małgorzata; Krynicka, Justyna; Drzeniecka-Osiadacz, Anetta

    2017-01-01

    We have investigated the relationship between the inflow of air masses and the ragweed pollen concentration in SW Poland (Wrocław) for a 10-year period of 2005-2014. The HYSPLIT trajectory model was used to verify whether episodes of high concentrations can be related to regions outside of the main known ragweed centres in Europe, like Pannonian Plain, northern Italy and Ukraine. Furthermore, we used two different meteorological data sets (the global GDAS data set and from the WRF mesoscale model; the meteorological parameters were: U and V wind components, temperature and relative humidity) into HYSPLIT to evaluate the influence of meteorological input on calculated trajectories for high concentration ragweed episodes. The results show that the episodes of high pollen concentration (above 20 pm(-3)) represent a great part of total recorded ragweed pollen in Wrocław, but occur rarely and not in all years. High pollen episodes are connected with air masses coming from south and south-west Europe, which confirms the existence of expected ragweed centres but showed that other centres near Wrocław are not present. The HYSPLIT simulations with two different meteorological inputs indicated that footprint studies on ragweed benefit from a higher resolution meteorological data sets.

  11. Rapid detection and differentiation of important Campylobacter spp. in poultry samples by dot blot and PCR.

    Science.gov (United States)

    Fontanot, Marco; Iacumin, Lucilla; Cecchini, Francesca; Comi, Giuseppe; Manzano, Marisa

    2014-10-01

    The detection of Campylobacter, the most commonly reported cause of foodborne gastroenteritis in the European Union, is very important for human health. The most commonly recognised risk factor for infection is the handling and/or consumption of undercooked poultry meat. The methods typically applied to evaluate the presence/absence of Campylobacter in food samples are direct plating and/or enrichment culture based on the Horizontal Method for Detection and Enumeration of Campylobacter spp. (ISO 10272-1B: 2006) and PCR. Molecular methods also allow for the detection of cells that are viable but cannot be cultivated on agar media and that decrease the time required for species identification. The current study proposes the use of two molecular methods for species identification: dot blot and PCR. The dot blot method had a sensitivity of 25 ng for detection of DNA extracted from a pure culture using a digoxigenin-labelled probe for hybridisation; the target DNA was extracted from the enrichment broth at 24 h. PCR was performed using a pair of sensitive and specific primers for the detection of Campylobacter jejuni and Campylobacter coli after 24 h of enrichment in Preston broth. The initial samples were contaminated by 5 × 10 C. jejuni cells/g and 1.5 × 10(2)C. coli cells/g, thus the number of cells present in the enrichment broth at 0 h was 1 or 3 cell/g, respectively.

  12. Evaluación de las pruebas dot blot y aglutinación de látex para el diagnóstico de cisticercosis en Perú

    Directory of Open Access Journals (Sweden)

    Eduardo Miranda-Ulloa

    Full Text Available Con el objetivo de evaluar las pruebas dot blot y aglutinación de látex para la detección de cisticercosis humana con antígeno de líquido de cisticerco de Taenia solium, se usaron 125 sueros humanos, de los cuales 60 procedían de personas con cisticercosis confirmada por Western Blot, 45 de personas con otras enfermedades parasitarias y 20 de personas aparentemente sanas. La concentración óptima del antígeno para impregnar las tiras dot blot fue de 0,01 ug/uL, y para impregnar las partículas de látex fue de 0,092 ug/uL. Para la prueba dot blot se encontró una sensibilidad del 100% y especificidad del 87,7%; para la aglutinación de látex una sensibilidad del 93,3% y especificidad del 89,2%. Ambas pruebas podrían ser de utilidad y factibles de implementar como alternativas de diagnóstico serológico en laboratorios de áreas endémicas del Perú

  13. Evaluación de las pruebas dot blot y aglutinación de látex para el diagnóstico de cisticercosis en Perú

    Directory of Open Access Journals (Sweden)

    Eduardo Miranda-Ulloa

    2014-06-01

    Full Text Available Con el objetivo de evaluar las pruebas dot blot y aglutinación de látex para la detección de cisticercosis humana con antígeno de líquido de cisticerco de Taenia solium, se usaron 125 sueros humanos, de los cuales 60 procedían de personas con cisticercosis confirmada por Western Blot, 45 de personas con otras enfermedades parasitarias y 20 de personas aparentemente sanas. La concentración óptima del antígeno para impregnar las tiras dot blot fue de 0,01 ug/uL, y para impregnar las partículas de látex fue de 0,092 ug/uL. Para la prueba dot blot se encontró una sensibilidad del 100% y especificidad del 87,7%; para la aglutinación de látex una sensibilidad del 93,3% y especificidad del 89,2%. Ambas pruebas podrían ser de utilidad y factibles de implementar como alternativas de diagnóstico serológico en laboratorios de áreas endémicas del Perú

  14. 巢湖西半湖主要入湖河流水质的模糊评价及结果分析%Fuzzy Evaluation and Results Analysis of Water Quality in Major Rivers Feeding Western Chaohu Lake

    Institute of Scientific and Technical Information of China (English)

    沈文静; 孙世群

    2011-01-01

    This paper applied the fuzzy comprehensive evaluation method tO the major rivers feeding the western Chaohu Lake. A corresponding fuzzy coefficient matrix was established and the weights to evaluate the water quality were identified in the study of the major rivers feeding the western Chaobu Lake. The final result was compared with the one ohtained by using the single factor assessment method. The results show that the water quality of most sections of the major rivers feeding the western Chaohu Lake belong to class V and bad class V. These research results by different methods are in conformity to the facts, but small differences still exist in some sections. Compared with the fuzzy comprehensive evaluation and single factor evaluation for water quality evaluation system, water quality with multiple factors can be reflected objectively and comprehensively. It provides basis for the formulation of environmental protection policies, plans for preventing and controlling water pollution as well as referential value for further research.%文章对巢湖西半湖主要入湖河流采用模糊综合评价法进行评价。通过建立相应的模糊系数矩阵及确定权重并综合评价模糊运算,最终将所得结果与单因子评价指数所得结果相比较。结果表明:巢湖西半湖入湖河流大多数断面属于V和劣V类水,两种方法的评价结果基本吻合,个别断面存在微小分歧。通过将模糊综合评价法和单因子评价法进行比较分析,客观全面地反映出了多种因子复合影响下的水质状况,为制定环保政策和水污染防治规划起到借鉴作用。

  15. The nonmarine Lower Cretaceous of the North American Western Interior foreland basin: New biostratigraphic results from ostracod correlations and early mammals, and their implications for paleontology and geology of the basin—An overview

    Science.gov (United States)

    Sames, Benjamin; Cifelli, Richard L.; Schudack, Michael E.

    2010-08-01

    The timespan represented by the hiatus between nonmarine Upper Jurassic (Early Berriasian?) and unconformably overlying Lower Cretaceous deposits throughout the North American Western Interior foreland basin has been under discussion for the entire 20th century and remains controversial to date. Ongoing research in revision of Early Cretaceous nonmarine ostracods of some respective North American formations leads to a breakthrough concerning the verification of their biostratigraphic utility as well as their subsequent application. These ostracods are not as endemic as hitherto believed and can be used for supraregional and regional correlation, as well as improvement of the age determination of North American units. New results strongly suggest a maximum age of Late Berriasian to Valanginian (˜ 142-138 Ma) for the lower part of the Lakota (Black Hills area, South Dakota) and Cedar Mountain (Utah) formations. A pre-Aptian maximum age for the Lakota Formation is supported by early mammals. These biostratigraphic results affect the correlatable formations as well, and therefore have broad implications on basin-related geologic and paleontologic topics that are overviewed and discussed herein. The central issue hampering an integrated synthesis of the foreland basin is its yet imprecise chronostratigraphic framework and documentation. Temporal relationships between the gologic processes of the basin and their control factors are still insufficiently calibrated or controversial. Detailed ongoing revision of North American Early Cretaceous nonmarine ostracods demonstrates their applicability, utility, and further potential as tool for improvement of the chronostratigraphy of the Western Interior foreland basin at both small and large scales. These ostracods also foster understanding of animal (e.g. early mammals and dinosaurs) and plant (angiosperms) evolution on the North American continent and show promise of providing age determinations for single-sample horizons in

  16. A simple explanation for a case of incompatibility with the reading frame theory in Duchenne muscular dystrophy: failure to detect an aberrant restriction fragment in Southern blot analysis.

    Science.gov (United States)

    Patria, S Y; Takeshima, Y; Suminaga, R; Nakamura, H; Iwasaki, R; Minagawa, T; Matsuo, M

    1999-09-01

    According to the translational reading frame theory, Duchenne muscular dystrophy (DMD) patients harbor out-of-frame deletion mutations in the dystrophin gene. We identified a Japanese DMD case who appeared to have an in-frame deletion of exons 46-54 that was disclosed by Southern blot analysis using a dystrophin cDNA as a probe. Analysis of dystrophin mRNA in skeletal muscle revealed the presence of an out-of-frame deletion of exons 46-53. In agreement with this result, the region encompassing exon 54 could be amplified from genomic DNA by polymerase chain reaction (PCR). Furthermore, re-analysis by Southern blot using an exon specific probe disclosed that a HindIII fragment containing exon 54 was present at aberrant size, leading to the incorrect conclusion that exon 54 had been deleted. Thus, this particular DMD case does not constitute an exception to the reading frame theory.

  17. Comparison of Southern blot analysis with isotopic and nonisotopic in situ hybridization for the detection of human papillomavirus sequences in invasive carcinoma of the uterine cervix.

    Science.gov (United States)

    D'Amato, L; Pilotti, S; Rotola, A; Di Luca, D; Cassai, E; Rilke, F

    1992-03-01

    To compare the efficiency of hybridization methods for the detection of HPV genome, 22 cases of invasive squamous cell carcinoma of the uterine cervix were analyzed by Southern blot analysis and in situ hybridization carried out with 35S- and biotin-labeled probes. These cases contained from less than one to as many as 50 copies per cell of HPV 16 and 18 types. To increase the sensitivity of biotinylated probes, a silver enhancement procedure of the peroxidase reaction product was applied. Results showed that in situ hybridization performed with isotopic probes is as sensitive as Southern blot analysis and is more sensitive than that performed with biotin-labeled probe. However, the application of the silver enhancement procedure increases the percentage of HPV-positive cases from 27 to 50%.

  18. Quantitative Expression Analysis in Brassica napus by Northern Blot Analysis and Reverse Transcription-Quantitative PCR in a Complex Experimental Setting.

    Science.gov (United States)

    Rumlow, Annekathrin; Keunen, Els; Klein, Jan; Pallmann, Philip; Riemenschneider, Anja; Cuypers, Ann; Papenbrock, Jutta

    Analysis of gene expression is one of the major ways to better understand plant reactions to changes in environmental conditions. The comparison of many different factors influencing plant growth challenges the gene expression analysis for specific gene-targeted experiments, especially with regard to the choice of suitable reference genes. The aim of this study is to compare expression results obtained by Northern blot, semi-quantitative PCR and RT-qPCR, and to identify a reliable set of reference genes for oilseed rape (Brassica napus L.) suitable for comparing gene expression under complex experimental conditions. We investigated the influence of several factors such as sulfur deficiency, different time points during the day, varying light conditions, and their interaction on gene expression in oilseed rape plants. The expression of selected reference genes was indeed influenced under these conditions in different ways. Therefore, a recently developed algorithm, called GrayNorm, was applied to validate a set of reference genes for normalizing results obtained by Northern blot analysis. After careful comparison of the three methods mentioned above, Northern blot analysis seems to be a reliable and cost-effective alternative for gene expression analysis under a complex growth regime. For using this method in a quantitative way a number of references was validated revealing that for our experiment a set of three references provides an appropriate normalization. Semi-quantitative PCR was prone to many handling errors and difficult to control while RT-qPCR was very sensitive to expression fluctuations of the reference genes.

  19. The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and northern blot analysis.

    Science.gov (United States)

    Katsuyama, M; Nishigaki, N; Sugimoto, Y; Morimoto, K; Negishi, M; Narumiya, S; Ichikawa, A

    1995-09-25

    A functional cDNA clone for the mouse prostaglandin (PG) E receptor EP2 subtype was isolated from a mouse cDNA library. The mouse EP2 receptor consists of 362 amino acid residues with seven putative transmembrane domains. [3H]PGE2 bound specifically to the membrane of Chinese hamster ovary cells stably expressing the cloned receptor. This binding was displaced by unlabeled prostanoids in the order of PGE2 = PGE1 > iloprost, a stable PGI2 agonist > PGF2 alpha > PGD2. Binding was also inhibited by butaprost (an EP2 agonist) and to a lesser extent by M&B 28767 (an EP3 agonist), but not by sulprostone (an EP1 and EP3 agonist) or SC-19220 (an EP1 antagonist). PGE2 and butaprost increased the cAMP level in the Chinese hamster ovary cells in a concentration-dependent manner. Northern blot analysis revealed that EP2 mRNA is expressed most abundantly in the uterus, followed by the spleen, lung, thymus, ileum, liver, and stomach.

  20. A simple dot-blot-Sirius red-based assay for collagen quantification.

    Science.gov (United States)

    Rodríguez-Rodríguez, Pilar; Arribas, Silvia M; de Pablo, Angel Luis López; González, M Carmen; Abderrahim, Fatima; Condezo-Hoyos, Luis

    2013-08-01

    The assessment of collagen content in tissues is important in biomedical research, since this protein is altered in numerous diseases. Hydroxyproline and Sirius red based assays are the most common methods for collagen quantification. However, these procedures have some pitfalls, such as the requirement of oxygen-free medium or expensive equipment and large sample size or being unsuitable for hydrolyzed collagen, respectively. Our objective was to develop a specific, versatile, and user-friendly quantitative method applicable to small tissue samples and extracts obtained from elastin purification, therefore, suitable for simultaneous quantification of elastin. This method is based on the binding of Sirius red to collagen present in a sample immobilized on a PVDF membrane, as in the dot-blot technique, and quantified by a scanner and image analysis software. Sample loading, Sirius red concentration, temperature and incubation time, type of standard substance, albumin interference, and quantification time are optimized. The method enabled the quantification of (1) intact collagen in several rat tissue homogenates, including small resistance-sized arteries, (2) partially hydrolyzed collagen obtained from NaOH extracts, compatible with elastin purification, and (3) for the detection of differences in collagen content between hypertensive and normotensive rats. We conclude that the developed technique can be widely used since it is versatile (quantifies intact and hydrolyzed collagen), requires small sample volumes, is user-friendly (low-cost, easy to use, minimum toxic materials, and reduced time of test), and is specific (minimal interference with serum albumin).

  1. Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose.

    Science.gov (United States)

    Rozier, C; Mache, R

    1984-10-11

    Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20 degrees C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloroplast 30S ribosomal subunits. A set of only 7 proteins was bound to chloroplast rRNA in stringent conditions: chloroplast S6, S10, S11, S14, S15, S17 and S22. They also bound to E. coli 16S rRNA. This set includes 4 chloroplast-synthesized proteins: S6, S11, S15 and S22. The core particles obtained after treatment by LiCl of chloroplast 30S ribosomal subunit contained 3 proteins (S6, S10 and S14) which are included in the set of 7 binding proteins. This set of proteins probably play a part in the early steps of the assembly of the chloroplast 30S ribosomal subunit.

  2. A novel methylation PCR that offers standardized determination of FMR1 methylation and CGG repeat length without southern blot analysis.

    Science.gov (United States)

    Grasso, Marina; Boon, Elles M J; Filipovic-Sadic, Stela; van Bunderen, Patrick A; Gennaro, Elena; Cao, Ru; Latham, Gary J; Hadd, Andrew G; Coviello, Domenico A

    2014-01-01

    Fragile X syndrome and associated disorders are characterized by the number of CGG repeats and methylation status of the FMR1 gene for which Southern blot (SB) historically has been required for analysis. This study describes a simple PCR-only workflow (mPCR) to replace SB analysis, that incorporates novel procedural controls, treatment of the DNA in separate control and methylation-sensitive restriction endonuclease reactions, amplification with labeled primers, and two-color amplicon sizing by capillary electrophoresis. mPCR was evaluated in two independent laboratories with 76 residual clinical samples that represented typical and challenging fragile X alleles in both males and females. mPCR enabled superior size resolution and analytical sensitivity for size and methylation mosaicism compared to SB. Full mutation mosaicism was detected down to 1% in a background of 99% normal allele with 50- to 100-fold less DNA than required for SB. A low level of full mutation mosaicism in one sample was detected using mPCR but not observed using SB. Overall, the sensitivity for detection of full mutation alleles was 100% (95% CI: 89%-100%) with an accuracy of 99% (95% CI: 93%-100%). mPCR analysis of DNA from individuals with Klinefelter and Turner syndromes, and DNA from sperm and blood, were consistent with SB. As such, mPCR enables accurate, sensitive, and standardized methods of FMR1 analysis that can harmonize results across different laboratories.

  3. Cytotoxic activity, DNA damage, cellular uptake, apoptosis and western blot analysis of ruthenium(II) polypyridyl complex against human lung decarcinoma A549 cell.

    Science.gov (United States)

    Lai, Shang-Hai; Jiang, Guang-Bin; Yao, Jun-Hua; Li, Wei; Han, Bing-Jie; Zhang, Cheng; Zeng, Chuan-Chuan; Liu, Yun-Jun

    2015-11-01

    A new ruthenium(II) polypyridyl complex [Ru(dmp)2(pddppn)](ClO4)2Ru1 was synthesized and characterized. The cytotoxic activity in vitro of the complex was evaluated by MTT method. Ru1 shows high effect on the inhibition of the cell growth against BEL-7402, HeLa, MG-63 and A549 cells with low IC50 values of 1.6±0.4, 9.0±0.8, 1.5±0.2 and 1.5±0.3 μM, respectively. The cellular uptake indicates that Ru1 can enter into the cytoplasm and accumulate in the cell nuclei. Ru1 can induce apoptosis in A549 cells and enhance the levels of reactive oxygen species (ROS) and induce the decrease of mitochondrial membrane potential. In addition, Ru1 can down-regulate the levels of Bcl-2, Bcl-x, Bak, and Bim expression and up-regulate the expression of Bag-1 and Bad. The complex induces apoptosis of A549 cells through an intrinsic ROS-mediated mitochondrial dysfunction pathway, which was accompanied by regulating the expression of caspases and Bcl-2 family proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Estimation of hepatitis E virus (HEV) pig seroprevalence using Elisa and Western blot and comparison between human and pig HEV sequences in Belgium.

    NARCIS (Netherlands)

    Thiry, D.; Mauroy, A.; Saegerman, C.; Thomas, I.; Wautier, M.; Miry, C.; Czaplicki, G.; Berkvens, D.; Praet, N.; Poel, van der W.H.M.; Cariolet, R.; Brochier, B.; Thiry, E.

    2014-01-01

    Zoonotic transmission of hepatitis E virus (HEV) is of special concern, particularly in high income countries were waterborne infections are less frequent than in developing countries. High HEV seroprevalences can be found in European pig populations. The aims of this study were to obtain prevalence

  5. Ruthenium(II) complexes: DNA-binding, cytotoxicity, apoptosis, cellular localization, cell cycle arrest, reactive oxygen species, mitochondrial membrane potential and western blot analysis.

    Science.gov (United States)

    Li, Wei; Jiang, Guang-Bin; Yao, Jun-Hua; Wang, Xiu-Zhen; Wang, Ji; Han, Bing-Jie; Xie, Yang-Yin; Lin, Gan-Jian; Huang, Hong-Liang; Liu, Yun-Jun

    2014-11-01

    The aim of our study was to investigate DNA-binding and cytotoxic activity of the four new Ru(II) polypyridyl complexes [Ru(dmb)₂(HMHPIP)](ClO₄)₂ (1), [Ru(bpy)₂(HMHPIP)](ClO₄)₂ (2), [Ru(phen)₂(HMHPIP)](ClO₄)₂ (3) and [Ru(dmp)₂(HMHPIP)](ClO₄)₂ (4). The complexes interact with DNA through intercalative mode and show relatively high cytotoxic activity against A549 cells, no cytotoxicity toward MG-63 cells. Complexes 1-4 can enhance the levels of ROS in A549 cells and induce the decrease of the mitochondrial membrane potential. These complexes inhibit the cell growth in A549 cells at G0/G1 or S phase. Complex 3 activated caspase 7, and down-regulated the expression of the anti-apoptotic protein Bcl-2. Complexes 1-4 induce apoptosis in A549 cells through ROS-mediated mitochondrial dysfunction pathway. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

  6. A Western Blot-based Investigation of the Yeast Secretory Pathway Designed for an Intermediate-Level Undergraduate Cell Biology Laboratory

    Science.gov (United States)

    Hood-DeGrenier, Jennifer K.

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in…

  7. Detection of PrPSc in Formalin Fixed Paraffin Embedded Tissue by Western Blot Differentiates Classical Scrapie, Nor98 Scrapie, and BSE

    Science.gov (United States)

    Transmissible spongiform encephalopathies including bovine spongiform encephalopathy and scrapie are fatal neurodegenerative disorders associated with the presence of an infectious abnormal isoform of normal mammalian proteins called prions (PrP**Sc). Identification of PrP**Sc in the CNS is typicall...

  8. Western Blot und realtime PCR- basierte Identifizierung möglicher Hypertonie assoziierter Gene an einem definiertem Hypotoniemodell an Wistar-Kyoto Ratten

    OpenAIRE

    Furundzija, Vesna

    2010-01-01

    Essential hypertension is a disturbance of blood pressure regulation and can be conditioned by a number of factors. The causes can be traced back to a variety of environmental factors and a range of genes relevant to blood pressure. A number of genes have been identified as possible candidates, although it is probable that others exist. The goal of the present work lies both in verification but also in the discovery of new differentially expressed genes that regulate blood pressure. ...

  9. Westernblot检测奶牛生殖系统朊蛋白的分布%Distribution of cellular prion protein in cow reproductive system revealed by Western blotting

    Institute of Scientific and Technical Information of China (English)

    张太翔; 田国宁; 张金玲; 田国华; 赵德明

    2008-01-01

    传染性海绵状脑病(TSEs)是一种可引起人和动物一系列神经退化性疾病的一种新型传染病,包括羊瘙痒病(Scrapie)、牛海绵状脑病(BSE)、鹿的慢性消耗性疾病(CWD)、水貂海绵状脑病(MSE)、猫的疯猫病(FSE)、人克雅氏病(CJD)。TSE疾病起始于正常的PrPc发生错误折叠,形成富含β片层的、抗蛋白酶水解的PrPSc。人们已经认识到PrPc向PrPSc转化是疾病发生和发展的关键。错误折叠的PrPSc有很强的聚合倾向,先形成淀粉样的小纤维,进一步聚合形成淀粉样斑块,最后发展为可被临床诊断的大脑海绵状退化变性病症。

  10. A Western Blot-based Investigation of the Yeast Secretory Pathway Designed for an Intermediate-Level Undergraduate Cell Biology Laboratory

    Science.gov (United States)

    Hood-DeGrenier, Jennifer K.

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in…

  11. Detection of classical and atypical/Nor98 scrapie by the paraffin-embedded tissue blot method.

    Science.gov (United States)

    Wemheuer, W M; Benestad, S L; Wrede, A; Wemheuer, W E; Brenig, B; Bratberg, B; Schulz-Schaeffer, W J

    2009-05-30

    The paraffin-embedded tissue (PET) blot method was used to investigate sections of the central nervous system and lymphatic tissues from 24 cases of classical scrapie and 25 cases of atypical/Nor98 scrapie in sheep and four healthy control sheep. The PET blot detected deposits of PrP(Sc) in the brain tissue of all 49 sheep with scrapie but no PrP(Sc) labelling could be detected in the control sheep. By contrast, not all the atypical/Nor98 scrapie cases were detectable by immunohistochemistry. The high sensitivity of the PET blot method made it possible to observe that in some atypical/Nor98 cases, deposits of PrP(Sc) may be restricted to supratentorial brain structures and that the diagnosis may be missed when only testing the obex area, where deposits are common in classical scrapie, and the cerebellar structures, where deposits are considered to be common in atypical/Nor98 cases.

  12. Carbonaceous aerosols in the Western Mediterranean during summertime and their contribution to the aerosol optical properties at ground level: First results of the ChArMEx-ADRIMED 2013 intensive campaign in Corsica

    Science.gov (United States)

    Sciare, Jean; Dulac, Francois; Feron, Anais; Crenn, Vincent; Sarda Esteve, Roland; Baisnee, Dominique; Bonnaire, Nicolas; Hamonou, Eric; Mallet, Marc; Lambert, Dominique; Nicolas, Jose B.; Bourrianne, Thierry; Petit, Jean-Eudes; Favez, Olivier; Canonaco, Francesco; Prevot, Andre; Mocnik, Grisa; Drinovec, Luka; Marpillat, Alexandre; Serrie, Wilfrid

    2014-05-01

    As part of the Chemistry-Aerosol Mediterranean Experiment (ChArMEx, http://charmex.lsce.ipsl.fr/), the CORSiCA (http://www.obs-mip.fr/corsica) and the ANR-ADRIMED programs, a large set of real-time measurements of carbonaceous aerosols was deployed in June 2013 at the Cape Corsica atmospheric supersite (http://gaw.empa.ch/gawsis/reports.asp?StationID=2076203042). Submicron organic aerosols (OA) were monitored every 30 min using an Aerosol Chemical Speciation Monitor (ACSM; Aerodyne Res. Inc. MA, USA); Fine (PM2.5) Organic Carbon (OC) and Elemental Carbon (EC) were measured every 2h using an OCEC Sunset Field Instrument (Sunset Lab, OR, USA) and every 12h using a low-vol (Leckel) filter sampler running at 2.3m3/h. Equivalent Black Carbon (BC) was monitored using two Aethalometers (models AE31 and AE33, Magee Scientific, US & Aerosol d.o.o., Slovenia) and a MAAP instrument (Thermo). Quality control of this large dataset was performed through chemical mass closure studies (using co-located SMPS and TEOM-FDMS) and direct comparisons with other real-time instruments running in parallel (Particle-Into-Liquid-Sampler-Ion-Chromatograph for ions, filter sampling, ...). Source apportionment of OA was then performed using the SourceFinder software (SoFi v4.5, http://www.psi.ch/acsm-stations/me-2) allowing the distinction between hydrogen- and oxygen-like organic aerosols (HOA and OOA, respectively) and highlighting the major contribution of secondary OA in the Western Mediterranean during summer. Using this time-resolved chemical information, reconstruction of the optical aerosol properties were performed and compared with integrating nephelometer (Model 3563, TSI, US) and photoacoustic extinctiometer (PAX, DMT, US) measurements performed in parallel. Results of these different closure studies (chemical/physical/optical) are presented and discussed here in details. They highlight the central role of carbonaceous aerosols on the optical properties of aerosols at ground level

  13. Testing for HTLV 1 and HTLV 2 among blood donors in Western Saudi Arabia: prevalence and cost considerations.

    Science.gov (United States)

    Hindawi, S; Badawi, M; Fouda, F; Mallah, B; Mallah, B; Rajab, H; Madani, T A

    2017-06-28

    Screening all blood donors for human T-cell lymphotropic viruses 1 and 2 (HTLV 1 and HTLV 2) is mandatory in Saudi Arabia. The aim of this study is to evaluate the results and costs associated with the current testing policy for HTLV 1 and HTLV 2 in blood donors at King Abdulaziz University Hospital (KAUH), Jeddah. Donor-testing results from Blood Transfusion Services at KAUH were reviewed over a 10-year period, from January 2006 through December 2015. All donors were screened using chemiluminescent microparticle immunoassay. Reactive samples were then tested by Western blot for confirmation. Costs associated with testing were calculated. Data of 107 419 donations in the study period were reviewed. Saudi nationals constituted 51 168 donors (47·6%). Of 107 419 blood donors tested for HTLV 1 and HTLV 2 antibody, and 95 (0·088%) donors were reactive to screening tests. None of the samples found to be reactive to screening tests was positive by Western blot. The average cost of testing was US$ 171 870 per year. No donors were confirmed to have HTLV 1 and HTLV 2 in this cohort exceeding 100 000 donors. We propose changes to the policy mandating universal testing by replacing it with universal leukodepletion coupled with targeted screening to donors coming from endemic area or donors at risk. Such changes are expected to lead to a reduction of testing cost without affecting safety. © 2017 British Blood Transfusion Society.

  14. Patterns of Limnohabitans microdiversity across a large set of freshwater habitats as revealed by Reverse Line Blot Hybridization.

    Directory of Open Access Journals (Sweden)

    Jan Jezbera

    Full Text Available Among abundant freshwater Betaproteobacteria, only few groups are considered to be of central ecological importance. One of them is the well-studied genus Limnohabitans and mainly its R-BT subcluster, investigated previously mainly by fluorescence in situ hybridization methods. We designed, based on sequences from a large Limnohabitans culture collection, 18 RLBH (Reverse Line Blot Hybridization probes specific for different groups within the genus Limnohabitans by targeting diagnostic sequences on their 16 S-23 S rRNA ITS regions. The developed probes covered in sum 92% of the available isolates. This set of probes was applied to environmental DNA originating from 161 different European standing freshwater habitats to reveal the microdiversity (intra-genus patterns of the Limnohabitans genus along a pH gradient. Investigated habitats differed in various physicochemical parameters, and represented a very broad range of standing freshwater habitats. The Limnohabitans microdiversity, assessed as number of RLBH-defined groups detected, increased significantly along the gradient of rising pH of habitats. 14 out of 18 probes returned detection signals that allowed predictions on the distribution of distinct Limnohabitans groups. Most probe-defined Limnohabitans groups showed preferences for alkaline habitats, one for acidic, and some seemed to lack preferences. Complete niche-separation was indicated for some of the probe-targeted groups. Moreover, bimodal distributions observed for some groups of Limnohabitans, suggested further niche separation between genotypes within the same probe-defined group. Statistical analyses suggested that different environmental parameters such as pH, conductivity, oxygen and altitude influenced the distribution of distinct groups. The results of our study do not support the hypothesis that the wide ecological distribution of Limnohabitans bacteria in standing freshwater habitats results from generalist adaptations of

  15. Zinc Blotting Assay for Detection of Zinc-Binding Prolamin in Barley (Hordeum vulgare) Grain

    DEFF Research Database (Denmark)

    Uddin, Mohammad Nasir; Langkilde, Ane; Vincze, Éva

    2014-01-01

    In plants, zinc is commonly found bound to proteins. In barley (Hordeum vulgare), major storage proteins are alcohol-soluble prolamins known as hordeins, and some of them have the potential to bind or store zinc. 65Zn overlay and blotting techniques have been widely used for detecting zinc...

  16. Different domains of Bacillus thuringiensis delta-endotoxins can bind to insect midgut membrane proteins on ligand blots

    NARCIS (Netherlands)

    Maagd, de R.A.; Klei, van der H.; Bakker, P.L.; Stiekema, W.J.; Bosch, D.

    1996-01-01

    We investigated the role of the constituent domains of the CryIA(b) and CryIA(c) δ-endotoxins in binding to midgut epithelial cell membrane proteins of Spodoptera exigua and Manduca sexta on ligand blots. A collection of wild- type and CryIC-CryIA hybrid toxins was used for this purpose. As demonstr

  17. Detection of Babesia and Theileria species infection in cattle from Portugal using a reverse line blotting method.

    Science.gov (United States)

    Silva, M G; Marques, P X; Oliva, A

    2010-12-15

    Babesiosis and Theileriosis are tick-borne diseases widespread in tropical and sub-tropical regions with high economic impact worldwide. In Portugal there are at least 4 tick vectors known to be competent for the transmission of Babesia and Theileria sp. identified: Rhipicephalus bursa, Rhipicephalus (Boophilus) annulatus, Ixodes ricinus and Haemaphysalis punctata. All these potential Babesia and Theileria tick vectors are widely distributed in Portugal, although they are predominant in the Southern region. In this study, 1104 cattle blood samples were randomly collected from Central and Southern regions of Portugal and analyzed by PCR-reverse line blotting (RLB) for the detection of Babesia and Theileria sp. Testing indicated that 74.7% of the bovines tested were positive for either Babesia and/or Theileria sp. In addition, five different apicomplexan species, namely, Theileria buffeli, Theileria annulata, Babesia divergens, Babesia bovis, and Babesia bigemina were detected by RLB among the bovines tested. T. buffeli was the most frequently found species, being present in 69.9% of the positive samples either as single infections (52.4%), or as mixed infections (17.5%). The Babesia specie most frequently found was B. divergens, detected in 4.2% of the infected bovines. Overall, infected bovines were found in all regions tested; however the highest number of infected bovines was observed in Évora district (96.2%) and in cattle from Limousin breeds (81.7%). The results indicate widespread Babesia and Theileria infections in Portuguese bovines, suggesting the need for improved control of ticks and tick-borne diseases.

  18. Results of exploratory drilling at the western portion of Los Azufres, Michoacan, Mexico, geothermal field; Resultados de la perforacion exploratoria en la porcion occidental del campo geotermico de Los Azufres, Michoacan, Mexico

    Energy Technology Data Exchange (ETDEWEB)

    Casarrubias Unzueta, Zenon [Gerencia de Proyectos Geotermoelectricos de la Comision Federal de Electricidad, Morelia (Mexico)

    1996-09-01

    The geological characteristics of the western portion of the Los Azufres, Michoacan, Mexico geothermal field were defined on the basis of EAZ-1 and EAZ-2 exploratory wells. A maximum temperature of 187 degrees celsius at 1995 m depth, with a pressure of 188 kg/cm{sup 2} were registered at EAZ-1 well. A permeable zone was detected at 1333 m with a measured temperature of 133 degrees celsius. The presence of laumontite at 1450 m depth, corresponds to a forming temperature similar to the measured one. Well EAZ-2 is also situated in the western sector of Los Azufres. During drilling, at 2197 m depth, a temperature of 222 degrees celsius and a pressure of 188 kg/cm{sup 2} were measured. Seventeen days later, 243 degrees celsius were registered at the same depth. The presence of a thermal anomaly, initially interpreted as an interest area by the existence of a low resistivity zone, was confirmed with the results above mentioned. The mineralogical analysis from 1400 m depth to the bottom of well EAZ-2 detected the existence of epidote, epidote+chlorite, epidote+amphybole, epidote+zoisite, quartz and unidentified clay+epidote, this assemblage suggest temperatures over 230 degrees celsius with a pressure of 85 bar at this interval. The temperatures estimated by fluid inclusions confirmed this interpretation. The results suggest the continuity of the Los Azufres hydrothermal system to the west, between AZ-3 and EAZ-2 wells. However, the NW sector is not connected to permeable zones associated with producing faults. [Espanol] Los resultados de la perforacion de los pozos exploratorios EAZ-1 y EAZ-2, permitieron definir las condiciones geologicas del yacimiento geotermico de Los Azufres, Michoacan, Mexico hacia la extension occidental del campo. En el pozo EAZ-1, localizado al NW del campo, se midio una temperatura maxima de 187 grados celsius, a una profundidad de 1995 m, con una presion de 188 kg/cm{sup 2}. Se registro una zona permeable a 1333 m, con una temperatura de 131

  19. North-south gradients in plasma concentrations of B-vitamins and other components of one-carbon metabolism in Western Europe : results from the European Prospective Investigation into Cancer and Nutrition (EPIC) Study

    NARCIS (Netherlands)

    Eussen, Simone J. P. M.; Nilsen, Roy M.; Midttun, Oivind; Hustad, Steinar; IJssennagger, Noortje; Meyer, Klaus; Fredriksen, Ase; Ulvik, Arve; Ueland, Per M.; Brennan, Paul; Johansson, Mattias; Bueno-de-Mesquita, Bas; Vineis, Paolo; Chuang, Shu-Chun; Boutron-Ruault, Marie Christine; Dossus, Laure; Perquier, Florence; Overvad, Kim; Teucher, Birgit; Grote, Verena A.; Trichopoulou, Antonia; Adarakis, George; Plada, Maria; Sieri, Sabina; Tumino, Rosario; Santucci de Magistris, Maria; Ros, Martine M.; Peeters, Petra H. M.; Luisa Redondo, Maria; Zamora-Ros, Raul; Chirlaque, Maria-Dolores; Ardanaz, Eva; Sonestedt, Emily; Ericson, Ulrika; Schneede, Joern; van Guelpen, Bethany; Wark, Petra A.; Gallo, Valentina; Norat, Teresa; Riboli, Elio; Vollset, Stein Emil

    2013-01-01

    Different lifestyle patterns across Europe may influence plasma concentrations of B-vitamins and one-carbon metabolites and their relation to chronic disease. Comparison of published data on one-carbon metabolites in Western European regions is difficult due to differences in sampling procedures and

  20. New research results on “La Plaza de Toros” of Cerro de San Pelayo” (Martimor, Salamanca: a tumulus burial with inhumation at the beginning of the Firts Millenium in the Western area of the North Meseta

    Directory of Open Access Journals (Sweden)

    López Jiménez, Óscar

    2004-06-01

    Full Text Available The aim of this work is to present the latest results obtained from the systematic revision of the materials from this unique site. We present here the burial level in its complete context and the first results of the analyses of this assemblage. We have documented in it human bones, elements of fauna possibly associated with a funerary feast, storage pottery and the well-known geometric painted vessel. The results obtained by analysis of the distribution and post-depositional processes are also very interesting, allowing us to recreate the space in which seem to be represented symbolic and prestige elements that indicate important social changes in these societies. All this, together with the calibration of the radiocarbon dates taken from the tumulus, offers a more complete archaeological documentation of this little known area in the South-western of the Northern Meseta.

    En este trabajo pretendemos adelantar los últimos resultados obtenidos de la revisión sistemática realizada sobre los materiales de este yacimiento de características únicas hasta ahora en la región. Avanzamos aquí la presentación del nivel de enterramiento en su contexto completo y los primeros resultados de los análisis de este conjunto. En él documentamos restos humanos, elementos de fauna posiblemente asociados a un banquete funerario, elementos de almacenaje y el ya conocido vaso pintado con decoración geométrica. Los resultados obtenidos por los análisis de dispersión y procesos postdeposicionales son igualmente de gran interés, permitiéndonos recrear ese espacio en el que parecen estar representados elementos simbólicos y de prestigio que indican importantes cambios en las sociedades de aquel momento. Todo ello, unido a la recalibración de las fechas radiocarbónicas del interior del túmulo, ofrece una completa documentación arqueológica de un momento poco conocido en el área sudoccidental de la Meseta Norte.

  1. Western Retrospections and Outlook

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    This year marks the 10-year anniversary of the strategy on development of China’s western region. With a land area of 6.85 million square km, accounting for 71.4 percent of the country’s total, the western region has been an indispensable part in achieving China’s overall prosperity and

  2. A combined RT-PCR and dot-blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius).

    Science.gov (United States)

    Lopez-Jimena, B; Cherif, N; Garcia-Rosado, E; Infante, C; Cano, I; Castro, D; Hammami, S; Borrego, J J; Alonso, M C

    2010-10-01

    To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin-labelled probes resulted in the identification of both genotypes separately. The application of the RT-PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46.87% of viral nervous necrosis virus carriers. The combination of RT-PCR and blot hybridization increases the detection rate up to 90.62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56.25%). This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

  3. Differentiation of Enterococcus faecium from Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains by PCR and dot-blot hybridisation.

    Science.gov (United States)

    Langa, S; Fernández, A; Martín, R; Reviriego, C; Marín, M L; Fernández, L; Rodríguez, J M

    2003-12-01

    Variations in length and sequence of the 16S/23S spacer region of Enterococcus faecium provided the basis for development of simple PCR and dot-blot hybridisation assays that enabled the differentiation of potentially probiotic Enterococcus faecium strains from Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. Such assays may be useful for differentiation of yoghurt starter cultures and enterococcal strains when they are simultaneously present in probiotic food products.

  4. Toxocariasis Resulting in Seeming Allergy

    Directory of Open Access Journals (Sweden)

    Rosanna Qualizza

    2009-09-01

    Full Text Available Toxocara canis is an intestinal nematode affecting dogs and cats that causes human infestations by ingestion of embryonated eggs excreted in dogs' faeces. Humans are transport hosts, in whom the larvae do not develop to adult worms, but may migrate to various tissues and organs, and survive for several years, giving rise to several clinical symptoms, which include allergy-like presentations. We report three cases presenting as dermatitis, rhinitis, asthma, and conjunctivitis which were diagnosed and unsuccessfully treated as allergy. The correct diagnosis was established after detecting anti-Toxocara antibodies by Western blotting. All clinical symptoms showed improvement after starting treatment with mebendazole and subsequent courses of the antiparasitic drug resulted in full recovery. This suggests the possible role of Toxocara canis in inducing chronic symptoms of allergic type. This is particularly important for asthma, where it has been demonstrated that Toxocara canis infection causes allergic inflammation in the lungs associated with bronchial hyperreactivity. On the other hand, in our patients with asthma and with dermatitis the positive results from allergy tests were a confounding factor in delaying the correct diagnosis, which was finally obtained by the detection of antibodies to Toxocara canis.

  5. Molecular diagnosis of Theileria and Babesia species infecting cattle in Northern Spain using reverse line blot macroarrays

    Directory of Open Access Journals (Sweden)

    Juste Ramón A

    2006-05-01

    Full Text Available Abstract Background Piroplasmosis in cattle is caused by tick-borne haemoprotozoan parasites of the genera Theileria and Babesia. Molecular detection techniques offer higher sensitivity and specificity than microscopy examination methods and serological tests. A reverse line blot (RLB macroarray that included generic and species-specific probes for Theileria annulata, Theileria buffeli, Babesia bovis, Babesia bigemina, Babesia divergens and Babesia major was used to study the presence and identity of the piroplasm species infecting 263 bovine blood samples from 79 farms, most of them in Northern Spain. Microscopy examination of blood smears and haematology were also performed whenever possible to identify animals with parasitaemia. Results RLB hybridisation identified infection in 54.0% of the samples, whereas only 28.8% were positive by microscopy examination. The most frequently found species was T. buffeli, present in 42.6% of the samples. T. annulata was found in 22 samples (8.4% from 12 farms, including 9 farms (14 samples located in Northern Spain where presence of the vector is not very common. Babesia infections were less frequently detected: B. major was found in 3.0% of the samples, B. bigemina in 2.7%, B. bovis in 2.3% and B. divergens in 1.1%. Mixed infections were detected in 14 samples, accounting for six different combinations of species. Conclusion This is the first report in which B. major and B. divergens have been detected in Spain using molecular identification techniques and the first time that B. bovis has been detected in Northern Spain. The detection of T. annulata in Northern Spain suggests that the distribution of Mediterranean theileriosis might be changing. Samples with positive RLB hybridisation but negative microscopy had haematology values within the normal ranges suggesting that they corresponded to chronic carriers that may serve as reservoirs of the infection. In this sense, sensitive and specific laboratorial

  6. A Comparison of Antibacterial Activity of Selected Thyme (Thymus) Species by Means of the Dot Blot Test with Direct Bioautographic Detection.

    Science.gov (United States)

    Orłowska, Marta; Kowalska, Teresa; Sajewicz, Mieczysław; Jesionek, Wioleta; Choma, Irena M; Majer-Dziedzic, Barbara; Szymczak, Grażyna; Waksmundzka-Hajnos, Monika

    2015-01-01

    Bioautography carried out with the aid of thin-layer chromatographic adsorbents can be used to assess antibacterial activity in samples of different origin. It can either be used as a simple and cost-effective detection method applied to a developed chromatogram, or to the dot blot test performed on a chromatographic plate, where total antibacterial activity of a sample is scrutinized. It was an aim of this study to compare antibacterial activity of 18 thyme (Thymus) specimens and species (originating from the same gardening plot and harvested in the same period of time) by means of a dot blot test with direct bioautography. A two-step extraction of herbal material was applied, and at step two the polar fraction of secondary metabolites was obtained under the earlier optimized extraction conditions [methanol-water (27+73, v/v), 130°C]. This fraction was then tested for its antibacterial activity against Bacillus subtilis bacteria. It was established that all investigated extracts exhibited antibacterial activity, yet distinct differences were perceived in the size of the bacterial growth inhibition zones among the compared thyme species. Based on the results obtained, T. citriodorus "golden dwarf" (sample No. 5) and T. marschallianus (sample No. 6) were selected as promising targets for further investigations and possible inclusion in a herbal pharmacopeia, which is an essential scientific novelty of this study.

  7. Western Food in China

    Institute of Scientific and Technical Information of China (English)

    1997-01-01

    AS the Chinese saying goes, "Ask for local custom when you enter a foreign country." Western food’s first introduction to China in the 17th century was accompanied with its adoption to Chinese dining habits. Western food was introduced into China in large scale during the mid-19th and early 20th centuries. However, as early as the 17th century Western missionaries and envoys were introducing food from their homeland to upper-class Chinese as a means of paying tribute or

  8. Violence the Western way.

    Science.gov (United States)

    Roth, B E

    1997-10-01

    Despite the quiet revolution in response to changing conceptualizations of gender in psychoanalysis, the Western has remained the domain of aggressive phallic masculinity. The iconic imagery of the Western, when combined with its narrative trajectory, is used to tell stories of violent encounters between men. The acceptance of the genre, and its duplication by other cultures and film makers, indicates that the Westerns' imagery and moral solutions tap into some basic deep structures of anxiety and pleasure in violence between men. As long as societies require subtle sublimations of aggressive and violent drives, it is likely that men will seek imaginary regressive experiences to discharge frustrations.

  9. Detection of Rickettsia in Rhipicephalus sanguineus ticks and Ctenocephalides felis fleas from southeastern Tunisia by reverse line blot assay.

    Science.gov (United States)

    Khrouf, Fatma; M'Ghirbi, Youmna; Znazen, Abir; Ben Jemaa, Mounir; Hammami, Adnene; Bouattour, Ali

    2014-01-01

    Ticks (n = 663) and fleas (n = 470) collected from domestic animals from southeastern Tunisia were screened for Rickettsia infection using reverse line blot assay. Evidence of spotted fever group Rickettsia was obtained. We detected Rickettsia felis in fleas, Rickettsia massiliae Bar 29 and the Rickettsia conorii Israeli spotted fever strain in ticks, and Rickettsia conorii subsp. conorii and Rickettsia spp. in both arthropods. The sensitivity of the adopted technique allowed the identification of a new association between fleas and R. conorii subsp. conorii species. The presence of these vector-borne Rickettsia infections should be considered when diagnosing this disease in humans in Tunisia.

  10. Opportunities for silvicultural treatment in western Oregon.

    Science.gov (United States)

    Colin D. MacLean

    1980-01-01

    A recent Forest Survey inventory of western Oregon has been analyzed to determine the extent of physical opportunities to increase wood production through silvicultural treatment. Results are presented by owner group and by geographic unit.

  11. RT-PCR and Northern blot analysis in search for a putative Paramecium beta-adrenergic receptor.

    Science.gov (United States)

    Płatek, A; Wiejak, J; Wyroba, E

    1999-01-01

    RT-PCR and Northern blot analysis were performed in order to search for a putative beta-adrenergic receptor (beta-AR) in Paramecium using several beta2-adrenergic-specific molecular probes. Under strictly defined RT-PCR conditions DNA species of expected molecular size about 360 bp were generated with the primers corresponding to the universal mammalian beta2-AR sequence tagged sites (located within the 4th and the 6th transmembrane regions of the receptor). This RT-PCR product hybridized in Southern blot analysis with the oligonucleotide probe designed to the highly conservative beta2-AR region involved in G-proteins interaction and located within the amplified region. Northern hybridization was performed on Paramecium total RNA and mRNA with human beta2-AR cDNA and two oligonucleotide probes: the first included Phe 290 involved in agonist binding (Strader et al., 1995) and the second was the backward RT-PCR primer. All these probes revealed the presence of about 2 kb mRNA which is consistent with the size of beta2-AR transcripts found in higher eukaryotes.

  12. Modification of T-cell antigenic properties of tetanus toxoid by SDS-PAGE separation. Implications for T-cell blotting

    DEFF Research Database (Denmark)

    Christensen, C B; Theander, T G

    1997-01-01

    Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20% of the ......Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20...... that SDS-PAGE alters the ability of TT to induce T-cell proliferation, possibly due to unpolymerized acrylamide binding to proteins during SDS-PAGE. The use of SDS-PAGE T-cell blotting in the screening for T-cell antigens must therefore be reconsidered. We suggest the use of SDS-Agarose Gel Electrophoresis...

  13. Mere end blot pirringer

    DEFF Research Database (Denmark)

    2006-01-01

    Med udgangspunkt i en række cases på vellykkede oplevelsesøkonomiske forretningsmodeller argumenterer artiklen for, at oplevelsesprodukter skal bygge på et klart tema, som forbrugeren kan koble sig sanse- og følelsesmæssigt op på. Forbrugeren skal kunne omsætte produktets pirringer til egne erfar...

  14. Mere end blot pirringer

    DEFF Research Database (Denmark)

    Jantzen, Christian; Østergaard, Per

    2006-01-01

    Med udgangspunkt i en række cases på vellykkede oplevelsesøkonomiske forretningsmodeller argumenterer artiklen for, at oplevelsesprodukter skal bygge på et klart tema, som forbrugeren kan koble sig sanse- og følelsesmæssigt op på. Forbrugeren skal kunne omsætte produktets pirringer til egne erfar...

  15. Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

    Directory of Open Access Journals (Sweden)

    Fang Ding

    Full Text Available 'Candidatus Liberibacter asiaticus' (CaLas, a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB. Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant.

  16. Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

    Science.gov (United States)

    Ding, Fang; Duan, Yongping; Paul, Cristina; Brlansky, Ronald H; Hartung, John S

    2015-01-01

    'Candidatus Liberibacter asiaticus' (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant.

  17. Duff mound consumption and cambium injury for centuries-old western larch from prescribed burning in western Montana

    Science.gov (United States)

    Michael G. Harrington

    2012-01-01

    Western larch is one of the most fire-adapted conifers in western North America. Its historical perpetuation depended upon regular fire disturbances, which creates open stand conditions and mineral seedbeds. A stand of 200- to 500-year-old larch in western Montana with deep duff mounds resulting from an unusually long 150-year fire-free period was mechanically thinned...

  18. Rings dominate western Gulf

    Science.gov (United States)

    Vidal L., Francisco V.; Vidal L., Victor M. V.; Molero, José María Pérez

    Surface and deep circulation of the central and western Gulf of Mexico is controlled by interactions of rings of water pinched from the gulf's Loop Current. The discovery was made by Mexican oceanographers who are preparing a full-color, 8-volume oceanographic atlas of the gulf.Anticyclonic warm-core rings pinch off the Loop Current at a rate of about one to two per year, the scientists of the Grupo de Estudios Oceanográficos of the Instituto de Investigaciones Eléctricas (GEO-IIE) found. The rings migrate west until they collide with the continental shelf break of the western gulf, almost always between 22° and 23°N latitude. On their westward travel they transfer angular momentum and vorticity to the surrounding water, generating cyclonic circulations and vortex pairs that completely dominate the entire surface and deep circulation of the central and western gulf.

  19. The application of a photon-counting camera in very sensitive, bioluminescence-enhanced detection systems for protein blotting. Ultrasensitive detection systems for protein blotting and DNA hybridization, II.

    Science.gov (United States)

    Hauber, R; Miska, W; Schleinkofer, L; Geiger, R

    1988-03-01

    A relatively simple, very sensitive bioluminescence-enhanced detection system for protein blots was described recently. This method utilizes antibodies conjugated with alkaline phosphatase. Alkaline phosphatase releases D-luciferin (Photinus pyralis) from D-luciferin-O-phosphate. Liberated D-luciferin reacts with luciferase, ATP and oxygen with light emission. The light produced is measured with a very sensitive photon counting camera (Argus 100), permitting visualization and localization of the alkaline phosphatase-conjugated antibodies on nitrocellulose sheets. Under non-optimized conditions the limit of detection is at present 5 to 500 fg of protein (rabbit immunoglobulin G), corresponding to 30 to 3 amol. The method is therefore 10(5) times more sensitive than other used at present.

  20. IN WESTERN SIBERIA

    Directory of Open Access Journals (Sweden)

    Chizhikov Il'ja Aleksandrovich

    2012-10-01

    Full Text Available The paper presents the results of the field study of the subsidence following the implementation of one of the road building methods. The field study was performed in the area alongside oil and gas field access roads built in the loose marshy ground (peat. The paper provides a description of the geosynthetics installation technology. Outdated road building technologies designated for weak road base conditions feature high consumption of materials and energy and substantial anthropogenic impact on the environment. These technologies comprise complete or partial replacement of the ground by sand, road metal, or macrofragmental material combined with timber as the reinforcing element of the base (a plank road. The objective of the field study was to develop an ecologically safe technology capable of reducing the anthropogenic impact on the environment and improving the strength and the operating safety of the road structure. The field study has proven that the aforementioned objective may be attained through the application of geosynthetic materials designated for the improvement of the bearing capacity of the weak road base. Geosynthetic materials and structures of the road subgrade ensure high speed of construction, high quality and reliability of the road structure, reduction in the consumption of materials and energy to assure the ecological safety of construction of oil and gas field access roads. Earlier, the author used the qualimetry method to assess the ecological safety. This method is used to assess the ecological safety in points. The assessments have proven that the reinforced base technology is preferable to the method of complete or partial replacement of the ground. Projection of the subsistence development pattern that makes it possible to evaluate the safety and the serviceability of the proposed solution is also an important efficiency factor. The field study involved a section of the oil and gas field access road outstretched

  1. Western Area Power Administration combined power system financial statements, 30 September 1995 and 1994

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-12-31

    The attached report presents the results of the independent certified public accountant`s audit of the Department of Energy`s Western Area Power Administration`s (Western) combined financial statements as of September 30, 1995. The auditors have expressed an unqualified opinion on Western`s 1995 statements. Their reports on Western`s internal control structure and on compliance with laws and regulations are also provided.

  2. China's Western Priority

    Institute of Scientific and Technical Information of China (English)

    YU SHUJUN

    2010-01-01

    @@ "Western Development" has become a buzzword in China over the past decade. It has appeared almost everywhere: in government docu-ments, media reports and even ordinary people's conversations. It has become a national campaign in the new century, with a wide variety of resources--human, financial and material-- flowing to the westem part of the country.

  3. Imprinting mutations in Angelman syndrome detected by Southern blotting using a probe containing exon {alpha} of SNRPN

    Energy Technology Data Exchange (ETDEWEB)

    Beuten, J.; Sutcliffe, J.S.; Nakao, M. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are associated with paternal and maternal deficiencies respectively, of gene expression within human chromosome 15q11-q13, and are caused by deletion, uniparental disomy (UPD), or other mutations. The SNRPN gene maps in this region, is paternally expressed, and is a candidate gene for PWS. Southern blotting using methylation-sensitive enzymes and a genomic DNA probe from the CpG island containing exon {alpha} of the SNRPN gene reveals methylation specific for the maternal allele. In cases of the usual deletions or UPD, the probe detects absence of an unmethylated allele in PWS and absence of a methylated allele in AS. We have analyzed 21 nondeletion/nonUPD AS patients with this probe and found evidence for an imprinting mutation (absence of a methylated allele) in 3 patients. Southern blotting with methylation-sensitive enzymes using the exon {alpha} probe, like use of the PW71 probe, should detect abnormalities in all known PWS cases and in 3 of the 4 forms of AS: deletion, UPD and imprinting mutations. This analysis provides a valuable diagnostic approach for PWS and AS. In efforts to localize the imprinting mutations in AS, one patient was found with failure to inherit a dinucleotide repeat polymorphism near probe 189-1 (D15S13). Analysis of this locus in AS families and CEPH families demonstrates a polymorphism that impairs amplification and a different polymorphism involving absence of hybridization to the 189-1 probe. The functional significance, if any, of deletion of the 189-1 region is unclear.

  4. Is Western Australia.

    Science.gov (United States)

    Shanmugakumar, Sharanyaa; Playford, Denese; Burkitt, Tessa; Tennant, Marc; Bowles, Tom

    2016-03-31

    Objective Despite public interest in the rural workforce, there are few published data on the geographical distribution of Australia's rural surgeons, their practice skill set, career stage or work-life balance (on-call burden). Similarly, there has not been a peer-reviewed skills audit of rural training opportunities for surgical trainees. The present study undertook this baseline assessment for Western Australia (WA), which has some of the most remote practice areas in Australia.Methods Hospital staff from all WA Country Health Service hospitals with surgical service (20 of 89 rural health services) were contacted by telephone. A total of 18 of 20 provided complete data. The study questionnaire explored hospital and practice locations of practicing rural surgeons, on-call rosters, career stage, practice skill set and the availability of surgical training positions. Data were tabulated in excel and geographic information system geocoded. Descriptive statistics were calculated in Excel.Results Of the seven health regions for rural Western Australia, two (28.6%) were served by resident surgeons at a ratio consistent with Royal Australasian College of Surgeons (RACS) guidelines. General surgery was offered in 16 (89%) hospitals. In total, 16 (89%) hospitals were served by fly-in, fly-out (FIFO) surgical services. Two hospitals with resident surgeons did not use FIFO services, but all hospitals without resident surgeons were served by FIFO surgical specialists. The majority of resident surgeons (62.5%) and FIFO surgeons (43.2%) were perceived to be mid-career by hospital staff members. Three hospitals (16.7%) offered all eight of the identified surgical skill sets, but 16 (89%) offered general surgery.Conclusions Relatively few resident rural surgeons are servicing large areas of WA, assisted by the widespread provision of FIFO surgical services. The present audit demonstrates strength in general surgical skills throughout regional WA, and augers well for the training

  5. Microstructural, modal and geochemical changes as a result of granodiorite mylonitisation – a case study from the Rolovská shear zone (Čierna hora Mts, Western Carpathians, Slovakia

    Directory of Open Access Journals (Sweden)

    Farkašovský Roman

    2016-09-01

    Full Text Available Strong tectonic remobilisation and shear zone development are typical features of the easternmost part of the Veporicum tectonic unit in the Western Carpathians. The granodiorite mylonites in the area of the Rolovská shear zone (Čierna hora Mts underwent a complex polystage evolution during the Hercynian and Alpine orogenies. Deformation during the latter reached greenschist facies under metamorphic conditions. Mylonites are macroscopically foliated rocks with a stretching lineation and shear bands. Structurally different mylonite types, ranging from protomylonites to ulramylonites with typical grainsize reduction from the margins towards the shear zone centre, have been assessed. The modal mineralogy of the different mylonite types changes considerably. Typical is a progressive decrease in feldspar content and simultaneously the quartz and white mica content increases from protomylonites towards the most strongly deformed ultramylonites. The deformation had a brittle character in less deformed rocks and a ductile one in more deformed tectonites. Obvious chemical changes occur in mesomylonites and ultramylonites. During mylonitisation, the original biotite granodiorite was depleted of Mg, Fe, Na, Ca and Ba, while K, Rb and mainly Si increased considerably. Other (major and trace elements reflect erratic behaviour due to lateral mobility. Chemical changes indicate the breakdown and subsequent recrystallisation of biotite and feldspars and, in turn, the crystallisation of albite and sericite. REE decrease in ultramylonites due to the breakup of accessory minerals during deformation and alteration.

  6. Contemporary Issues of Studying of Western European and Russian Mindset (According to the Results of Russian Nationwide Conference “National Identity through Language and Literature. Characteristics of Conceptosphere of a National Culture”

    Directory of Open Access Journals (Sweden)

    Ratsiburskaya L. V.

    2015-01-01

    Full Text Available He work of the Russian nationwide conference “National identity through language and literature. Characteristics of conceptoshere of national culture” is analyzed in the article. Previous theoretical sources on the issue in question are summarized. The matters represented in the considered scientific forum are generalized. Diachronic analysis of national cultural consciousness as well as complex cognitive-based approach are used to investigate the issue. Special attention is paid to the study of linguistic world-image as exemplified in fiction, folklore, religious texts, business papers, scholarly works and media texts, as well as to the research of intercultural communication in the era of multiculturalism and globalization. As long as terms “cultural code”, “national code”, and “fictional code” are not precisely defined, the problem of terminological field was noted, as well as the issue of linguo-cognitive interpretation of methods for representing country image in linguistic consciousness. The attention was paid to Russian as a foreign language, to perception and transforming of European cultural codes by American and western European writers in synchronous and diachronic ways. Geopolitical factor of North and South that matters both in global (European and local (in a particular countries context was noted, particularly the issue of German distinction of the modern European mindset was reviewed. Lots of speaker papers mirrored interdisciplinary links that is of importance for contemporary scholarship in the context of globalization and multicultural society. Up-to-date schools of thought in the field of linguistics, literary studies and journalism were represented.

  7. Communicating with Westerners

    Institute of Scientific and Technical Information of China (English)

    Sarah; Callicott

    2007-01-01

    <正>The majority response from my students when asked "what do you want to learn from my class?" is "How do I communicate better with westerners?" My students also have other questions such as "How do I improve my oral English?" and "How is America different from China?" These questions can be answered in many different ways, but hopefully I will give you a couple of ideas to get you started.

  8. Dot Blot para determinar la identidad antigénica en vacunas conjugadas contra Streptococcus pneumoniae serotipo 19F

    Directory of Open Access Journals (Sweden)

    Osmir Cabrera-Blanco

    2017-04-01

    Full Text Available Las autoridades regulatorias recomiendan el uso de técnicas de Resonancia Magnética Nuclear o técnicas serológicas para la determinación de la identidad de los antígenos presentes en las vacunas conjugadas. Con la aparición de las vacunas conjugadas multivalentes, se ha hecho necesario recurrir a técnicas inmunoquímicas con la utilización de anticuerpos monoclonales para aumentar la sensibilidad en la determinación de la identidad de los antígenos en dichas vacunas conjugadas. El objetivo del presente trabajo fue establecer las condiciones óptimas de trabajo que permitieran utilizar la técnica del Dot Blot para determinar la identidad de los antígenos en vacunas conjugadas de Streptococcus pneumoniae serotipo 19F. Para ello se estudiaron los tiempos de incubación, la influencia del reactivo en la solución de bloqueo; también las concentraciones óptimas del anticuerpo monoclonal y de los ingredientes farmacéuticos activos, así como los volúmenes de aplicación óptimos para estos y vacunas. Se utilizó un anticuerpo monoclonal contra el polisacárido capsular del serotipo 19F de neumococo. Las muestras empleadas en este trabajo fueron lotes de ingredientes farmacéuticos activos de conjugados de polisacárido capsular 19F y lotes de un candidato vacunal cubano conjugado heptavalente contra neumococos. Los resultados mostraron que para la determinación de la identidad antigénica fueron suficientes 10 µL de muestras de los principios activos a una concentración de 125 µg/mL e igual volumen para las vacunas heptavalentes. Quedó demostrado que una concentración de 1 µg/mL para el anticuerpo monoclonal y tiempos de incubación de 30 min a 37 °C fueron suficientes para la determinación. Estos resultados permiten concluir que quedaron establecidas las condiciones óptimas de trabajo para determinar la identidad antigénica por Dot Blot del polisacárido capsular de S. pneumoniae serotipo 19F presente en las vacunas

  9. RNA interference tools for the western flower thrips, Frankliniella occidentalis.

    Science.gov (United States)

    Badillo-Vargas, Ismael E; Rotenberg, Dorith; Schneweis, Brandi A; Whitfield, Anna E

    2015-05-01

    The insect order Thysanoptera is exclusively comprised of small insects commonly known as thrips. The western flower thrips, Frankliniella occidentalis, is an economically important pest amongst thysanopterans due to extensive feeding damage and tospovirus transmission to hundreds of plant species worldwide. Geographically-distinct populations of F. occidentalis have developed resistance against many types of traditional chemical insecticides, and as such, management of thrips and tospoviruses are a persistent challenge in agriculture. Molecular methods for defining the role(s) of specific genes in thrips-tospovirus interactions and for assessing their potential as gene targets in thrips management strategies is currently lacking. The goal of this work was to develop an RNA interference (RNAi) tool that enables functional genomic assays and to evaluate RNAi for its potential as a biologically-based approach for controlling F. occidentalis. Using a microinjection system, we delivered double-stranded RNA (dsRNA) directly to the hemocoel of female thrips to target the vacuolar ATP synthase subunit B (V-ATPase-B) gene of F. occidentalis. Gene expression analysis using real-time quantitative reverse transcriptase-PCR (qRT-PCR) revealed significant reductions of V-ATPase-B transcripts at 2 and 3 days post-injection (dpi) with dsRNA of V-ATPase-B compared to injection with dsRNA of GFP. Furthermore, the effect of knockdown of the V-ATPase-B gene in females at these two time points was mirrored by the decreased abundance of V-ATPase-B protein as determined by quantitative analysis of Western blots. Reduction in V-ATPase-B expression in thrips resulted in increased female mortality and reduced fertility, i.e., number of viable offspring produced. Survivorship decreased significantly by six dpi compared to the dsRNA-GFP control group, which continued decreasing significantly until the end of the bioassay. Surviving female thrips injected with dsRNA-V-ATPase-B produced

  10. Coral Paleo-Uplift History Overlying a Very Shallow AD 2007 Megathrust Rupture of the Western Solomons Forearc: Deficit of Interseismic Subsidence Results in Net Long-Term Uplift

    Science.gov (United States)

    Taylor, F. W.; Thirumalai, K.; Lavier, L. L.; Frohlich, C.; Shen, C.; WU, C.; Sun, H.; Papabatu, A. K.

    2013-12-01

    The processes of forearc deformation and their relation to the megathrust earthquake cycle provide critical insights regarding longer-term arc tectonic evolution and the earthquake cycle itself. At the Western Solomon Islands arc, Ranongga and other islands sit over the seismogenic megathrust as close as 4 km from the trench where the Australian plate converges at ~80-100 mm/yr. Coral reefs fringe the coasts and record the relative sea level changes accompanying both long-term tectonic uplift and the shorter-term earthquake cycle. The AD 2007 Mw 8.1 megathrust rupture zone dips from 1-2 km to ~25 km deep beneath islands and caused uplift that we measured at coasts along a profile normal to the arc trend. Vertical components of the 2007 displacement show the expected fairly smooth uplift profile typical of elastic strain release. In contrast, along the same profile the amounts of longer-term net uplift of the mid-Holocene (~6 ka) coral shoreline are more variable. Likewise, the elevations of individual coral paleo-shorelines recording a series of individual uplifts (as young as 600 yr old with ~2.25 m uplift) also indicate variable amounts of uplift along the profile when compared to the 2007 coseismic uplift. For 50 -100 yr before 2007, all sites along the same profile subsided at rates on the order of 1-3 cm/yr, all of which equaled or exceeded the upward growth rates of corals. However, the amounts of pre-2007 subsidence are highly variable along the profile and do not conform well to the smoother profile of the 2007 displacements. Where total uplift since the mid-Holocene is smaller, pre-2007 subsidence and 2007 uplift may be relatively large. Where total uplift since the mid-Holocene is larger, 2007 coseismic uplift may be large while pre-2007 subsidence is relatively small. Therefore, the differences in longer-term uplift rates appear tied to variable subsidence during the interseismic period rather than being primarily a coseismic phenomenon. Furthermore

  11. "Enzyme-Linked Immunotransfer Blot Analysis of Somatic and Excretory- Secretory Antigens of Fasciola hepatica in Diagnosis of Human Fasciolosis"

    Directory of Open Access Journals (Sweden)

    MB Rokni

    2004-07-01

    Full Text Available The liver fluke Fasciola hepatica causes fascioliasis, a liver disease in most part of the world and particularly in north of Iran. Diagnosis of the diseases is anchored in coprological manner but serological methods are preferable due to some obscurities. In this study, sera obtained from human patients infected with Fasciola hepatica were tested by the enzymelinked immunotrotransfer blot (EITB technique with the parasite s somatic and excretory-secretory (ES antigens in order to evaluate the diagnostic potential of the assay. The study included sera from 40 patients infected with F. hepatica, 20 infected with hydatidosis, 6 with toxocariasis, 10 with strongyloidiasis, 10 with amoebiasis, 5 with malaria and 30 normal controls. By this assay, most pf the serum samples from humans with fascioliasis recognized two antigenic polypeptides of 27 and 29 kDa using both antigens. The sensitivity, specificity, positive and negative predictive values for somatic antigen were 91.0%, 96.2%, 95.2% and 92.7% respectively, while these parameters as for ES antigen were 95.2%, 98.0%, 97.5% and 96.2%, correspondingly. Totally, two cases of reactions for the first antigen and one for the latter were verified. The study suggests that the 27 and 29 kDa bands for two antigens in EITB test could be considered for the immunodiagnosis of human fascioliasis.

  12. Development of rapid, sensitive and non-radioactive tissue-blot diagnostic method for the detection of citrus greening.

    Science.gov (United States)

    Nageswara-Rao, Madhugiri; Miyata, Shin-Ichi; Ghosh, Dilip; Irey, Mike; Garnsey, Stephen M; Gowda, Siddarame

    2013-01-01

    Citrus huanglongbing (HLB or citrus greening) is one of the most devastating diseases of citrus worldwide. The disease is caused by Gram-negative, phloem-limited α-proteobacterium, 'Candidatus Liberibacter asiaticus', vectored by the psyllid, Diaphorina citri Kuwayama. Citrus plants infected by the HLB bacterium may not show visible symptoms sometimes for years following infection and non-uniform distribution within the tree makes the detection of the pathogen very difficult. Efficient management of HLB disease requires rapid and sensitive detection early in the infection followed by eradication of the source of pathogen and the vector. The polymerase chain reaction (PCR) based method is most commonly employed for screening the infected/suspected HLB plants and psyllids. This is time consuming, cumbersome and not practical for screening large number of samples in the field. To overcome this, we developed a simple, sensitive, non-radioactive, tissue-blot diagnostic method for early detection and screening of HLB disease. Digoxigenin labeled molecular probes specific to 'Ca. L. asiaticus' nucleotide sequences have been developed and used for the detection of the pathogen of the HLB disease. The copy number of the target genes was also assessed using real-time PCR experiments and the optimized real-time PCR protocol allowed positive 'Ca. L. asiaticus' detection in citrus samples infected with 'Ca. L. asiaticus' bacterium.

  13. Loss of AND-34/BCAR3 expression in mice results in rupture of the adult lens

    Science.gov (United States)

    Near, Richard I.; Smith, Richard S.; Toselli, Paul A.; Freddo, Thomas F.; Bloom, Alexander B.; Vanden Borre, Pierre; Seldin, David C.

    2009-01-01

    Purpose AND-34/BCAR3 (Breast Cancer Anti-Estrogen Resistance 3) associates with the focal adhesion adaptor protein, p130CAS/BCAR1. Expression of AND-34 regulates epithelial cell growth pattern, motility, and growth factor dependence. We sought to establish the effects of the loss of AND-34 expression in a mammalian organism. Methods AND-34−/− mice were generated by homologous recombination. Histopathology, in situ hybridization, and western blotting were performed on murine tissues. Results Western analyses confirmed total loss of expression in AND-34−/− splenic lymphocytes. Mice lacking AND-34 are fertile and have normal longevity. While AND-34 is widely expressed in wild type mice, histologic analysis of multiple organs in AND-34−/− mice is unremarkable and analyses of lymphocyte development show no overt changes. A small percentage of AND-34−/− mice show distinctive small white eye lesions resulting from the migration of ruptured cortical lens tissue into the anterior chamber. Following initial vacuolization and liquefaction of the lens cortex first observed at postnatal day three, posterior lens rupture occurs in all AND-34−/− mice, beginning as early as three weeks and seen in all mice at three months. Western blot analysis and in situ hybridization confirmed the presence of AND-34 RNA and protein in lens epithelial cells, particularly at the lens equator. Prior data link AND-34 expression to the activation of Akt signaling. While Akt Ser 473 phosphorylation was readily detectable in AND-34+/+ lens epithelial cells, it was markedly reduced in the AND-34−/− lens epithelium. Basal levels of p130Cas phosphorylation were higher in AND-34+/+ than in AND-34−/− lens epithelium. Conclusions These results demonstrate the loss of AND-34 dysregulates focal adhesion complex signaling in lens epithelial cells and suggest that AND-34-mediated signaling is required for maintenance of the structural integrity of the adult ocular lens. PMID:19365570

  14. Whole genomic sequencing of RT98 mitochondria derived from Oryza rufipogon and northern blot analysis to uncover a cytoplasmic male sterility-associated gene.

    Science.gov (United States)

    Igarashi, Keisuke; Kazama, Tomohiko; Motomura, Keiji; Toriyama, Kinya

    2013-02-01

    Cytoplasmic male sterility (CMS) is a maternally inherited trait resulting in the failure to produce functional pollen and is often observed when an alien cytoplasm is transferred into a cultivated species. An RT98A CMS line and an RT98C fertility restorer line were obtained by successive backcrossing between Oryza rufipogon W1109 and Oryza sativa cultivar Taichung 65. To uncover the CMS-associated mitochondrial genes, we determined the complete sequence of the RT98-CMS mitochondrial genome using next-generation pyrosequencing, and searched new open reading frames (orfs) absent in a reported mitochondrial genome of O. sativa Nipponbare. Then, six candidates were selected for the CMS-associated genes based on the criteria in which they were chimeric in structure or encoded a peptide with transmembrane domains. One of the candidates, orf113, showed different transcript sizes between RT98A and RT98C on Northern blot analysis. The orf113 gene was shown to be co-transcribed with atp4 and cox3 encoding ATP synthase F0 subunit 4 and Cyt c oxidase subunit 3, respectively, and their transcripts were distinctly processed in the presence of a fertility restorer gene. Our results indicate that orf113 is a CMS-associated gene of RT98-CMS.

  15. Storage and stability of IgG and IgM monoclonal antibodies dried on filter paper and utility in Neisseria meningitidis serotyping by Dot-blot ELISA

    Directory of Open Access Journals (Sweden)

    Yto André Y

    2008-03-01

    Full Text Available Abstract Background A simple filter paper method was developed for, the transport and storage of monoclonal antibodies (Mabs at room temperature or -20°C after spotting on filter paper, for subsequent serotyping of outer membrane antigens of N.meningitidis by dot-blot ELISA. Methods Monoclonal antibodies (Mabs were spotted within a 0.5–1 cm diameter area of Whatman grade 903 paper, which were stored individually at room temperature or at -20°C. These MAbs were stored and analyzed after periods of one week, 4 weeks, 12 months, or 13 years in the case of frozen Mab aliquots, or after 4 weeks at -20°C or at room temperature (RT in the case of Mabs dried on filter paper strips. Assays were performed in parallel using dot-blot ELISA. In addition to the MAbs specific for serotyping class 1, 2 or 3, we used a larger number of Mabs for polysaccharides, lipooligosaccharides (LOS, class 5 and cross-reactive antigens for native outer membrane of N.meningitidis. The Mabs dried on filter paper were eluted with phosphate-buffered saline (PBS containing 0.2% gelatin. Results Mabs of the isotypes IgG and IgM dried on filter papers were not affected by duration of storage. The detection by serotyping Mabs was generally consistent for dried filter paper MAb samples stored frozen for over 1 year at -20°C, and although decreased reactive antibody titers were found after storage, this did not interfere with the specificity of the Mabs used after 13 years as dry spots on filter paper. Conclusion The use of filter paper is an inexpensive and convenient method for collecting, storing, and transporting Mab samples for serotyping studies. In addition, the samples occupy little space and can be readily transported without freezing. The efficiency of using immunoglobulin G (IgG or M (IgM eluted was found to be consistent with measurement of IgG or IgM titers in most corresponding, ascites Mabs stored frozen for over 1 year. The application of meningococcal typing

  16. Western Opera in China

    Institute of Scientific and Technical Information of China (English)

    1992-01-01

    “ALTHOUGH the road islong and leads far,I’llsearch hard for truth.″Ithink these lines by Qu Yuan(c.340-278 B.C.)are most appropriatein describing Western opera inChina.Opera originated from musicaldrama in Italy.From Dafne by Flo-rentine composer Jacopo Peri in1597,opera has a history of nearly400 years,if we do not count folk,church or court music and danceor song dramas in Europe.Afterspreading from Italy to Austria,France,Germany,Britain,northernEuropean countries and Russia,op-era has developed many national

  17. Utility of Schistosoma bovis Adult Worm Antigens for Diagnosis of Human Schistosomiasis by Enzyme-Linked Immunosorbent Assay and Electroimmunotransfer Blot Techniques

    Science.gov (United States)

    Pardo, J.; Carranza, C.; Turrientes, M. C.; Arellano, J. L. Pérez; Vélez, R. López; Ramajo, V.; Muro, A.

    2004-01-01

    Immunodiagnostic methods based on the detection of antibodies continue to be the most effective and practical methods for the diagnosis of imported schistosomiasis. Schistosoma bovis is a species whose final natural hosts are bovines, ovines, caprines, and small wild ruminants. Different studies have demonstrated the analogies existing between S. bovis and other Schistosoma species which affect humans. The objective of this work was to evaluate the utility of S. bovis adult worm antigens (AWA) for the diagnosis of imported human schistosomiasis by enzyme-linked immunosorbent assay (ELISA) and electroimmunotransfer blotting (EITB) techniques. By detecting eggs, the ELISA for S. bovis AWA was able to definitively detect imported cases with a sensitivity of 94%. The specificity of the ELISA for S. bovis AWA was 97%. There were no differences between the results of the S. bovis AWA ELISA for patients infected with Schistosoma mansoni and those infected with Schistosoma haematobium. The EITB technique showed bands of 85, 37, and 20 kDa, which are characteristic of infections with Schistosoma spp. Specific bands to indicate infection by different species of Schistosoma have not been detected. The combined use of the ELISA for S. bovis AWA and EITB increased the global sensitivity of the study to 97%. Our findings suggest that the ELISA for S. bovis AWA is a useful test for the immunodiagnosis of imported schistosomiasis and that EITB for detecting S. bovis AWA permits the confirmation of diagnosis when the ELISA for S. bovis AWA is positive. PMID:15539523

  18. Inverse PCR and Quantitative PCR as Alternative Methods to Southern Blotting Analysis to Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants.

    Science.gov (United States)

    Stefano, Biricolti; Patrizia, Bogani; Matteo, Cerboneschi; Massimo, Gori

    2016-06-01

    One of the major unanswered questions with respect to the commercial use of genetic transformation in woody plants is the stability of the transgene expression over several decades within the same individual. Gene expression is strongly affected by the copy number which has been integrated into the plant genome and by the local DNA features close to the integration sites. Because woody plants cannot be subjected to selfing or backcrossing to modify the transgenic allelic structure without affecting the valuable traits of the cultivar, molecular characterization of the transformation event is therefore crucial. After assessing the transgene copy number of a set of apple transgenic clones with Southern blotting, we describe two alternative methods: the first is based on inverse PCR (i-PCR) and the second on the quantitative PCR (q-PCR). The methods produced comparable results with the exception of the data regarding a high copy number clone, but while the q-PCR-based system is rapid and easily adaptable to high throughput systems, the i-PCR-based method can provide information regarding the transformation event and the characteristics of the sequences flanking the transgenic construct.

  19. Application of the Reverse Line Blot Assay for the Molecular Detection of Theileria and Babesia sp. in Sheep and Goat Blood Samples from Pakistan

    Directory of Open Access Journals (Sweden)

    A Rasul

    2013-06-01

    Full Text Available Background: The present study was designed to detect the presence of tick-borne parasites (Theileria and Babesia spp. in 196 blood samples collected from apparently healthy sheep and goats from two provinces, Punjab and Khyber Pukhtoon Khwa, in Pakistan.Methods: Reverse line blot (RLB assay was applied for the parasitic detection by the amplification of hypervariable V4 region of the 18S ribosomal RNA (rRNA gene. A membrane with covalently linked generic and species specific oligonucleotide probes was used for the hybridization of amplified PCR products.Results: Parasites were detected in 16% of the ruminant blood samples under study. Two Theileria species, T. lestoquardi and T. ovis, were identified in samples. 25, of the total 32, infected animals were from Khyber Pukhtoon Khwa.Conclusion: Sheep were more prone to tick borne haemoprotozans as 81% infected samples were sheep as compared to 19% goats (P > 0.001. Risk factor analysis revealed that male (P = 0.03, ani­mals infested by ticks (P = 0.03 and herd composed of sheep only (P = 0.001 were more infected by blood parasites.

  20. [Western diet and Alzheimer's disease].

    Science.gov (United States)

    Berrino, Franco

    2002-01-01

    Alzheimer Disease, characterised by a global impairment of cognitive functions, is more and more common in Western societies, both because of longer life expectancy and, probably, because of increasing incidence. Several hints suggest that this degenerative disease is linked to western diet, characterised by excessive dietary intake of sugar, refined carbohydrates (with high glycaemic index), and animal product (with high content of saturated fats), and decreased intake of unrefined seeds--cereals, legumes, and oleaginous seeds--and other vegetables (with high content of fibres, vitamins, polyphenols and other antioxidant substances, phytoestrogens) and, in several populations, of sea food (rich in n-3 fatty acids). It has been hypothesised, in fact, that AD, may be promoted by insulin resistance, decreased endothelial production of nitric oxide, free radical excess, inflammatory metabolites, homocysteine, and oestrogen deficiency. AD, therefore, could theoretically be prevented (or delayed) by relatively simple dietary measures aimed at increasing insulin sensitivity (trough reduction of refined sugars and saturated fats from meat and dairy products), the ratio between n-3 and n-6 fatty acids (e.g. from fish and respectively seed oils), antioxidant vitamins, folic acid, vitamin B6, phytoestrogens (vegetables, whole cereals, and legumes, including soy products), vitamin B12 (bivalve molluscs, liver), and Cr, K, Mg, and Si salts. This comprehensive improvement of diet would fit with all the mechanistic hypotheses cited above. Several studies, on the contrary, are presently exploring monofactorial preventive strategies with specific vitamin supplementation or hormonal drugs, without, however, appreciable results.

  1. Long-term implications of T-cell receptor gene rearrangement analysis by Southern blot in patients with cutaneous T-cell lymphoma.

    Science.gov (United States)

    Guitart, Joan; Camisa, Charles; Ehrlich, Michelle; Bergfeld, Wilma F

    2003-05-01

    T-cell clonality analysis by Southern blot (TSB) in skin biopsy specimens suggestive of mycosis fungoides may be helpful in confirming the diagnosis of a cutaneous lymphoma. However, there are no data available regarding the long-term prognostic implication of such results. We sought to determine the long-term prognostic significance of TSB results from skin biopsy specimens of patients with mycosis fungoides. We reviewed the records from the Cleveland Clinic Foundation and Northwestern University Medical Center for cases of biopsy-proven mycosis fungoides with results available for skin biopsy TSB from 1987 to 1990. The detection of clonality by TSB correlates with a higher TNM stage (median stage for positive TSB, IIb vs negative TSB, Ib; P <.05), but not with age at presentation (62 vs 59 years) or duration of disease before presentation (6.2 vs 5.9 years). Although the long-term survival was not significantly different between the 2 groups, there was a trend for patients with positive TSB to die earlier (5-year survival of 67% vs 87%). Disease progression did not correlate with TSB results. Higher clonality rates were noted among patients with biopsy specimens showing a denser lymphoid infiltrate and a higher grade of cytologic atypia. Detection of clonality with TSB requires a significant clonal burden. Although clonality can be detected in patients with patches and plaques (T1 and T2) most cases with positive results were obtained from patients with advanced disease (T3 and T4). In our experience, detection of clonality by TSB does not correlate with disease progression and does not carry long-term prognostic implications.

  2. Western Parenting: A Personal Perspective

    Institute of Scientific and Technical Information of China (English)

    Paul Ward

    2011-01-01

    Bonding: Western fathers are keen to take an equal role in parenting. They recognise their responsibility as a new dad, and are committed early to sharing every family task with the mother. It is common to see a proud western father walking outside, on their own, with their tiny baby. Western dad's know that this time is special, and the mother needs to relax and feel confident to trust the dad with the baby.

  3. Geological history of the Western north pacific.

    Science.gov (United States)

    Fischer, A G; Heezen, B C; Boyce, R E; Bury, D; Douglas, R G; Garrison, R E; Kling, S A; Krasheninnikov, V; Lisitzin, A P; Pimm, A C

    1970-06-05

    A considerable portion of the abyssal floor of the western North Pacific was already receiving pelagic sediment in late Jurassic time. Carbonate sediments were later replaced by abyssal clays as the basin deepened and bottom waters became more aggressive. The resulting facies boundary, which can be recognized on seismic profiles, is broadly transgressive; it ranges in age from mid-Cretaceous in the western Pacific to Oligocene in the central Pacific. Cherts are encountered at and below the major facies boundary and appear to have been formed by postdepositional processes.

  4. The Western Denmark Cardiac Computed Tomography Registry

    DEFF Research Database (Denmark)

    Nielsen, Lene Hüche; Nørgaard, Bjarne Linde; Tilsted, Hans Henrik;

    2015-01-01

    -CCTR, showed that coronary CT angiographies accounted for only 23% of all nonregistered cardiac CTs, indicating >90% completeness of coronary CT angiographies in the WDHR-CCTR. The completeness of individual variables varied substantially (range: 0%-100%), but was >85% for more than 70% of all variables. Using......BACKGROUND: As a subregistry to the Western Denmark Heart Registry (WDHR), the Western Denmark Cardiac Computed Tomography Registry (WDHR-CCTR) is a clinical database established in 2008 to monitor and improve the quality of cardiac computed tomography (CT) in Western Denmark. OBJECTIVE: We...... expected numbers; and 4) positive predictive values as well as negative predictive values of 19 main patient and procedure variables. RESULTS: By December 31, 2012, almost 22,000 cardiac CTs with up to 40 variables for each procedure have been registered. Of these, 87% were coronary CT angiography...

  5. Detection of mutation in embB gene of Mycobacterium tuberculosis from clinical isolates of tuberculous patients in China by means of reverse-dot blot hybridization

    Institute of Scientific and Technical Information of China (English)

    XUE QIONG WU; YANG LU; JIAN QIN LIANG; JUN XIAN ZHANG; GUANG YU ZHANG; CUI HUAN L(U); HONG MIN LI; BEI CHUAN DING

    2006-01-01

    The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol (EMB) resistance of the clinical isolates of tuberculous patients in China was investigated by reversedot blot hybridization (RDBH) in addition to evaluating the clinical value with application of PCR-RD-BH technique to detect EMB resistance. In the present study, the genotypes of the 258 bp fragments of embB genes from 196 clinical isolates of M. tuberculosis were analysed with RDBH and DNA sequencing. It was demonstrated that 60 out of 91 phenotypically EMB-resistant isolates (65.9%) showed 5 types of missense mutations at codon 306 of embB gene, resulting in the replacement of the Met residue of the wild type strain with Val, Ile or Leu residues. In these mutations, the GTP mutation (38/91,41.8% ) and the ATA mutation ( 16/91, 17.6% ) were the most encountered genotypes. The embB mutation at codon 306 could also be found in 69 isolates of phenotypically EMB-sensitive but resistant to other anti-tuberculous drugs, but no such gene mutation could be found in 36 strains of drug-sensitive isolates. Meanwhile, the concordance with the results of DNA sequencing for one wide-type probe and 5 probes for specific mutations was 100%. It was concluded that the EMB-resistance occurring in most M. tuberculosis is due to appearance of embB mutation at codon 306, and the PCR-RDBH assay was proved to be a rapid, simple and reliable method for the detection of gene mutations, which might be a good alternative for the drug-resistance screening.

  6. Evaluation of the Punch-it™ NA-Sample kit for detecting microbial DNA in blood culture bottles using PCR-reverse blot hybridization assay.

    Science.gov (United States)

    Kim, Jungho; Wang, Hye-Young; Kim, Seoyong; Park, Soon Deok; Yu, Kwangmin; Kim, Hyo Youl; Uh, Young; Lee, Hyeyoung

    2016-09-01

    DNA extraction efficiency affects the success of PCR-based method applications. The Punch-it™ NA-Sample kit for extracting DNA by using paper chromatography is technically easy to use and requires just two reagents and only 10min to complete. The Punch-it™ NA-Sample kit could be offered as a rapid, accurate, and convenient method for extracting bacterial and fungal DNA from blood culture bottles. We compared the efficiencies of the commercial kit (Punch-it™ NA-Sample kit) and an in-house conventional boiling method with Chelex-100 resin for DNA extraction from blood culture bottles. The efficiency of the two DNA extraction methods was assessed by PCR-reverse blot hybridization assay (PCR-REBA, REBA Sepsis-ID) for detecting Gram positive (GP) bacteria, Gram negative (GN) bacteria, and Candida species with 196 positive and 200 negative blood culture bottles. The detection limits of the two DNA extraction methods were 10(3)CFU/mL for GP bacteria, 10(3)CFU/mL for GN bacteria, and 10(4)CFU/mL for Candida. The sensitivity and specificity of the Punch-it™ NA-Sample kit by REBA Sepsis-ID were 95.4% (187/196) and 100% (200/200), respectively. The overall agreement of the two DNA extraction methods was 98.9% (392/396). Three of four samples showing discrepant results between the two extraction methods were more accurately matched up with the Punch-it™ NA-Sample kit based on conventional culture methods. The results indicated that the Punch-it™ NA-Sample kit extracted bacterial and fungal DNA in blood culture bottles and allowed extracted DNA to be used in molecular assay. Copyright © 2016. Published by Elsevier B.V.

  7. Stumpage market integration in western national forests

    Science.gov (United States)

    Jean M. Daniels

    2011-01-01

    This study presents results of statistical tests for stumpage market integration on 62 national forests in the Western United States. Quarterly stumpage prices from 1984 to 2007 obtained from cut and sold reports for USDA Forest Service Regions 1, 4, 5, and 6 (Northern, Intermountain, Pacific Southwest, and Pacific Northwest, respectively) were analyzed to establish...

  8. Efficacy of SISTA South Africa on sexual behavior, HIV stigma and relationship control among isiXhosa women in the Western Cape Province, South Africa: Results of a Randomized Controlled Trial

    Science.gov (United States)

    Wingood, Gina M.; Reddy, Priscilla; Lang, Delia L.; Saleh-Onoya, Dorina; Braxton, Nikia; Sifunda, Sibusiso; DiClemente, Ralph J

    2013-01-01

    Background The HIV epidemic has a devastating impact among South-African women. The current study evaluated the efficacy of SISTA South Africa, a culturally congruent HIV intervention for isiXhosa women in South Africa that was adapted from SISTA, an HIV intervention for African American women. Methods A randomized controlled trial recruited 342 isiXhosa women 18 – 35 years of age. Participants were randomized to the general health comparison or the SISTA South Africa intervention. Xhosa speaking peer health educators tailored the SISTA South Africa curriculum, while maintaining the core elements of the original SiSTA intervention. Participants completed assessments at baseline and 6 months follow-up. Results Relative to participants in the comparison, participants in the HIV intervention reduced the frequency of unprotected vaginal intercourse acts (adjusted mean difference = 1.06; p = .02), were more likely to report not desiring dry sex (AOR = .229; 95% CI = .10, .47; p = .0001) and, were more likely to perceive that their main sexual partner did not desire dry sex (AOR = .24; 95% CI = .11, .52; p = .0001). Additionally, women randomized to the intervention also reported an increase in HIV knowledge, greater relationship control and had more accepting attitudes towards HIV stigma. The HIV intervention did not reduce STI incidence. Conclusions This trial demonstrates that an HIV intervention which is adapted to enhance its gender and cultural relevance for rural isiXhosa women can reduce self-reported sexual risk behaviors and enhance mediators of HIV among this vulnerable population. PMID:23673889

  9. Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization.

    Science.gov (United States)

    Bekker, Cornelis P J; de Vos, Sander; Taoufik, Amar; Sparagano, Olivier A E; Jongejan, Frans

    2002-10-22

    The detection of Anaplasma and Ehrlichia species is usually based on species-specific PCR assays, since no assay is yet available which can detect and identify these species simultaneously. To this end, we developed a reverse line blot (RLB) assay for simultaneous detection and identification of Anaplasma and Ehrlichia species in domestic ruminants and ticks. In a PCR the hypervariable V1 region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for members of the genera Anaplasma and Ehrlichia [Int. J. Syst. Evol. Microbiol. 51 (2001) 2145]. Amplified PCR products from blood of domestic ruminants or Amblyomma variegatum tick samples were hybridized onto a membrane to which eight species-specific oligonucleotide probes and one Ehrlichia and Anaplasma catch-all oligonucleotide probe were covalently linked. No DNA was amplified from uninfected blood, nor from other hemoparasites such as Theileria annulata, or Babesia bigemina. The species-specific probes did not cross-react with DNA amplified from other species. E. ruminantium, A. ovis and another Ehrlichia were identified by RLB in blood samples collected from small ruminants in Mozambique. Finally, A. variegatum ticks were tested after feeding on E. ruminantium infected sheep. E. ruminantium could be detected in adult ticks even if feeding of nymphs was carried out 3.5 years post-infection. In conclusion, the developed species-specific oligonucleotide probes used in an RLB assay can simultaneously detect and identify several Ehrlichia and Anaplasma species. However, as no quantitative data for the detection limit are available yet, only positive results are interpretable at this stage.

  10. A comparison of DNA extraction protocols from blood spotted on FTA cards for the detection of tick-borne pathogens by Reverse Line Blot hybridization.

    Science.gov (United States)

    Hailemariam, Zerihun; Ahmed, Jabbar Sabir; Clausen, Peter-Henning; Nijhof, Ard Menzo

    2017-01-01

    An essential step in the molecular detection of tick-borne pathogens (TBPs) in blood is the extraction of DNA. When cooled storage of blood under field conditions prior to DNA extraction in a dedicated laboratory is not possible, the storage of blood on filter paper forms a promising alternative. We evaluated six DNA extraction methods from blood spotted on FTA Classic(®) cards (FTA cards), to determine the optimal protocol for the subsequent molecular detection of TBPs by PCR and the Reverse Line Blot hybridization assay (RLB). Ten-fold serial dilutions of bovine blood infected with Babesia bovis, Theileria mutans, Anaplasma marginale or Ehrlichia ruminantium were made by dilution with uninfected blood and spotted on FTA cards. Subsequently, DNA was extracted from FTA cards using six different DNA extraction protocols. DNA was also isolated from whole blood dilutions using a commercial kit. PCR/RLB results showed that washing of 3mm discs punched from FTA cards with FTA purification reagent followed by DNA extraction using Chelex(®) resin was the most sensitive procedure. The detection limit could be improved when more discs were used as starting material for the DNA extraction, whereby the use of sixteen 3mm discs proved to be most practical. The presented best practice method for the extraction of DNA from blood spotted on FTA cards will facilitate epidemiological studies on TBPs. It may be particularly useful for field studies where a cold chain is absent. Copyright © 2016 Elsevier GmbH. All rights reserved.

  11. Development of a Rapid Reverse Blot Hybridization Assay for Detection of Clinically Relevant Antibiotic Resistance Genes in Blood Cultures Testing Positive for Gram-Negative Bacteria.

    Science.gov (United States)

    Wang, Hye-Young; Yoo, Gilsung; Kim, Juwon; Uh, Young; Song, Wonkeun; Kim, Jong Bae; Lee, Hyeyoung

    2017-01-01

    Rapid and accurate identification of the causative pathogens of bloodstream infections is crucial for the prompt initiation of appropriate antimicrobial therapy to decrease the related morbidity and mortality rates. The aim of this study was to evaluate the performance of a newly developed PCR-reverse blot hybridization assay (REBA) for the rapid detection of Gram-negative bacteria (GNB) and their extended-spectrum β-lactamase (ESBL), AmpC β-lactamase, and carbapenemase resistance genes directly from the blood culture bottles. The REBA-EAC (ESBL, AmpC β-lactamase, carbapenemase) assay was performed on 327 isolates that were confirmed to have an ESBL producer phenotype, 200 positive blood culture (PBCs) specimens, and 200 negative blood culture specimens. The concordance rate between the results of REBA-EAC assay and ESBL phenotypic test was 94.2%. The sensitivity, specificity, positive predictive value, and negative predictive value of the REBA-EAC assay for GNB identification in blood culture specimens were 100% (95% CI 0.938-1.000, P < 0.001), 100% (95% CI 0.986-1.000, P < 0.001), 100% (95% CI 0.938-1.000, P < 0.001), and 100% (95% CI 0.986-1.000, P < 0.001), respectively. All 17 EAC-producing GNB isolates from the 73 PBCs were detected by the REBA-EAC assay. The REBA-EAC assay allowed easy differentiation between EAC and non-EAC genes in all isolates. Moreover, the REBA-EAC assay was a rapid and reliable method for identifying GNB and their β-lactamase resistance genes in PBCs. Thus, this assay may provide essential information for accelerating therapeutic decisions to achieve earlier appropriate antibiotic treatment during the acute phase of bloodstream infection.

  12. Accurate and Practical Identification of 20 Fusarium Species by Seven-Locus Sequence Analysis and Reverse Line Blot Hybridization, and an In Vitro Antifungal Susceptibility Study▿†

    Science.gov (United States)

    Wang, He; Xiao, Meng; Kong, Fanrong; Chen, Sharon; Dou, Hong-Tao; Sorrell, Tania; Li, Ruo-Yu; Xu, Ying-Chun

    2011-01-01

    Eleven reference and 25 clinical isolates of Fusarium were subject to multilocus DNA sequence analysis to determine the species and haplotypes of the fusarial isolates from Beijing and Shandong, China. Seven loci were analyzed: the translation elongation factor 1 alpha gene (EF-1α); the nuclear rRNA internal transcribed spacer (ITS), large subunit (LSU), and intergenic spacer (IGS) regions; the second largest subunit of the RNA polymerase gene (RPB2); the calmodulin gene (CAM); and the mitochondrial small subunit (mtSSU) rRNA gene. We also evaluated an IGS-targeted PCR/reverse line blot (RLB) assay for species/haplotype identification of Fusarium. Twenty Fusarium species and seven species complexes were identified. Of 25 clinical isolates (10 species), the Gibberella (Fusarium) fujikuroi species complex was the commonest (40%) and was followed by the Fusarium solani species complex (FSSC) (36%) and the F. incarnatum-F. equiseti species complex (12%). Six FSSC isolates were identified to the species level as FSSC-3+4, and three as FSSC-5. Twenty-nine IGS, 27 EF-1α, 26 RPB2, 24 CAM, 18 ITS, 19 LSU, and 18 mtSSU haplotypes were identified; 29 were unique, and haplotypes for 24 clinical strains were novel. By parsimony informative character analysis, the IGS locus was the most phylogenetically informative, and the rRNA gene regions were the least. Results by RLB were concordant with multilocus sequence analysis for all isolates. Amphotericin B was the most active drug against all species. Voriconazole MICs were high (>8 μg/ml) for 15 (42%) isolates, including FSSC. Analysis of larger numbers of isolates is required to determine the clinical utility of the seven-locus sequence analysis and RLB assay in species classification of fusaria. PMID:21389150

  13. Development of a Rapid Reverse Blot Hybridization Assay for Detection of Clinically Relevant Antibiotic Resistance Genes in Blood Cultures Testing Positive for Gram-Negative Bacteria

    Science.gov (United States)

    Wang, Hye-young; Yoo, Gilsung; Kim, Juwon; Uh, Young; Song, Wonkeun; Kim, Jong Bae; Lee, Hyeyoung

    2017-01-01

    Rapid and accurate identification of the causative pathogens of bloodstream infections is crucial for the prompt initiation of appropriate antimicrobial therapy to decrease the related morbidity and mortality rates. The aim of this study was to evaluate the performance of a newly developed PCR-reverse blot hybridization assay (REBA) for the rapid detection of Gram-negative bacteria (GNB) and their extended-spectrum β-lactamase (ESBL), AmpC β-lactamase, and carbapenemase resistance genes directly from the blood culture bottles. The REBA-EAC (ESBL, AmpC β-lactamase, carbapenemase) assay was performed on 327 isolates that were confirmed to have an ESBL producer phenotype, 200 positive blood culture (PBCs) specimens, and 200 negative blood culture specimens. The concordance rate between the results of REBA-EAC assay and ESBL phenotypic test was 94.2%. The sensitivity, specificity, positive predictive value, and negative predictive value of the REBA-EAC assay for GNB identification in blood culture specimens were 100% (95% CI 0.938–1.000, P < 0.001), 100% (95% CI 0.986–1.000, P < 0.001), 100% (95% CI 0.938–1.000, P < 0.001), and 100% (95% CI 0.986–1.000, P < 0.001), respectively. All 17 EAC-producing GNB isolates from the 73 PBCs were detected by the REBA-EAC assay. The REBA-EAC assay allowed easy differentiation between EAC and non-EAC genes in all isolates. Moreover, the REBA-EAC assay was a rapid and reliable method for identifying GNB and their β-lactamase resistance genes in PBCs. Thus, this assay may provide essential information for accelerating therapeutic decisions to achieve earlier appropriate antibiotic treatment during the acute phase of bloodstream infection. PMID:28232823

  14. Globalisation and western music historiography

    Directory of Open Access Journals (Sweden)

    Romanou Katy

    2015-01-01

    Full Text Available Globalisation of musicology and music history aims to fuse the divisions created during Western music’s acme, and is referred to as “post-European historical thinking”. Therefore, “post” and “pre” European historical thinking have much in common. One aspect of this process of fragmentation was that music history was separated from theory and that Western Music Histories succeeded General Music Histories (a development described in some detail in the article. Connecting global music history with “post-European” historical thinking is one among numerous indications of Western awareness that European culture has reached some sort of a terminal phase. Concurrently, countries that have been developing by following Western Europe as a prototype, are leading today some past phase of Western development, which, with the ideas of cultural relativism prevailing, are not considered inferior.

  15. Mathematics in middle schools in Western European countries

    NARCIS (Netherlands)

    Pelgrum, W.J.

    1988-01-01

    The International Association for the Evaluation of Educational Achievement has conducted a number of cross-national studies in which Western European countries participated. Results from the Second International Mathematics Study regarding the content and outcomes of this study in some Western Euro

  16. Evaluation of the seasonal and annual abortifacient risk of western juniper trees on Oregon rangelands: Abortion risk of western juniper trees

    Science.gov (United States)

    Western juniper trees can cause late term abortions in cattle, similar to ponderosa pine trees. Analyses of western juniper trees from 35 locations across the state of Oregon suggest that western juniper trees in all areas present an abortion risk in pregnant cattle. Results from this study demonstr...

  17. Soviet Policy Toward Western Europe Objectives, Instruments, Results.

    Science.gov (United States)

    1986-02-01

    grain should be del’vered "only if Russia was willing to pay a political price-the destruction of the Berlin Wall.ൔ’ In 1965, Fritz Erler , the floor...Ostpolitik, Cambridge University Press, New York, 1981, p. 123. 122Fritz Erler , "The Alliance and the Future of Germany," Foreign Affairs, Vol. 43, No. 3

  18. Western Disturbances: A review

    Science.gov (United States)

    Dimri, A. P.; Niyogi, D.; Barros, A. P.; Ridley, J.; Mohanty, U. C.; Yasunari, T.; Sikka, D. R.

    2015-06-01

    Cyclonic storms associated with the midlatitude Subtropical Westerly Jet (SWJ), referred to as Western Disturbances (WDs), play a critical role in the meteorology of the Indian subcontinent. WDs embedded in the southward propagating SWJ produce extreme precipitation over northern India and are further enhanced over the Himalayas due to orographic land-atmosphere interactions. During December, January, and February, WD snowfall is the dominant precipitation input to establish and sustain regional snowpack, replenishing regional water resources. Spring melt is the major source of runoff to northern Indian rivers and can be linked to important hydrologic processes from aquifer recharge to flashfloods. Understanding the dynamical structure, evolution-decay, and interaction of WDs with the Himalayas is therefore necessary to improve knowledge which has wide ranging socioeconomic implications beyond short-term disaster response including cold season agricultural activities, management of water resources, and development of vulnerability-adaptive measures. In addition, WD wintertime precipitation provides critical mass input to existing glaciers and modulates the albedo characteristics of the Himalayas and Tibetan Plateau, affecting large-scale circulation and the onset of the succeeding Indian Summer Monsoon. Assessing the impacts of climate variability and change on the Indian subcontinent requires fundamental understanding of the dynamics of WDs. In particular, projected changes in the structure of the SWJ will influence evolution-decay processes of the WDs and impact Himalayan regional water availability. This review synthesizes past research on WDs with a perspective to provide a comprehensive assessment of the state of knowledge to assist both researchers and policymakers, and context for future research.

  19. Seabird Colonies in Western Greenland

    DEFF Research Database (Denmark)

    Boertmann, D.; Mosbech, A.; Falk, K.;

    surveys of seabird colonies are needed, due to a lack of information or because the present information probably is outdated. The most immediate threats to the colonial seabirds in western Greenland during the breeding time is hunting and egging. Oil pollution is a minor threat to-day, but will increase...... if offshore areas with oil potential are explored and developed. Tab. 6 gives an overview of each species sensitivity to oil spills and the capacity to recover, as well as a comparison of the western Greenland population numbers to the North Atlantic population numbers. The most significant western Greenland...

  20. Results,Effects and Countermeasures of Rural Human Resource Development in Western Minority Areas:A Case Study of Three Prefecture-level States of Southern Xinjiang Uygur Autonomous Region%西部民族地区农村人力资源开发:成效、影响及对策--以新疆南疆三地州为例

    Institute of Scientific and Technical Information of China (English)

    潘明明; 龚新蜀; 李光明

    2016-01-01

    Rural human resource development is not only an important measure of sustainable precise poverty alleviation ,but also a critical route to rural comprehensive well-off society in western minority areas .Based on three prefecture-level states in southern Xinjiang Uygur autono-mous region ,this paper systematically analyzes the effectiveness ,influencing factors and counter-measures of rural human resource development in western minority areas .It draws the results as follows :the overall effectiveness of rural human resource development in western minority re-gions experiences a slowly growth trend ,and shifts from low level to medium level after 2009 ;the effectiveness in terms of rural labor migration and human resources quality improves signifi-cantly ,but the effectiveness in terms of human resources size and structure adjustment worsens constantly .Individual and family characteristics of farmers (family income ,the number of chil-dren and the consciousness ﹠ behavior of human resource development ) ,rural development con-ditions (government support ,basic education ,skills training ,cultural environment and opportu-nities for migrant workers ) and rural social security system (labor rights ,legal popularization and security order ) play an important role in the results of rural human resource development . Through guiding peasants to establish the consciousness of human resource development ,west-ern minority regions should place emphasis on soft and hard environment construction of rural human resource development and increasingly perfect institutional guarantee mechanism of hu-man resource development ,to improve the results of rural human resource development .%人力资源开发不仅是西部民族地区可持续精准扶贫的重要举措,也是西部民族地区农村全面建成小康社会的根本途径。文章以新疆南疆三地州为例,对西部民族地区农村人力资源开发成效、影响因素和实施对策展开系统研究。结

  1. Experiencias en Western Australia

    Directory of Open Access Journals (Sweden)

    M.A. Pérez Fernández

    2008-01-01

    Full Text Available Décadas de uso inadecuado de los recursos naturales en Australia han llevado a la extinción de numerosas especies autóctonas. Aprendiendo de sus propios errores, se han inicido recientemente diferentes proyectos de conservación en los que participan diversos agentes interesados. La Región de los Central Ranges, en el Desierto de Gibson, pertenece al pueblo aborigen Ngaanyatjarra. En los años 90 se llevó a cabo una campaña de recolección de organismos, patrocinada por el Museo de Western Australia y el Departamento de Conservación Ambiental (DEC, en la que participaron miembros de la comunidad Ngaanyatjarra, conocedores y cuidadores del territorio. El resultado científico se tradujo en la identificación de dos nuevas especies, así como numerosas nuevas citas de plantas y animales para el territorio. La minería es una de las actividades más impactantes en Australia, pero la concienciación social ha llevado a que las compañías desarrollen importantes campañas de protección de especies. El mulgara (Dasycercus cristicaula ocupaba zonas que hoy en día se dedican a la minería, y de las que prácticamente ha desaparecido. Un programa de investigación financiado por la empresa Resolute Resources y dirigido por el Departamento de Conservación y Manejo del Territorio (CALM ha permitido identificar poblaciones de este marsupial carnívoro y diseñar un programa de manejo cuyo objetivo es evitar actuaciones incompatibles con su actividad biológica. El resultado más relevante en ambas iniciativas ha sido la colaboración entre diferentes agentes implicados, con intercambio de conocimientos y experiencias. Especialmente importante ha sido la posibilidad de diseñar planes de manejo y actuación sobre el territorio, orientados a la preservación de valores naturales y culturales antiguos.

  2. Western Military Culture and Counterinsurgency:

    African Journals Online (AJOL)

    francois

    large-scale battles using massive force.4 According to well-known military ... a conflict, he could therefore take advantage of all elements of Western military .... development of industrial total warfare had led to a rigid attitude towards conflicts.

  3. Western Pacific Typhoon Aircraft Fixes

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Western Pacific typhoon aircraft reconnaissance data from the years 1946 - 1965 and 1978, excluding 1952, were transcribed from original documents, or copy of...

  4. Western Hemisphere Knowledge Partnerships

    Science.gov (United States)

    Malone, T. F.

    2001-05-01

    , and application of knowledge concerning the nature of -- and interaction among -- matter, living organisms, energy, information, and human behavior. This strategy calls for innovative partnerships among the physical, biological, health, and social sciences, engineering, and the humanities. New kinds of partnership must also be forged among academia, business and industry, governments, and nongovernmental organizations. Geophysicists can play an important role in these partnerships. A focus for these partnerships is to manage the individual economic productivity that drives both human development and global change. As world population approaches stability during the twenty-first century, individual economic productivity will be the critical link between the human and the natural systems on planet Earth. AGU is among a core group of individuals and institutions proposing Western Hemisphere Knowledge Partnerships (WHKP) to test the hypothesis that knowledge, broadly construed, is an important organizing principle in choosing a path into the future. The WHKP agenda includes: (1) life-long learning, (2) the health and resilience of natural ecosystems, (3) eco-efficiency in economic production and consumption, (4) extension of national income accounts, (5) environmentally benign sources of energy, (6) delivery of health care, (7) intellectual property rights, and (8) networks for action by local communities.Collaboratories and distance education technologies will be major tools. A panel of experts will explore this proposal.

  5. Western Australian food security project

    Directory of Open Access Journals (Sweden)

    Maycock Bruce

    2007-08-01

    Full Text Available Abstract Background The aim of the Western Australian (WA Food Security Project was to conduct a preliminary investigation into issues relating to food security in one region within the Perth metropolitan area in Western Australia. The first phase of the project involved a food audit in one lower income area that was typical of the region, to identify the range, variety and availability of foods in the region. Methods A comprehensive food audit survey was provided to all food outlet owners/operators in one lower socio-economic region within the City of Mandurah (n = 132 outlets. The purpose of the survey was to investigate the range, variety and availability of foods in the Mandurah region as well as examining specific in-store characteristics such as the types of clientele and in-store promotions offered. Surveys were competed for 99 outlets (response rate = 75%. Results The range of foods available were predominantly pre-prepared with more than half of the outlets pre-preparing the majority of their food. Sandwiches and rolls were the most popular items sold in the outlets surveyed (n = 51 outlets followed by pastries such as pies, sausage rolls and pasties (n = 33 outlets. Outlets considered their healthiest food options were sandwiches or rolls (n = 51 outlets, salads (n- = 50 outlets, fruit and vegetables (n = 40 outlets, seafood (n = 27 outlets, meats such as chicken (n = 26 outlets and hot foods such as curries, soups or quiches (n = 23 outlets. The majority of outlets surveyed considered pre-prepared food including sandwiches, rolls and salads, as healthy food options regardless of the content of the filling or dressings used. Few outlets (n = 28% offered a choice of bread type other than white or wholemeal. High fat pastries and dressings were popular client choices (n = 77% as were carbonated drinks (n = 88% and flavoured milks (n = 46%. Conclusion These findings clearly indicate the need for further investigation of the impact of

  6. Matrix-assisted laser desorption-ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures.

    Science.gov (United States)

    Lamer, S; Jungblut, P R

    2001-03-10

    In theory, peptide mass fingerprinting by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower microl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.

  7. PURIFICATION AND ANALYSIS OF CARBOHYDRATE CLUSTER FROM ERYTHROPOIETIN RECOMBINANT EXPRESSION RESULT ON THE LEAVEN SYSTEM OF Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Adi Santoso

    2011-04-01

    Full Text Available For clinical purposes, pure protein and identification of carbohydrate structure from erythropoietin recombinant are needed. Purification was done with His-Trap affinity chromatography method and continued with gel filtration chromatography column to get purer protein. The carbohydrate cluster from the resulting pure protein then can be recognized by using N- and O-glycosidase and can be compared to EPO recombinant from mammal cells. The result showed similarity on the declining trend of the protein molecule’s weight, which could be seen using the Western blot method. Pure oligosaccharide was hydrolyzed to produce various monosaccharide through incubation with HCl 4 N in 100 oC temperature for 6 hours and the result was applied on high intensity liquid chromatography incubator to learn the composition of its monosaccharide.

  8. Pottery ethnoarchaeology in Western Ethiopia

    Directory of Open Access Journals (Sweden)

    González Ruibal, Alfredo

    2005-12-01

    Full Text Available The results of three ethnoarchaeological field seasons carried out among the Berta, Gumuz, Mao and Kwama of western Ethiopia are presented here. Fieldwork focused on the gathering of general data on the material culture of Benishangul- Gumuz, and particularly on pottery and vernacular architecture. The data relating to production, distribution and consumption of pottery are addressed in this article. The peoples studied are organised on egalitarian lines and practise a slash-and-burn agriculture.

    Se presentan los resultados de tres campañas etnoarqueológicas llevadas a cabo entre los Berta, Gumuz, Mao y Kwama de Etiopía. El trabajo se centró en la recogida de datos generales sobre la cultura material de la región de Benishangul-Gumuz y en particular en la cerámica y la arquitectura vernácula. Aquí se tratan los datos relativos a la producción, distribución y consumo de cerámica. Los pueblos estudiados se organizan en comunidades igualitarias y practican una agricultura de roza y quema.

  9. 单抗免疫斑点法和组织印迹法检测侵染蝴蝶兰的建兰花叶病毒%Detection of CymMV in Orchids Tissue by Dot-ELISA and Tissue Blot-ELISA with Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    董晓辉; 孟春梅; 黎军英; 吴建祥; 荣松; 张超; 洪健

    2009-01-01

    Dot-ELISA and tissue blot-ELISA with monoclonal antibody to detect Cymbidium mosaic virus (CymMV) were established. 8000-dilution CymMV monoclonal antibody could be adopted to detect CymMV infecting leaf extract within a dilution limitation of 1:10240. CymMV could still be detected in the eighth print by tissue blot-ELISA . Similar results were shown between the fifth print by tissue blot-ELISA and 80-dilution of infected leaf extract by dot-ELISA. Comparison tests showed that the commonly used tissue blot-ELISA was less sensitive than dot-ELISA. It could illustrate that CymMV monoclonal antibody was more specific and sensitive than the polyclonal antibodies. The improved tissue blot-ELISA is more simple than dot-ELISA, and is suitable for fast detection with a large number of samples in the field.%应用抗建兰花叶病毒(CymMV)的单克隆抗体,建立了快速检测蝴蝶兰病样的免疫斑点法(Dot-ELISA)和组织印迹法(Tissue blot-ELISA).CymMV单抗稀释8000倍时,Dot-ELISA可检出病毒粗汁液的最大稀释度为1:10240;Tissue blot-ELISA中样品1次平切后1~5次印迹与Dot-ELISA样品1:80稀释结果相当,6~8次印迹与Dot-ELISA 1:320稀释结果相当,前8次印迹均可以得到满意的检测效果.Tissue blot-ELISA的灵敏度略低于Dot-ELISA,使用单抗的结果比多抗特异性强,操作更为简便,适合大量兰花样品的快速检测.

  10. Geochemistry of sediment cores of the western equatorial Indian Ocean

    Digital Repository Service at National Institute of Oceanography (India)

    Murty, P; Cronan, D.S.; Rao, Ch.M.; Paropkari, A.L.; Topgi, R.S.; Guptha, M.V.S.; Colley, N.

    Geochemical investigations including partition analysis have been carried out on nine sediment cores from the western equatorial Indian Ocean. The results show that a core from the Arabian Sea exhibits a greater terrigenous influence than cores from...

  11. Anti-Taenia solium metacestode IgG antibodies in serum samples from inhabitants of a central-western region of Brazil

    Directory of Open Access Journals (Sweden)

    Oliveira Heliana B. de

    2006-01-01

    Full Text Available A total of 354 serum samples from inhabitants who frequent the Clinical Laboratory in Catalão, Goiás, in the central-western region of Brazil, were collected from June to August, 2002. The samples were evaluated by indirect immunofluorescence antibody tests and an enzyme linked immunosorbent assay in order to detect anti-Taenia solium metacestode IgG antibodies. Reactive and inconclusive samples were tested by Western blotting (WB. Considering WB as a confirmation, the frequency of antibodies in the serum samples of the above population was 11.3% (CI 5.09 - 17.51. The immunodominant bands most frequently recognized in WB were 64-68 kDa (97.5% and 47-52 kDa (80%. The percentage of seropositivity to cysticercosis was significantly higher for individuals residing in areas without sewage systems (p < 0.0001. In conclusion, the results indicate a probable endemic situation of cysticercosis in this population. These results reinforce the urgent need for control and prevention measures to be taken by the local public health services.

  12. Relevance of Ara h1, Ara h2 and Ara h3 in peanut-allergic patients, as determined by immunoglobulin E Western blotting, basophil-histamine release and intracutaneous testing: Ara h2 is the most important peanut allergen

    NARCIS (Netherlands)

    Koppelman, S.J.; Wensing, M.; Ertmann, M.; Knulst, A.C.; Knol, E.F.

    2004-01-01

    Background: A number of allergenic proteins in peanut has been described and the relative importance of these allergens is yet to be determined. Objectives: We have investigated the relevance of previously identified peanut allergens in well-characterized peanut-allergic patients by in vitro, ex viv

  13. Relevance of Ara h1, Ara h2 and Ara h3 in peanut-allergic patients, as determined by immunoglobulin E Western blotting, basophil-histamine release and intracutaneous testing: Ara h2 is the most important peanut allergen

    NARCIS (Netherlands)

    Koppelman, S.J.; Wensing, M.; Ertmann, M.; Knulst, A.C.; Knol, E.F.

    2004-01-01

    Background: A number of allergenic proteins in peanut has been described and the relative importance of these allergens is yet to be determined. Objectives: We have investigated the relevance of previously identified peanut allergens in well-characterized peanut-allergic patients by in vitro, ex viv

  14. Study on the Relationship between Western Blot Assay and the Infection of HIV%蛋白免疫印迹法试验结果与HIV感染关系的探讨

    Institute of Scientific and Technical Information of China (English)

    胡秀杰; 包小兵; 宋艳文

    2005-01-01

    目的通过对蛋白免疫印迹法(WB)试验结果的观察,确定个体是否感染HIV以及HIV感染状况.方法对50份抗-HIV初筛阳性血清使用WB法进行确认实验.结果在被检测样品中,HIV-1型确认试验阳性45例,占90%;HIV-1型确认试验阳性,且提示HIV-2阳性感染1例,占2%;HIV-1型确认试验弱阳性(不确定)2例,占4%;HIV-1型确认试验阴性2例,占4%.结论WB法试验为确定个体有无HIV感染以及HIV感染状况提供依据.

  15. FINITE ELEMENT NUMERICAL SIMULATION OF LAND SUBSIDENCE AND GROUNDWATER EXPLOITATION BASED ON VISCO-ELASTIC PLASTIC BLOT'S CONSOLIDATION THEORY

    Institute of Scientific and Technical Information of China (English)

    LUO Zu-jiang; ZENG Feng

    2011-01-01

    The land subsidence due to groundwater exploitation has an obvious hysteretic nature with respect to the decrease of the under groundwater level,and the uneven settlement often causes ground fissures.To study these important features,a visco-elastic plastic constitutive relationship with consideration of the coupling of seepage and soil deformation is proposed,and a finite element model with variable coefficients based on the Biot's consolidation theory is built.With the groundwater exploitation and the land subsidence control in Cangzhou City,Hebei Province as an example,the variations of the under groundwater level and the development of the land subsidence due to the groundwater exploitation are simulated and ground fissures are predicted by the horizontaldisplacement calculation.The results show that the lag time between the land subsidence and the under groundwater level descent is about a month,and the simulated results of fissures agree well with the observed data.The model can well reveal the characterization of the interaction between the land subsidence and the groundwater exploitation.

  16. Western Canada SAGD drilling and completions performance

    Energy Technology Data Exchange (ETDEWEB)

    Turchin, S.; Tucker, R. [Ziff Energy Group (Canada)

    2011-07-01

    In the heavy oil industry, steam assisted gravity drainage (SAGD) is a thermal recovery method used to enhance oil recovery. In 2009, Ziff Energy carried out a study on SAGD drilling and completions performance in Western Canada. This paper presents the methodology used to assess drilling performances and the results obtained. This study was conducted on 159 SAGD well pairs and 1,833 delineation wells in the Western Canadian Sedimentary Basin from late 2004 to fall 2008. The drilling performance assessment was calculated from several aspects including well quality, drilling and completions cost performance and drilling time analysis. This study provided a detailed analysis of drilling and completions costs of SAGD which can help companies to improve their performance.

  17. mima. Western Emioni

    African Journals Online (AJOL)

    many other African countries (3, 4). Given such uncontrolled ... wide to curb the ever expanding population, ... Distributions (CBD), and social marketing ... contraceptive method mix in Ethiopia is said to bébroad. ... to identify levels of family planning awareness and use ... are diagnosed, prioritized and intervened. Results.

  18. Western Area Power Administration. Combined power system financial statements

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1998-02-26

    This report presents the results of the independent certified public accountants` audit of the Western Area Power Administration`s combined power system statements of assets, Federal investment and liabilities, and the related combined statements of revenues, expenses and accumulated net revenues, and cash flows. The auditors` report on Westerns internal control structure disclosed three new reportable conditions concerning the lack of: (1) a reconciliation of stores inventory from subsidiary ledgers to summary financial information, (2) communication of interest during construction and related adjustments to interest on Federal investment, and (3) a system to prevent and detect power billing errors. None of the conditions were considered to be material weaknesses. Western provided concurrence and corrective action plans. The auditors` report on Western`s compliance with laws and regulations also disclosed two new instances of noncompliance. Western failed to calculate nonreimbursable expenses in accordance with the Grand Canyon Protection Act and had an unexplained difference in gross Federal investment balances used to calculate interest on Federal investment. Western provided concurrence and corrective action plans for the instances.

  19. Studying Summer Season Drought in Western Russia

    Directory of Open Access Journals (Sweden)

    Anthony R. Lupo

    2014-01-01

    Full Text Available During the 2010 summer, a severe drought impacted Western Russia, including regions surrounding Moscow and Belgorod (about 700 km south of Moscow. The drought was accompanied by high temperatures. Moscow recorded 37.8°C (100°F for the first time in over 130 years of record keeping. The record heat, high humidity, dry weather, and smoke from forest fires caused increased human mortality rates in the Moscow region during the summer. The excessive heat and humidity in Western Russia were the result of atmospheric blocking from June through mid-August. The NCAR-NCEP reanalyses were used to examine blocking in the Eastern European and Western Russia sector during the spring and summer seasons from 1970 to 2012. We found that drier years were correlated with stronger and more persistent blocking during the spring and summer seasons. During these years, the Moscow region was drier in the summer and Belgorod during the spring seasons. In the Moscow region, the drier summers were correlated with transitions from El Niño to La Niña, but the opposite was true in the Belgorod region. Synoptic flow regimes were then analyzed and support the contention that dry years are associated with more blocking and El Niño transitions.

  20. Western Sport and Spiritualism

    Directory of Open Access Journals (Sweden)

    Kosiewicz Jerzy

    2014-06-01

    Full Text Available Sport activity of achievement-oriented (professional, Olympic, spectacular character is first of all exposition of rivalry and striving for variously understood sports success (resulting from measurable or discretionary criteria. It refers to winning a competition or taking another expected place as well as to other forms of satisfaction, such as financial gratification or social (political, ethnic, professional recognition. Spirituality is here neither an aim, nor an expected value - it constitutes rather an additional or redundant quality. A competitor focuses his/her attention first of all on the main aim assumed in planned or current rivalry. Emotional sensations which are experienced by athletes before, during or after competitions testify to mental and emotional stress which accompanies sports combat.

  1. ALIENS IN WESTERN STREAM ECOSYSTEMS

    Science.gov (United States)

    The USEPA's Environmental Monitoring and Assessment Program conducted a five year probability sample of permanent mapped streams in 12 western US states. The study design enables us to determine the extent of selected riparian invasive plants, alien aquatic vertebrates, and some ...

  2. The Shape of a Western

    DEFF Research Database (Denmark)

    Nielsen, Jakob Isak

    2007-01-01

    The article is written for an issue on the Western genre. By means of a comparative analysis of Winchester '73 (Anthony Mann, 1950) and The Man from Laramie (Anthony Mann, 1955) the article accounts for stylistic, narrative, generic and theoretical implications of the transition from Academy ratio...

  3. Western juniper in eastern Oregon.

    Science.gov (United States)

    Donald R. Gedney; David L. Azuma; Charles L. Bolsinger; Neil. McKay

    1999-01-01

    This report analyzes and summarizes a 1988 inventory of western juniper (Juniperus occidentalis Hook.) in eastern Oregon. This inventory, conducted by the Pacific Northwest Research Station of the USDA Forest Service, was intensified to meet increased need for more information about the juniper resource than was available in previous inventories. A...

  4. Remaking Education in Western Europe

    Science.gov (United States)

    Jones, Ken

    2005-01-01

    This article makes a contribution to discussion on the neo-liberal reshaping of education in Western Europe. It argues for a greater attentiveness on the part of education researchers to collective social actors such as trade unions and social movements. Making use of concepts from Gramsci and from Poulantzas, it suggests that such actors had a…

  5. Gendering Citizenship in Western Europe

    DEFF Research Database (Denmark)

    Siim, Birte; Lister, Ruth; Williams, Fiona

    The first part of the book clarifies the ways that the concept of citizenship has developed historically and is understood today in a range of Western European welfare states. It elaborates on the contempory framing of debates and struggles around citizenship. This provides a framework for thee p...

  6. Western Transitology and Chinese Reality

    DEFF Research Database (Denmark)

    Brødsgaard, Kjeld Erik

    It is the object of considerable debate in Western scholarship whether an authoritarian political order dominated by a strong communist party can continue to exist in China given the many challenges stemming from internal reform and the impact of globalization. Will China eventually turn democrat...

  7. On some Western European Aphids

    NARCIS (Netherlands)

    Hille Ris Lambers, D.

    1939-01-01

    1. The genus Atheroides Haliday Of this genus 3 Western European species are known : A. serrulatus Hal. (syn. festucae Mordv.?), A. hirtellus Hal. (syn. A. junci Laing) and A. brevicornis Laing. a) A. serrulatus Hal. is quite common. It lives on Poa annua often, but prefers species of grass with nar

  8. ALIENS IN WESTERN STREAM ECOSYSTEMS

    Science.gov (United States)

    The USEPA's Environmental Monitoring and Assessment Program conducted a five year probability sample of permanent mapped streams in 12 western US states. The study design enables us to determine the extent of selected riparian invasive plants, alien aquatic vertebrates, and some ...

  9. Shell Trumpets from Western Mexico

    Directory of Open Access Journals (Sweden)

    Robert Novella

    1991-11-01

    Full Text Available Marine shells have been used as musical instruments in almost all parts of the world (Izikowitz 1935, including Mesoamerica, where large univalves, also called conch shells in the literature, had a utilitarian function as trumpets. Their use is well documented in most cultural areas of Mesoamerica, as in Western Mexico, through their various occurrences in archaeological contexts and museums collections.

  10. Polymerase chain reaction and Southern blot-based analysis of the C9orf72 hexanucleotide repeat in different motor neuron diseases.

    Science.gov (United States)

    Hübers, Annemarie; Marroquin, Nicolai; Schmoll, Birgit; Vielhaber, Stefan; Just, Marlies; Mayer, Benjamin; Högel, Josef; Dorst, Johannes; Mertens, Thomas; Just, Walter; Aulitzky, Anna; Wais, Verena; Ludolph, Albert C; Kubisch, Christian; Weishaupt, Jochen H; Volk, Alexander E

    2014-05-01

    The GGGGCC-hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. This study determined the frequency of C9orf72 repeat expansions in different motor neuron diseases (amyotrophic lateral sclerosis (ALS), motor