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Sample records for addition western blotting

  1. Western blots.

    PubMed

    Freeman, Lita A

    2013-01-01

    Western analysis of apolipoproteins, lipoproteins, and proteins involved in lipoprotein metabolism can be challenging due to their size, hydrophobic nature, and, in some cases, low abundance. Here we describe a Western blotting method that has been used successfully for many proteins involved in lipoprotein metabolism, as well as intact LDL or HDL particles. Proteins or lipoprotein particles separated by gel electrophoresis are transferred to a PVDF membrane in a Hoefer TE22 transfer tank with Tris-Glycine-SDS-Methanol transfer buffer. The membrane is blocked with 3 % BSA/5 % milk to prevent nonspecific binding of antibody to the membrane and is then incubated with primary antibody that binds specifically to the protein of interest. After washing away unbound primary antibody, the membrane is then incubated with an HRP-labeled secondary antibody that binds primary antibody. After washing away unbound secondary antibody, the membrane is then incubated with a substrate for HRP, generating a chemiluminescent signal at the location of the protein of interest. The protein is visualized by exposing the membrane to an autoradiography film or an imaging device. Information on the use of several human antibodies, including apoA-I, A-II, apoB, apoC-II, apoC-III, apoD, apoL1, apoM, PON1, SAA, ABCA1, nitrotyrosine, and LCAT, is provided. This method can be used for Western blotting of virtually any protein as well as native lipoprotein particles. PMID:23912997

  2. The western blot

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. Single-cell western blotting

    PubMed Central

    Hughes, Alex J.; Spelke, Dawn P.; Xu, Zhuchen; Kang, Chi-Chih; Schaffer, David V.; Herr, Amy E.

    2014-01-01

    To measure cell-to-cell variation in protein-mediated functions — a hallmark of biological processes — we developed an approach to conduct ~103 concurrent single-cell western blots (scWesterns) in ~4 hours. A microscope slide supporting a 30 µm-thick photoactive polyacrylamide gel enables western blotting comprised of: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins, and antibody probing. We apply this scWestern to monitor single rat neural stem cell differentiation and responses to mitogen stimulation. The scWestern quantifies target proteins even with off-target antibody binding, multiplexes to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supports analyses of low starting cell numbers (~200) when integrated with fluorescence activated cell sorting. The scWestern thus overcomes limitations in single-cell protein analysis (i.e., antibody fidelity, sensitivity, and starting cell number) and constitutes a versatile tool for the study of complex cell populations at single-cell resolution. PMID:24880876

  4. Multiplexed Western Blotting Using Microchip Electrophoresis.

    PubMed

    Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

    2016-07-01

    Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps. PMID:27270033

  5. Single cell-resolution western blotting.

    PubMed

    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. PMID:27466711

  6. Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)

    PubMed Central

    Gilda, Jennifer E.; Ghosh, Rajeshwary; Cheah, Jenice X.; West, Toni M.; Bodine, Sue C.; Gomes, Aldrin V.

    2015-01-01

    Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis. PMID:26287535

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

    PubMed

    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.

  8. Improved semiquantitative Western blot technique with increased quantification range.

    PubMed

    Heidebrecht, F; Heidebrecht, A; Schulz, I; Behrens, S-E; Bader, A

    2009-06-30

    With the development of new interdisciplinary fields such as systems biology, the quantitative analysis of protein expression in biological samples gains more and more importance. Although the most common method for this is ELISA, Western blot also has advantages: The separation of proteins by size allows the evaluation of only specifically bound protein. This work examines the Western blot signal chain, determines some of the parameters relevant for quantitative analysis and proposes a mathematical model of the reaction kinetics. Using this model, a semiquantitative Western blot method for simultaneous quantification of different proteins using a hyperbolic calibration curve was developed. A program was written for the purpose of hyperbolic regression that allows quick determination of the calibration curve coefficients. This program can be used also for approximation of calibration curves in other applications such as ELISA, BCA or Bradford assays. PMID:19351538

  9. Western Blot of Stained Proteins from Dried Polyacrylamide Gels

    NASA Technical Reports Server (NTRS)

    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.

  10. Western blotting is useful in the salivary diagnosis of Helicobacter pylori infection

    PubMed Central

    Ballam, L; Mendall, M; Asante, M; Morris, J; Strachan, D; Whincup, P; Cook, D

    2000-01-01

    Background—The salivary diagnosis of Helicobacter pylori infection offers attractive possibilities for the epidemiological study of infection in children. Salivary enzyme linked immunosorbent assay (ELISA) is less reliable then serum ELISA, owing to variable transudation of immunoglobulin. In addition, children are more difficult to study because of lower specific serum antibody concentrations to H pylori. The performance of salivary western blotting in comparison with serum western blotting and serum ELISA was investigated in school children. Subjects and methods—Paired serum and saliva specimens were obtained from 665 school children aged 9–11 in 10 British towns. All saliva and serum specimens were first analysed by ELISA; subsequently, western blotting of both specimens was performed on 31 and 34 specimens, respectively, to establish the criteria for positivity for western blotting. The remaining 121 specimens were then tested blindly and saliva was compared with the serum. Results—The sensitivity and specificity of salivary ELISA in the 665 specimens was 32 of 50 (64%) and 530 of 691 (87%), respectively, when compared with serum ELISA. The western blotting validation was performed on 28 subjects with positive serum and positive salivary ELISA, 28 saliva positives with negative serum, 16 saliva negatives with positive serum, and 50 doubly negative subjects. Compared with serum western blots, the sensitivity and specificity of salivary western blots was 38 of 47 (81%) and 68 of 75 (91%), respectively. Using serum ELISA as the gold standard, the sensitivity and specificity were 32 of 44 (73%) and 72 of 78 (92%), respectively, the specificity being significantly higher than salivary ELISA (p < 0.001). Conclusion—Salivary western blotting for IgG is useful in the diagnosis of H pylori infection and is superior to ELISA. It also permits the identification of pathogenic strains. Key Words: Helicobacter pylori • western blotting • enzyme linked

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

    PubMed

    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.

  12. A high-affinity reversible protein stain for Western blots.

    PubMed

    Antharavally, Babu S; Carter, Brad; Bell, Peter A; Krishna Mallia, A

    2004-06-15

    We describe a reversible staining technique, using MemCode, a reversible protein stain by which proteins can be visualized on nitrocellulose and polyvinylidine fluoride (PVDF) membranes without being permanently fixed to the membrane itself. This allows subsequent immunoblot analysis of the proteins to be performed. The procedure is applicable only to protein blots on nitrocellulose and PVDF membranes. MemCode is a reversible protein stain composed of copper as a part of an organic complex that interacts noncovalently with proteins. MemCode shows rapid protein staining, taking 30s to 1 min for completion. The method is simple and utilizes convenient application conditions that are compatible with the matrix materials and the protein. The stain is more sensitive than any previously described dye-based universal protein staining system. The turquoise-blue-stained protein bands do not fade with time and are easy to photograph compared to those stained with Ponceau S. Absorbance in the blue region of the spectrum offers good properties for photo documentation and avoids interference from common biological chromophores. The stain on the protein is easily reversible in 2 min for nitrocellulose membrane and in 10 min for PVDF membrane with MemCode stain eraser. The stain is compatible with general Western blot detection systems, and membrane treatment with MemCode stain does not interfere with conventional chemiluminescent or chromogenic detection using horseradish peroxide and alkaline phosphatase substrates. The stain is also compatible with N-terminal sequence analysis of proteins.

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

    PubMed Central

    Ericson, Brad L.; Carlson, Darby J.

    2016-01-01

    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. PMID:27298753

  14. Western blotting by thin-film direct coating.

    PubMed

    Yen, Yi-Kuang; Jiang, Yi-Wei; Chang, Shih-Chung; Wang, An-Bang

    2014-05-20

    A novel thin-film direct coating (TDC) technique was developed to markedly reduce the amount of antibody required for Western blotting (WB). Automatic application of the technique for a few seconds easily and homogeneously coats the specific primary antibody on the polyvinylidene fluoride (PVDF) membrane. While conventional WB requires 0.4 μg of the primary antibody, the proposed technique only uses 4 × 10(-2) μg, which can be reduced further to 4 × 10(-5) μg by reducing the coater width. Moreover, the proposed process reduces antibody probing times from 60 to 10 min. The quantification capability of TDC WB showed high linearity within a 4-log2 dynamic range for detecting target antigen glutathione-S-transferase. Furthermore, TDC WB can specifically detect the extrinsic glutathione-S-transferase added in the Escherichia coli or 293T cell lysate with better staining sensitivity than conventional WB. TDC WB can also clearly probe the intrinsic β-actin, α-tubulin, and glyceraldehyde 3-phosphate dehydrogenase, which are usually used as control proteins in biological experiments. This novel technique has been shown to not only have valuable potential for increasing WB efficiency but also for providing significant material savings for future biomedical applications. PMID:24773468

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

    PubMed

    Heda, Ghanshyam D; Omotola, Oluwabukola B; Heda, Rajiv P; Avery, Jamie

    2016-09-01

    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

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

    PubMed Central

    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

  17. Multicenter evaluation of the novel ABN Western blot (immunoblot) system in comparison with an enzyme-linked immunosorbent assay and a different Western blot.

    PubMed

    Weber, B; Hess, G; Enzensberger, R; Harms, F; Evans, C J; Hamann, A; Doerr, H W

    1992-03-01

    A new, modular Western blot (immunoblot) system for human immunodeficiency virus (HIV) antibodies (ABN WesPage; Wellcome) was compared with enzyme immunoassays (Wellcome, Behringwerke, and Abbott) and with a U.S. Food and Drug Administration (FDA)-licensed Western blot (DuPont) in a multicenter study. A total of 649 serum samples from HIV patients at different stages of the disease, as well as from high-risk patients, from patients with conditions unrelated to AIDS, and from healthy blood donors, were used in the evaluation along with nine seroconversion panels. For evaluation of Western blot reactivity, both Centers for Disease Control (CDC) and FDA criteria were used. With the DuPont Western blot as the reference assay, the overall sensitivity and specificity of the ABN WesPage were 100 and 99.1%, respectively, when indeterminate results were not taken into account and when both tests were interpreted in accordance with CDC criteria. The DuPont Western blot detected significantly more antibodies to pol and gag gene products than the ABN WesPage. The ABN WesPage showed a higher positive rate of detection of viral envelope band gp160. When both Western blots were interpreted in accordance with CDC criteria, the ABN WesPage and the DuPont Western blot yielded 9.3 and 10.4% indeterminate results, respectively. When the DuPont Western blot was interpreted in accordance with the manufacturer's instructions (FDA criteria), 25.7% of the samples tested were regarded as indeterminate. The choice of interpretation criteria is of paramount importance for the evaluation of HIV Western blot patterns.

  18. Better management of Western blotting results using professional photo management software.

    PubMed

    Iorio-Morin, Christian; Germain, Pascale; Parent, Jean-Luc

    2013-04-01

    Western blotting is a proven technique essential to a significant proportion of molecular biology projects. However, as results accumulate over the years, managing data can become daunting. Recognizing that the needs of a scientist working with Western blotting results are conceptually the same as those of a professional photographer managing a summer's worth of wedding photos, we report here a new workflow for managing Western blotting results using professional photo management software. The workflow involves (i) scanning all film-based results; (ii) importing the scans into the software; (iii) processing the scans; (iv) tagging the files with metadata, and (v) creating appropriate "smart-albums." Advantages of this system include space savings (both on our hard drives and on our desks), safer archival, quicker access, and easier sharing of the results. In addition, metadata-based workflows improve cross-experiment discovery and enable questions like "show me all blots labelled with antibody X" or "show me all experiments featuring protein Y". As project size and breadth increase, workflows delegating results management to the computer will become more and more important so that scientists can keep focussing on science. PMID:23404762

  19. A streamlined Western blot exercise: An efficient and greener approach in the laboratory classroom.

    PubMed

    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 from staining and destaining, whereas with western blotting it is the times required for antibody incubations and the numerous wash steps. This laboratory exercise incorporates 2,2,2-trichloroethanol (TCE) into the SDS-PAGE gel allowing for visualization of migrated proteins in a matter of minutes, saving both the time and chemical waste associated with traditional Coomassie staining. Additionally, TCE staining does not affect protein transfer eliminating the requirement for duplicated gels for total protein and western analyses. Protein transfer can be confirmed immediately without the use of Ponceau S staining. Lastly, this western blot procedure has been further shortened by using an HRP-conjugated primary antibody, which eliminates the secondary antibody incubation and washes, and uses a colorimetric detection to allow for visualization by students without the need for specialized equipment. PMID:26153352

  20. Preparation of Cell Lysate from Mouse Oocytes for Western Blotting Analysis.

    PubMed

    Marangos, Petros

    2016-01-01

    Western Blotting has been used extensively for the identification of the protein factors that regulate mammalian oocyte meiosis. However, the limitations in collecting sufficient numbers of oocytes can hinder the efficiency of the technique. Here we provide a detailed protocol for the accurate preparation of mouse oocyte samples for Western Blotting analysis. PMID:27557583

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

    ERIC Educational Resources Information Center

    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…

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

    PubMed

    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 (p<0.05). Western 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 (p<0.1). Luminex xMAP combined with Western blot improves the diagnostic sensitivity of HIV infection at an early stage, and reduces the chances of missed diagnosis.

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

    PubMed

    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.

  4. [Analysis of new criteria for increasing the specificity of commercial Western blots].

    PubMed

    Gershy-Damet, G M; Koffi, K; Soro, B; Sanon, S; Traore, M; Assoa, A; N'Goran, K; N'Gom, A

    1992-01-01

    We examined the frequency of serum cross-reactivity on Western blot for HIV1 and HIV2. 661 patients with tuberculosis in Abidjan, and 4,899 asymptomatic persons for HIV1 and HIV2 infections were tested. All specimens positive on ELISA for HIV1 or HIV2 were further characterized by synthetic peptide based tests. Confirmed positive samples were tested by HIV1 and HIV2 specific Western blot criteres utilisis. Dual serologic reactivity on synthetic peptide tests was significantly more frequent in HIV positive patients with tuberculosis than asymptomatic subjects. Positive HIV1 Western blots were seen in 61%-86% of specimens positive for HIV2 only on synthetic peptide tests. [Cross-reactivity, to HIV2 Western blots by HIV1 positive specimens was significantly more frequent in patients with tuberculosis than in asymptomatic subjects.] Using recently recommended criteria for HIV1 and HIV2 Western blot interpretation (presence of 2 env bands) reduced the overall proportion of HIV1 positive specimens having a positive HIV2 Western blot from 39% to 14% and HIV2 positive specimens having a positive HIV1 Western blot from 31% to 8%.

  5. SDS-Polyacrylamide Electrophoresis and Western Blotting Applied to the Study of Asthma.

    PubMed

    García-Solaesa, Virginia; Abad, Sara Ciria

    2016-01-01

    Western blotting is used to analyze proteins after being separated by electrophoresis and subsequently electro-transferred to a membrane. Once immobilized, a specific protein can be identified through its reaction with a labeled antibody or antigen. It is a methodology commonly used in biomedical research such as asthma studies, to assess the pathways of inflammatory mediators involved in the disease.Here, we describe an example of western blotting to determine the factors involved in asthma. In this chapter, the methodology of western blotting is reviewed, paying attention on potential problems and giving interesting recommendations. PMID:27300534

  6. Microfluidic integration of Western blotting is enabled by electrotransfer-assisted sodium dodecyl sulfate dilution.

    PubMed

    Hou, Chenlu; Herr, Amy E

    2013-01-01

    We integrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent antibody probing in a single, monolithic microdevice to realize microfluidic Western blotting. A hurdle to successful on-chip Western blotting lies in restoring antibody recognition of previously sized (denatured, reduced) proteins. To surmount this hurdle, we locally dilute free SDS from SDS-protein complexes using differential electromigration of the species during electrotransfer between SDS-PAGE and blotting regions of a microchamber. Local dilution of SDS minimizes re-association of SDS with proteins offering means to restore antibody binding affinity to proteins after SDS-PAGE. To achieve automated, programmable operation in a single instrument, we utilize a 1 × 2 mm(2) glass microchamber photopatterned with spatially distinct, contiguous polyacrylamide regions for SDS-PAGE, electrotransfer, and antibody blotting. Optimization of both the SDS-PAGE and electrotransfer conditions yields transfer distances of <1 mm (40 s). The Western blot is completed in 180 s, with fully automated assay operation using programmable voltage control. After SDS-PAGE and electrotransfer, we observe ~80% capture of protein band mass on the blotting region for a model protein, C-reactive protein. This novel microfluidic Western blot approach introduces fine transport control for in-transit protein handling to form the basis for an automated, rapid alternative to conventional slab-gel Western blotting. PMID:23042290

  7. Detection and quantification of protein-protein interactions by far-western blotting.

    PubMed

    Jadwin, Joshua A; Mayer, Bruce J; Machida, Kazuya

    2015-01-01

    Far-western blotting is a convenient method to characterize protein-protein interactions, in which protein samples of interest are immobilized on a membrane and then probed with a non-antibody protein. In contrast to western blotting, which uses specific antibodies to detect target proteins, far-western blotting detects proteins on the basis of the presence or absence of binding sites for the protein probe. When specific modular protein binding domains are used as probes, this approach allows characterization of protein-protein interactions involved in biological processes such as signal transduction, including interactions regulated by posttranslational modification. We here describe a rapid and simple protocol for far-western blotting, in which GST-tagged Src homology 2 (SH2) domains are used to probe cellular proteins in a phosphorylation-dependent manner. We also present a batch quantification method that allows for the direct comparison of probe binding patterns.

  8. Using a large area CMOS APS for direct chemiluminescence detection in Western blotting electrophoresis

    NASA Astrophysics Data System (ADS)

    Esposito, Michela; Newcombe, Jane; Anaxagoras, Thalis; Allinson, Nigel M.; Wells, Kevin

    2012-03-01

    Western blotting electrophoretic sequencing is an analytical technique widely used in Functional Proteomics to detect, recognize and quantify specific labelled proteins in biological samples. A commonly used label for western blotting is Enhanced ChemiLuminescence (ECL) reagents based on fluorescent light emission of Luminol at 425nm. Film emulsion is the conventional detection medium, but is characterized by non-linear response and limited dynamic range. Several western blotting digital imaging systems have being developed, mainly based on the use of cooled Charge Coupled Devices (CCDs) and single avalanche diodes that address these issues. Even so these systems present key drawbacks, such as a low frame rate and require operation at low temperature. Direct optical detection using Complementary Metal Oxide Semiconductor (CMOS) Active Pixel Sensors (APS)could represent a suitable digital alternative for this application. In this paper the authors demonstrate the viability of direct chemiluminescent light detection in western blotting electrophoresis using a CMOS APS at room temperature. Furthermore, in recent years, improvements in fabrication techniques have made available reliable processes for very large imagers, which can be now scaled up to wafer size, allowing direct contact imaging of full size western blotting samples. We propose using a novel wafer scale APS (12.8 cm×13.2 cm), with an array architecture using two different pixel geometries that can deliver an inherently low noise and high dynamic range image at the same time representing a dramatic improvement with respect to the current western blotting imaging systems.

  9. Charge standards as reference points in two-dimensional western blot patterns

    SciTech Connect

    Zhang, J.S.; Tollaksen, S.L.; Giometti, C.S.

    1986-01-01

    High-resolution two-dimensional electrophoresis can be used to separate hundreds or even thousands of proteins from complex mixtures. Specific proteins in 2DE patterns can be identified by using Western Blotting. Correlation between a single stained spot on a nitrocellulose blot and the same spot in a stained 2DE gel pattern can be difficult since no reference points are available on the blot. Inclusion of a protein or proteins to serve as reference or standard points in both the blotted pattern and the stained gel pattern would make pattern recognition much more rapid and reliable. In this paper the use of a commercial charge standard (carbamylated creatine phosphokinase mix) was used to produce reference points in both gel patterns and corresponding nitrocellulose blots to simplify recognition of individual protein spots in complicated gel patterns. 8 refs., 2 figs.

  10. Investigation of immunofluorescence cross-reactions against Trichinella spiralis by western blot (immunoblot) analysis.

    PubMed Central

    Robert, F; Weil, B; Kassis, N; Dupouy-Camet, J

    1996-01-01

    Immunofluorescence cross-reactions in Trichinella spiralis serodiagnosis are sometimes difficult to identify. We compared the results of an indirect immunofluorescence assay and the profiles obtained by Western blot (immunoblot) analysis for three groups of patients: 10 T. spiralis-infected patients, 10 patients with autoimmune diseases, and 7 patients with parasitic diseases other than trichinellosis. The degree of immunofluorescence cross-reaction was variable. Western blotting allowed us to differentiate Trichinella infection from other parasitic diseases. In 3 of 10 serum samples from patients with autoimmune diseases, bands which had the same sizes as Trichinella bands were observed, and they could correspond to shared epitopes such as heat shock proteins. PMID:8877138

  11. A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

    PubMed Central

    Eaton, Samantha L.; Hurtado, Maica Llavero; Oldknow, Karla J.; Graham, Laura C.; Marchant, Thomas W.; Gillingwater, Thomas H.; Farquharson, Colin; Wishart, Thomas M.

    2014-01-01

    The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term “western blot” suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many “optimized” western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies. PMID:25490604

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

    ERIC Educational Resources Information Center

    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…

  13. Post-obstruction rat sperm autoantigens identified by two-dimensional gel electrophoresis and western blotting.

    PubMed

    Flickinger, C J; Bush, L A; Williams, M V; Naaby-Hansen, S; Howards, S S; Herr, J C

    1999-05-01

    Although antisperm autoantibody responses to obstruction of the male reproductive system have been documented, information on the nature of the cognate sperm autoantigens has been limited. In the present study, the patterns of sperm autoantigens recognized by sera from rats after obstruction of the vas deferens or epididymis were studied by high resolution two-dimensional (2-D) gel electrophoresis and western blotting. Comparisons of patterns of autoantigens stained on 2-D western blots of sera from prepubertal vasectomy, prepubertal epididymal ligation and adult vasectomy groups revealed both similarities and differences. Sera from sham-operated animals showed no detectable reaction or much lighter staining of a small number of spots. Visualization of sperm autoantigens on 2-D western blots supported the hypothesis that there is a relatively small set of sperm proteins that can be regarded as dominant post-obstruction sperm autoantigens because they are recognized by multiple post-obstruction sera. The 2-D analysis revealed previously undetected distinctions in the autoantigens recognized after adult and prepubertal vasectomy, as well as variations with the site of obstruction. These differences in the response may be due in part to changes in antigens of spermatozoa in different parts of the tract and at different ages, as well as variations in exposure of sperm cell proteins to the immune system resulting from the sites of spermatic granulomas. Preparative 2-D gels and western blotting with post-obstruction sera are now being used to identify specific sperm autoantigens by microsequencing of selected proteins. PMID:10392780

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

    EPA Science Inventory

    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...

  15. Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis.

    PubMed

    Liu, Yu-Ting; Toyokuni, Shinya

    2009-01-01

    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.

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

    ERIC Educational Resources Information Center

    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…

  17. Nanogold Immunodetection Detection Systems for the Identification of Autoantigens by Western Blotting.

    PubMed

    Moore, Jacen S; Scofield, R Hal

    2015-01-01

    Nanogold-conjugated immunodetection systems are now widely and commercially available for use in a number of research applications including electron microscopy, light microscopy, and western blotting. Nanogold clusters are small, uniform in size, and stable, unlike gold colloids historically used in protein detection. Covalent linkage of nanogold particles to secondary antibodies prevents dissociation of the gold particles during the staining process, making protein detection reliable, antigen specific, and highly sensitive. Nanogold labeling is extremely versatile and can be used in conjunction with other staining methodologies including Alexa Fluor immunofluorescence detection to perform coupled staining procedures. Silver enhancement increases the limits of sensitivity for nanogold staining, thus improving detection signals for antigens with reduced expression levels. Herein, we describe the use of nanogold-silver detection as an immunodetection system for standard western blotting of autoantigens.

  18. Western Blotting using the Invitrogen NuPage Novex Bis Tris minigels.

    PubMed

    Penna, Aubin; Cahalan, Michael

    2007-01-01

    Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence. Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer). The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method. PMID:18989435

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

    PubMed Central

    2011-01-01

    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. PMID:21247488

  20. Single-cell Western blotting after whole-cell imaging to assess cancer chemotherapeutic response.

    PubMed

    Kang, Chi-Chih; Lin, Jung-Ming G; Xu, Zhuchen; Kumar, Sanjay; Herr, Amy E

    2014-10-21

    Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors.

  1. Restricted specificity of the autoantibody response in Goodpasture's syndrome demonstrated by two-dimensional western blotting.

    PubMed

    Derry, C J; Dunn, M J; Rees, A J; Pusey, C D

    1991-12-01

    The autoantigen in Goodpasture's syndrome is known to be contained within the non-collagenous (NC1) domain of type IV collagen. We have examined the specificity of autoantibodies to glomerular basement membrane (GBM) using the technique of 2-D electrophoresis followed by Western blotting. Protein stains of 2-D gels of collagenase-digested human GBM revealed extensive charge and size heterogeneity. Major components were of mol. wt 24-30 kD and 43-56 kD, corresponding to monomeric and dimeric subunits of NCl. Western blotting of 2-D gels with IgG from patients with anti-GBM disease demonstrated that the most antigenic components migrated as cationic 28-kD monomers (pI 10) and similarly charged dimers, although other components were recognized less strongly. The mobility of the strongly antigenic polypeptides was different to that of the known alpha 1 and alpha 2 chains of type IV collagen. Autoantibodies from all 20 patients studied showed the same pattern of reactivity, regardless of their clinical features (in particular, the presence or absence of pulmonary haemorrhage) or HLA type. A monoclonal antibody (P1) to human GBM bound in a similar pattern, particularly recognizing the cationic components. 2-D gels of affinity-purified GBM from a P1 column showed enrichment of the 28-kD monomers, which were recognized by human autoantibodies on Western blotting. These results demonstrate that the autoimmune response in Goodpasture's syndrome is of restricted specificity, and support the suggestion that the major autoantigenic determinant is present on the novel alpha 3 chain of type IV collagen. PMID:1747953

  2. Detection of IgA and IgM antibodies to HIV-1 in neonates by radioimmune western blotting.

    PubMed Central

    Portincasa, P.; Conti, G.; Re, M. C.; Chezzi, C.

    1992-01-01

    OBJECTIVE--To detect infection with HIV-1 by IgA and IgM response at birth in children born to HIV-1 seropositive mothers. DESIGN--Western blotting and radioimmune western blotting on stored sera from infected and uninfected babies born to HIV-1 seropositive mothers. Sera were pretreated to remove IgG. SETTING--Parma and Bologna, Italy. SUBJECTS--12 infected and five uninfected babies born to HIV-1 seropositive mothers and three babies born to seronegative mothers. MAIN OUTCOME MEASURES--Effectiveness of western blotting and radioimmune western blotting in detecting antibodies to HIV-1 gene products. RESULTS--With conventional western blotting we found IgA class antibodies to HIV-1 proteins in serum from three out of 12 infected children; in two of these three the serum was collected at age 3 months (positive controls). Radioimmune western blotting detected both IgA and IgM antibodies in serum from all infected children tested, whereas all serum from uninfected children born to seropositive and seronegative mothers showed no such antibodies. CONCLUSION--Although the technique should be tested on more patients, radioimmune western blotting seems to be a valuable tool for serological diagnosis of congenital HIV-1 infection at birth in neonates born to seropositive mothers. Images FIG 1 FIG 2 PMID:1628052

  3. Evaluating dot and Western blots using image analysis and pixel quantification of electronic images.

    PubMed

    Vierck, J L; Bryne, K M; Dodson, M V

    2000-01-01

    Inexpensive computer imaging technology was used to assess levels of insulin-like growth factor-I (IGF-I) on dot blots (DB) and alpha-Actinin on Western blots (WB). In the first procedure, known IGF-I samples were dotted on nitrocellulose membranes using a vacuum manifold. After the DB were developed and dried, the images were digitized using an HP Deskscan II flat bed scanner, exported into Image-Pro Plus and analyzed by taking the combined mean of 45 degrees and 135 degrees sample lines drawn through each dot. Dot blots corresponding to a linear concentration range from 10 to 300 ng IGF-I were assessed by this method. In the second procedure, WB were scanned with a ScanJet 3c flat bed scanner and their backgrounds were clarified using Image-Pro Plus. A second image analysis program, Alpha Imager 2000, was then used to define the boundaries of protein bands, assess pixel number and density, and to obtain final numerical data for quantifying alpha-Actinin on the WB. Collectively, the results of these two studies suggest that specific proteins may be evaluated by using relatively inexpensive image analysis software systems via pixel quantification of electronic images. PMID:11549944

  4. Validation of Endothelin B Receptor Antibodies Reveals Two Distinct Receptor-related Bands on Western Blot

    PubMed Central

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

    2014-01-01

    Antibodies are important tools for the study of protein expression, but are often used without full validation. In this study, we use Western blots to characterize antibodies targeted to the N- (NT) or C-termini (CT) and the second (IL2) or third intracellular (IL3) loops 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 50kD 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 kD band, but failed to detect endogenous ETB in rat brain. Bands detected by the CT-targeted or IL3-targeted antibodies were found to be unrelated to ETB. Our findings show that functional ETB receptors can be detected at 50 kD or 37 kD on Western blot, with drastic differences in antibody affinity for these bands. The 37 kD band likely reflects ETB receptor processing, which appears to be dependent on cell type and/or culture condition. PMID:25232999

  5. Analyzing the Homeostasis of Signaling Proteins by a Combination of Western Blot and Fluorescence Correlation Spectroscopy

    PubMed Central

    Chung, Yi-Da; Sinzinger, Michael D.; Bovee-Geurts, Petra; Krause, Marina; Dinkla, Sip; Joosten, Irma; Koopman, Werner J.; Adjobo-Hermans, Merel J.W.; Brock, Roland

    2011-01-01

    The determination of intracellular protein concentrations is a prerequisite for understanding protein interaction networks in systems biology. Today, protein quantification is based either on mass spectrometry, which requires large cell numbers and sophisticated measurement protocols, or on quantitative Western blotting, which requires the expression and purification of a recombinant protein as a reference. Here, we present a method that uses a transiently expressed fluorescent fusion protein of the protein-of-interest as an easily accessible reference in small volumes of crude cell lysates. The concentration of the fusion protein is determined by fluorescence correlation spectroscopy, and this concentration is used to calibrate the intensity of bands on a Western blot. We applied this method to address cellular protein homeostasis by determining the concentrations of the plasma membrane-located transmembrane scaffolding protein LAT and soluble signaling proteins in naïve T cells and transformed T-cell lymphoma (Jurkat) cells (with the latter having nine times the volume of the former). Strikingly, the protein numbers of soluble proteins scaled with the cell volume, whereas that of the transmembrane protein LAT scaled with the membrane surface. This leads to significantly different stoichiometries of signaling proteins in transformed and naïve cells in concentration ranges that may translate directly into differences in complex formation. PMID:22261070

  6. Evaluating cytoplasmic and nuclear levels of inflammatory cytokines in cancer cells by western blotting.

    PubMed

    Gatla, Himavanth R; Singha, Bipradeb; Persaud, Valerie; Vancurova, Ivana

    2014-01-01

    Increased expression and cellular release of inflammatory cytokines, interleukin-8 (IL-8; CXCL8), and high mobility group box-1 (HMGB1) are associated with increased cell proliferation, angiogenesis, and metastasis during cancer progression. In prostate and ovarian cancer cells, increased levels of IL-8 and HMGB1 correlate with poor prognosis. We have recently shown that proteasome inhibition by bortezomib (BZ) specifically increases IL-8 release from metastatic prostate and ovarian cancer cells. In this chapter, we describe a protocol to analyze the cytoplasmic and nuclear levels of IL-8 and HMGB1 in prostate and ovarian cancer cells by western blotting. IL-8 is localized in the cytoplasm in both cell types, and its protein levels are significantly increased by BZ. In contrast, HMGB1 is localized in the nucleus, and BZ increases its nuclear levels only in ovarian cancer cells. The protocol includes isolation of cytoplasmic and nuclear extracts, followed by SDS electrophoresis and western blotting, and can be easily modified to analyze the cytoplasmic and nuclear cytokine levels in other cell types. PMID:24908314

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

    PubMed

    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.

  8. Evaluating cytoplasmic and nuclear levels of inflammatory cytokines in cancer cells by western blotting.

    PubMed

    Gatla, Himavanth R; Singha, Bipradeb; Persaud, Valerie; Vancurova, Ivana

    2014-01-01

    Increased expression and cellular release of inflammatory cytokines, interleukin-8 (IL-8; CXCL8), and high mobility group box-1 (HMGB1) are associated with increased cell proliferation, angiogenesis, and metastasis during cancer progression. In prostate and ovarian cancer cells, increased levels of IL-8 and HMGB1 correlate with poor prognosis. We have recently shown that proteasome inhibition by bortezomib (BZ) specifically increases IL-8 release from metastatic prostate and ovarian cancer cells. In this chapter, we describe a protocol to analyze the cytoplasmic and nuclear levels of IL-8 and HMGB1 in prostate and ovarian cancer cells by western blotting. IL-8 is localized in the cytoplasm in both cell types, and its protein levels are significantly increased by BZ. In contrast, HMGB1 is localized in the nucleus, and BZ increases its nuclear levels only in ovarian cancer cells. The protocol includes isolation of cytoplasmic and nuclear extracts, followed by SDS electrophoresis and western blotting, and can be easily modified to analyze the cytoplasmic and nuclear cytokine levels in other cell types.

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

    PubMed

    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

  10. V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

    PubMed Central

    Posch, Anton; Kohn, Jonathan; Oh, Kenneth; Hammond, Matt; Liu, Ning

    2013-01-01

    The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a "black box" because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as β-actin, β-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative and reliable. PMID:24429481

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

    PubMed

    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. PMID:27180586

  12. Detection of Hypoderma actaeon infestation in Cervus elaphus with ELISA and western blotting.

    PubMed

    Domínguez, Julia; Panadero, Rosario; de la Fuente-López, Concepción

    2010-07-01

    We used enzyme-linked immunosorbent assay (ELISA) and western blotting (WB) to evaluate the reactivity of first-stage larval extracts of Hypoderma lineatum with antibodies in sera from 76 red deer from an endemic area for Hypoderma actaeon. Antibodies in sera from deer infested with H. actaeon recognized hypodermin C from H. lineatum in both ELISA and WB assays. ELISA values were correlated with the epidemiology of the fly infestation in the area where the deer sera were collected. There was no clear relationship between anti-hypodermin C antibody levels and the age of the animals or the number of Hypoderma grubs found at necropsy in the hides of the deer. Our results suggest that first-instar antigens of H. lineatum can be used to diagnose natural infestations by H. actaeon by indicating the presence of first-stage larvae, which can help to accurately describe H. actaeon epidemiology. PMID:20688700

  13. Detection of Hypoderma actaeon infestation in Cervus elaphus with ELISA and western blotting.

    PubMed

    Domínguez, Julia; Panadero, Rosario; de la Fuente-López, Concepción

    2010-07-01

    We used enzyme-linked immunosorbent assay (ELISA) and western blotting (WB) to evaluate the reactivity of first-stage larval extracts of Hypoderma lineatum with antibodies in sera from 76 red deer from an endemic area for Hypoderma actaeon. Antibodies in sera from deer infested with H. actaeon recognized hypodermin C from H. lineatum in both ELISA and WB assays. ELISA values were correlated with the epidemiology of the fly infestation in the area where the deer sera were collected. There was no clear relationship between anti-hypodermin C antibody levels and the age of the animals or the number of Hypoderma grubs found at necropsy in the hides of the deer. Our results suggest that first-instar antigens of H. lineatum can be used to diagnose natural infestations by H. actaeon by indicating the presence of first-stage larvae, which can help to accurately describe H. actaeon epidemiology.

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

    PubMed Central

    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

  15. Detection of yellowhead virus and Chinese baculovirus in penaeid shrimp by the Western blot technique.

    PubMed

    Nadala, E C; Tapay, L M; Cao, S; Loh, P C

    1997-12-01

    The continuing threat posed by viral diseases in cultured shrimp calls for the development of detection technologies for monitoring the animals, especially broodstock. Two of the most highly pathogenic viruses of penaeid shrimp are the yellow-head virus (YHV) and Chinese baculovirus (CBV, also called white spot baculovirus). A Western blot (WB) protocol capable of detecting YHV and CBV in the hemolymph of infected shrimp was developed. The use of the hemolymph as material for virus detection allowed for sample collection without sacrificing the animals. This protocol was highly specific, rapid, and sensitive enough to detect the presence of the viruses before the appearance of overt symptoms. It was also useful for demonstrating the growth of both viruses in primary shrimp lymphoid cell cultures.

  16. 2-D Western blotting for evaluation of antibodies developed for detection of host cell protein.

    PubMed

    Berkelman, Tom; Harbers, Adriana; Bandhakavi, Sricharan

    2015-01-01

    Recombinant proteins generated for therapeutic use must be substantially free of residual host cell protein (HCP). The presence of host cell protein (HCP) is usually assayed by ELISA using a polyclonal antibody mixture raised against a population of proteins derived from the host cell background. This antibody should recognize as high a proportion as possible of the potential HCPs in a given sample. A recommended method for evaluating the assay involves two-dimensional electrophoretic separation followed by Western blotting.We present here a method using commercial anti-HCP antibody and samples derived from Chinese Hamster Ovary (CHO) cells. The 2-D electrophoresis procedure gives highly reproducible spot patterns and entire procedure can be completed in less than 2 days. Software analysis enables the straightforward generation of percent coverage values for the antibody when used to probe HCP-containing samples. PMID:25820736

  17. IgG-subclass-specific antibody reactivity to respiratory syncytial virus polypeptides investigated by western blot.

    PubMed

    Jankowski, M; Hornsleth, A; Olsen, P G

    1990-01-01

    IgG1 and IgG3 subclass-specific antibody reactivity (ABR) in serum samples obtained from infants and children in relation to acute lower respiratory disease caused by respiratory syncytial virus (RSV) infection was investigated by Western blot. IgG1 ABR was directed against the nucleocapsid polypeptides VPN and VPP as well as against the glycoproteins GP48 (F1) and GP90. IgG3 ABR was directed only against VPN and VPP. In infants, a low IgG1 reactivity against glycoproteins was observed. When serum samples obtained in the early convalescent phase were tested, ABR against GP48 and GP90 as well as against VPP differed with respect to RSV subtypes A and B. IgG1 ABR increased in the late convalescent phase, while IgG3 ABR decreased during this phase when serum samples from primary infections were tested.

  18. Study of anti-dengue NS1 antibody by western blot.

    PubMed

    Kuno, G; Vorndam, A V; Gubler, D J; Gómez, I

    1990-10-01

    The presence of anti-nonstructural protein (NS1) antibody in natural dengue infections has been suspected to be associated with development of dengue haemorrhagic fever (DHF). The Western blot technique was used to study the dynamics of the immune response to NS1 and to determine the frequency of anti-NS1 antibody among confirmed dengue patients in Indonesia, where DHF is common, and in Puerto Rico, where DHF is less frequently observed. Anti-NS1 antibody was rarely found in those with primary infections in either group. The antibody occurred at a significantly higher frequency in acute-phase serum samples from secondary infections in Indonesia than in those from Puerto Rico. No difference was observed, however, in Indonesian patients with secondary infection who had dengue fever or DHF.

  19. 2-D Western blotting for evaluation of antibodies developed for detection of host cell protein.

    PubMed

    Berkelman, Tom; Harbers, Adriana; Bandhakavi, Sricharan

    2015-01-01

    Recombinant proteins generated for therapeutic use must be substantially free of residual host cell protein (HCP). The presence of host cell protein (HCP) is usually assayed by ELISA using a polyclonal antibody mixture raised against a population of proteins derived from the host cell background. This antibody should recognize as high a proportion as possible of the potential HCPs in a given sample. A recommended method for evaluating the assay involves two-dimensional electrophoretic separation followed by Western blotting.We present here a method using commercial anti-HCP antibody and samples derived from Chinese Hamster Ovary (CHO) cells. The 2-D electrophoresis procedure gives highly reproducible spot patterns and entire procedure can be completed in less than 2 days. Software analysis enables the straightforward generation of percent coverage values for the antibody when used to probe HCP-containing samples.

  20. Strategies for laboratory HIV testing: an examination of alternative approaches not requiring Western blot.

    PubMed Central

    Sato, P. A.; Maskill, W. J.; Tamashiro, H.; Heymann, D. L.

    1994-01-01

    Advances in laboratory tests for antibodies to human immunodeficiency virus (HIV) have permitted the development of alternative HIV testing strategies that do not require use of the Western blot approach. Three strategies are proposed. In strategy I, sera are tested for HIV antibody using an enzyme-linked immunosorbent assay (ELISA)/rapid/simple (ERS) test; in strategy II, sera reactive in an initial ERS test are retested using a second ERS test; strategy III involves retesting with a third ERS test all sera reactive in two previous ERS tests. Where the objective is identification of asymptomatic HIV-infected individuals, strategy III is proposed where HIV prevalences in the study population are < or = 10%, and strategy II at prevalences > 10%. Strategy II is recommended where the diagnosis of HIV-related disease requires HIV testing. For serosurveillance, strategy II is recommended if the prevalence is < or = 10%, and strategy I if the prevalences are > 10%. Use of strategy I is recommended for transfusion and transplantation safety, at any prevalence. Lower-cost laboratory HIV testing will permit such testing to become more widely available. PMID:8131248

  1. Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

    NASA Astrophysics Data System (ADS)

    Parra-Belky, Karlett

    2002-11-01

    A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

  2. Protein quantification by chemiluminescent Western blotting: elimination of the antibody factor by dilution series and calibration curve.

    PubMed

    Charette, Steve J; Lambert, Herman; Nadeau, Philippe J; Landry, Jacques

    2010-02-28

    Quantification of chemiluminescent signals from a Western blot is routinely used to determine the increase or the decrease in protein expression or modification in cell or tissue extracts. However, although scientists readily agree that such a procedure is not quantitative, it is nonetheless used quantitatively in most publications without appropriate controls that would increase the accuracy of the measurement. Here we reexamined this aspect and found that the primary antibody itself influences the relation between the Western blot signal and the protein amount on the membrane. This relation is non-linear and varies from one antibody to another. In that context, we strongly encourage researchers to use dilution series and calibration curve when quantifying protein by Western blot using chemiluminescent signal.

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

    PubMed

    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%. PMID:22965727

  4. Stain-Free total protein staining is a superior loading control to β-actin for Western blots.

    PubMed

    Gilda, Jennifer E; Gomes, Aldrin V

    2013-09-15

    Semi-quantification of proteins using Western blots typically involves normalization against housekeeping genes such as β-actin. More recently, Ponceau S and Coomassie blue staining have both been shown to be suitable alternatives to housekeeping genes as loading controls. Stain-Free total protein staining offers the advantage of no staining or destaining steps. Evaluation of the use of Stain-Free staining as an alternative to β-actin or the protein stain Ponceau S showed that Stain-Free staining was superior to β-actin and as good as or better than Ponceau S staining as a loading control for Western blots. PMID:23747530

  5. Evaluation of the Aspergillus Western Blot IgG Kit for Diagnosis of Chronic Aspergillosis

    PubMed Central

    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.

    2014-01-01

    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. PMID:25392351

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

    PubMed

    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.

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

    PubMed Central

    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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. Interpretation criteria for standardized Western blots for three European species of Borrelia burgdorferi sensu lato.

    PubMed

    Hauser, U; Lehnert, G; Lobentanzer, R; Wilske, B

    1997-06-01

    Western blots (WBs; immunoblots) are a widely used tool for the serodiagnosis of Lyme borreliosis, but so far, no defined criteria for performance, analysis, and interpretation have been established in Europe. For the current study WBs were produced with strains PKa2 (Borrelia burgdorferi sensu stricto), PKo (Borrelia afzelii), and PBi (Borrelia garinii). To improve resolution we used gels of 17 cm in length. In a first step, 13 immunodominant proteins were identified with monoclonal antibodies. Then, the apparent molecular masses of all visually distinguishable bands were determined densitometrically. Approximately 40 bands of between 14 and 100 kDa were differentiated for each strain. From a study with 330 serum samples (from 189 patients with Lyme borreliosis and 141 controls), all observed bands were documented. To establish criteria for a positive WB result, the discriminating ability of a series of band combinations (interpretation rules) were evaluated separately for each strain (for immunoglobulin G [IgG] WB, > 40 combinations; for IgM WB, > 15 combinations). The following interpretation criteria resulting in specificities of greater than 96% were recommended: for IgG WB, at least one band of p83/100, p58, p56, OspC, p21, and p17a for PKa2; at least two bands of p83/100, p58, p43, p39, p30, OspC, p21, p17, and p14 for PKo; and at least one band of p83/100, p39, OspC, p21, and p17b for PBi; for IgM WB, at least one band of p39, OspC, and p17a or a strong p41 band for PKa2; at least one band of p39, OspC, and p17 or a strong p41 band for PKo; and at least one band of p39 and OspC or a strong p41 band for PBi. The overall sensitivity was the highest for PKo WB, followed by PBi and PKa2 WB, in decreasing order. Standardization of WB assays is necessary for comparison of results from different laboratories.

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

    PubMed

    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.

  11. Western Blot Assay Using Recombinant p26 Antigen for Detection of Equine Infectious Anemia Virus-Specific Antibodies▿

    PubMed Central

    Alvarez, I.; Gutierrez, G.; Ostlund, E.; Barrandeguy, M.; Trono, K.

    2007-01-01

    We analyzed the performance of a single-band Western blot (WB) test using recombinant p26 (rp26) capsid protein of equine infectious anemia virus. According to the results obtained, the rp26 WB test is a reliable confirmatory diagnostic tool to be used as a complementary test after an enzyme-linked immunosorbent assay or agar gel immunodiffusion test yielding doubtful results. PMID:17959820

  12. Western blot analysis to illustrate relative control levels of the lac and ara promoters in Escherichia coli.

    PubMed

    Nielsen, Brent L; Willis, Van C; Lin, Chin-Yo

    2007-03-01

    The lactose operon and its control is a fundamental transcriptional regulatory concept presented in introductory and many advanced molecular biology courses. Much is known about the positive and negative control mechanisms that govern levels of expression of this operon. One basic principle that is taught about the lac operon is that it is "leaky," meaning that the transcriptional control of the operon is not 100% efficient and that in wild-type cells, transcription from the promoter is never completely "off," but there is always some basal transcription. In contrast, the arabinose operon is often used as an example of a tightly controlled operon, and transcription from the ara promoter is very low in the absence of inducer. The relative levels of control of these two operons can be illustrated using Western blots of proteins expressed in the presence and absence of the appropriate inducers and antibodies against the gene products. Different times of growth and the addition of inducer can also be examined. The results are very dramatic and help to reinforce the principles of promoter control. PMID:21591073

  13. High resolution autoantibody detection by optimized protein G-horseradish peroxidase immunostaining in a western blotting assay.

    PubMed

    Hu, G R; Harrop, P; Warlow, R S; Gacis, M L; Walls, R S

    1997-03-28

    We report a procedure for optimisation of Western blotting using protein G-horseradish peroxidase (protein G-HRP) which avoids the false positive reactions often caused by second antibodies and increases the detection of autoantibodies by protein G conjugate. A number of modifications were investigated. Higher concentrations of serum and protein G-HRP at 1:5 to 1:10 and 1:100, respectively, increased the detection to the same order as that obtained with second antibody systems and gold staining with silver enhancement. The role of various detergents in the procedure was established. 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) in incubation with protein G-HRP increased the binding between protein G and immunoglobulin G. Addition of Tween-20 for blocking produced little background so that protein blockers could be avoided. Prolonged incubation with serum increased markedly the sensitivity of the procedure when compared with the recommended 2 h incubation period. Polyvinylidene difluoride membrane provided better transfer effect, lower background and higher mechanical strength than nitrocellulose membrane. The utilization of only one antibody-specific ligand increased the simplicity, reliability, economy, efficiency and specificity of the method. These modifications make this method significantly better for detection and screening for autoantibodies.

  14. Far-western blotting as a solution to the non-specificity of the anti-erythropoietin receptor antibody

    PubMed Central

    Fecková, Barbora; Kimáková, Patrícia; Ilkovičová, Lenka; Szentpéteriová, Erika; Debeljak, Nataša; Solárová, Zuzana; Sačková, Veronika; Šemeláková, Martina; Bhide, Mangesh; Solár, Peter

    2016-01-01

    The erythropoietin receptor (EpoR) is a member of the cytokine receptor family. The interaction between erythropoietin (Epo) and EpoR is important for the production and maturation of erythroid cells, resulting in the stimulation of hematopoiesis. The fact that EpoR was also detected in neoplastic cells has opened the question about the relevance of anemia treatment with recombinant Epo in cancer patients. Numerous studies have reported pro-stimulating and anti-apoptotic effects of Epo in cancer cells, thus demonstrating EpoR functionality in these cells. By contrast, a previous study claims the absence of EpoR in tumor cells. This apparent discrepancy is based, according to certain authors, on the use of non-specific anti-EpoR antibodies. With the aim of bypassing the direct detection of EpoR with an anti-EpoR antibody, the present authors propose a far-western blot methodology, which in addition, confirms the interaction of Epo with EpoR. Applying this technique, the presence of EpoR and its interaction with Epo in human ovarian adenocarcinoma A2780 and normal human umbilical vein endothelial cells was confirmed. Furthermore, modified immunoprecipitation of EpoR followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis confirmed a 57 kDa protein as a human Epo-interacting protein in both cell lines. PMID:27446474

  15. Densitometric analysis of Western blot (immunoblot) assays for human immunodeficiency virus antibodies and correlation with clinical status.

    PubMed Central

    Schmidt, G; Amiraian, K; Frey, H; Stevens, R W; Berns, D S

    1987-01-01

    Western blot assays for antibodies directed against components of human immunodeficiency virus (HIV) associated with acquired immunodeficiency syndrome (AIDS) were examined with a densitometer and integrator. Antibody responses to seven HIV proteins were determined from the areas under the peaks of bands on blots from 430 seropositive individuals. Antibody responses corresponded qualitatively and quantitatively with clinical status. The Western blot assays examined were done on single specimens from individuals in one of four clinical states: asymptomatic with no risk factor identified, asymptomatic with risk factor(s) identified, AIDS-related complex, and AIDS. The ratios of gp41 antibody to p24 antibody and of gp41 antibody to total HIV antibodies increased, and the number of total HIV antibodies decreased progressively in these populations. Parameters were assigned to characterize the typical response found in AIDS: gp41 antibody/p24 antibody ratio, greater than or equal to 2.0; gp41 antibody/total HIV antibodies ratio, greater than or equal to 0.30; and number of total HIV antibodies, less than or equal to 25.0 signal units. Parameter match increased with progression of clinical status. These parameters were applied in a brief follow-up study of 34 HIV-infected asymptomatic individuals who developed AIDS-related complex or AIDS. Initial specimens showed a stronger correlation than our population data base had predicted, suggesting that the parameters have prognostic value. Densitometric analysis of antibody responses on Western blot assays of single or serial specimens should prove useful to physicians in staging and monitoring HIV-infected individuals and in predicting which individuals will progress to AIDS. Images PMID:2444624

  16. Mycoplasma agassizii Strain Variation and Distinct Host Antibody Responses Explain Differences between Enzyme-Linked Immunosorbent Assays and Western Blot Assays ▿

    PubMed Central

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

    2010-01-01

    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 < 0.0001) for all antigens tested. The results confirm that strain variation is responsible for the observed differences between 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. PMID:20810678

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

    PubMed

    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

  18. Immunohistochemical and Western blot analyses of collar enamel in the jaw teeth of gars, Lepisosteus oculatus, an actinopterygian fish.

    PubMed

    Sasagawa, Ichiro; Ishiyama, Mikio; Yokosuka, Hiroyuki; Mikami, Masato; Shimokawa, Hitoyata; Uchida, Takashi

    2014-06-01

    Although most fish have no enamel layer in their teeth, those belonging to Lepisosteus (gars), an extant actinopterygian fish genus, do and so can be used to study amelogenesis. In order to examine the collar enamel matrix in gar teeth, we subjected gar teeth to light and electron microscopic immunohistochemical examinations using an antibody against bovine amelogenin (27 kDa) and antiserum against porcine amelogenin (25 kDa), as well as region-specific antibodies and antiserum against the C-terminus and middle region, and N-terminus of porcine amelogenin, respectively. The enamel matrix exhibited intense immunoreactivity to the anti-bovine amelogenin antibody and the anti-porcine amelogenin antiserum in addition to the C-terminal and middle region-specific antibodies, but not to the N-terminal-specific antiserum. These results suggest that the collar enamel matrix of gar teeth contains amelogenin-like proteins and that these proteins possess domains that closely resemble the C-terminal and middle regions of porcine amelogenin. Western blot analyses of the tooth germs of Lepisosteus were also performed. As a result, protein bands with molecular weights of 78 kDa and 65 kDa were clearly stained by the anti-bovine amelogenin antibody as well as the antiserum against porcine amelogenin and the middle-region-specific antibody. It is likely that the amelogenin-like proteins present in Lepisosteus do not correspond to the amelogenins found in mammals, although they do possess domains that are shared with mammalian amelogenins.

  19. Deep proteomic profiling of vasopressin-sensitive collecting duct cells. I. Virtual Western blots and molecular weight distributions.

    PubMed

    Yang, Chin-Rang; Tongyoo, Pumipat; Emamian, Milad; Sandoval, Pablo C; Raghuram, Viswanathan; Knepper, Mark A

    2015-12-15

    The mouse mpkCCD cell line is a continuous cultured epithelial cell line with characteristics of renal collecting duct principal cells. This line is widely used to study epithelial transport and its regulation. To provide a data resource useful for experimental design and interpretation in studies using mpkCCD cells, we have carried out "deep" proteomic profiling of these cells using three levels of fractionation (differential centrifugation, SDS-PAGE, and HPLC) followed by tandem mass spectrometry to identify and quantify proteins. The analysis of all resulting samples generated 34.6 gigabytes of spectral data. As a result, we identified 6,766 proteins in mpkCCD cells at a high level of stringency. These proteins are expressed over eight orders of magnitude of protein abundance. The data are provided to users as a public data base (https://helixweb.nih.gov/ESBL/Database/mpkFractions/). The mass spectrometry data were mapped back to their gel slices to generate "virtual Western blots" for each protein. For most of the 6,766 proteins, the apparent molecular weight from SDS-PAGE agreed closely with the calculated molecular weight. However, a substantial fraction (>15%) of proteins was found to run aberrantly, with much higher or much lower mobilities than predicted. These proteins were analyzed to identify mechanisms responsible for altered mobility on SDS-PAGE, including high or low isoelectric point, high or low hydrophobicity, physiological cleavage, residence in the lysosome, posttranslational modifications, and expression of alternative isoforms due to alternative exon usage. Additionally, this analysis identified a previously unrecognized isoform of aquaporin-2 with apparent molecular mass <20 kDa.

  20. Immunohistochemical and Western Blotting Analyses of Ganoine in the Ganoid Scales of Lepisosteus oculatus: an Actinopterygian Fish.

    PubMed

    Sasagawa, Ichiro; Oka, Shunya; Mikami, Masato; Yokosuka, Hiroyuki; Ishiyama, Mikio; Imai, Akane; Shimokawa, Hitoyata; Uchida, Takashi

    2016-05-01

    In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin. PMID:27139791

  1. A mass screening survey of cystic echinococcosis by ultrasonography, Western blotting, and ELISA among university students in Manisa, Turkey.

    PubMed

    Kilimcioğlu, Ali Ahmet; Girginkardeşler, Nogay; Korkmaz, Metin; Özkol, Mine; Düzgün, Fatih; Östan, Ipek; Pabuşcu, Yüksel; Dinç, Gönül; Ok, Ulgen Zeki

    2013-12-01

    Cystic echinococcosis (CE) is one of the most important zoonotic diseases in a wide geographic area, including Turkey. In the present project, a total of 4275 students from Celal Bayar University, Manisa, Turkey, were screened by ultrasonography (US) and specific antibodies for CE were examined by Western blotting (WB) and ELISA in finger prick blood samples of 2034 of 4275 volunteered students. We aimed to report the apparent prevalence of CE based on different diagnostic procedures and to compare WB and ELISA with US in diagnosis of CE in a mass screening setting. Six new cases were diagnosed as CE by US during the survey. In addition to these cases, three students were also detected to have been previously operated and pathologically confirmed for hepatic CE. US revealed parenchymal changes in these cases in concordance with their operation history; so, the prevalence of CE by US was calculated as 0.21% (9/4275) (95%CI, 0.11-0.39%) among university students in Manisa. Bands were detected at 8, 28, 32, 38, 42, 47, 70 and 90kDa by WB and the cases were considered to be positive for CE when at least three of the bands were seen together. Apparent prevalence of CE by ELISA and WB were found to be 2.11% (43/2034) (95%CI, 1.57-2.83%) and 0.25% (5/2034) (95%CI, 0.10-0.57%), respectively. Of the six US positive cases, WB was positive in only one case with two cysts in the liver. All of four cases with liver involvement were positive by ELISA. The high prevalence of CE among university students in Manisa indicated that CE is a major health problem in this area of Turkey. Our results supported that WB is rather difficult and not feasible as a mass screening test and may not be effective for confirmation especially in asymptomatic cases. As a result, we recommend US to be used initially in mass screening surveys for CE followed by confirmation by ELISA for suspected cases. Further examination primarily by chest X-ray followed by computed tomography and/or magnetic

  2. Detection of anti-HIV-1 IgG antibodies in whole saliva by GACELISA and Western blot assays.

    PubMed

    Matee, M I; Lyamuya, E F; Simon, E; Mbena, E C; Kagoma, C; Samaranayake, L P; Scheutz, F

    1996-05-01

    The present study, based on 158 HIV seropositives and 167 HIV seronegatives, demonstrates that saliva collected with the Omni-SAL device and tested with GACELISA (an IgG antibody capture ELISA) is an effective non-invasive alternative to serum for anti-HIV IgG antibody screening. The study also shows that a conventional serum Western blot kit can be used, with slight modifications, for confirmatory testing of saliva specimens. Collecting saliva with the Omni-SAL device had a very good acceptance rate among Tanzanian subjects, and although this diagnostic method is not yet known by the general public, 65% of the study participants preferred to give saliva instead of blood for HIV testing.

  3. An analysis of beta-lactam-derived antigens on spleen cell and serum proteins by ELISA and Western blotting.

    PubMed

    Warbrick, E V; Thomas, A L; Stejskal, V; Coleman, J W

    1995-11-01

    Penicillins and related beta-lactam antibiotics are known to conjugate to proteins to generate potentially antigenic (haptenic) determinants. In the present study, we used a rabbit polyclonal antibody raised against benzylpenicillin (BP) to investigate the capacity of six penicillins and one cephalosporin to generate haptenic groups in vitro on cultured mouse spleen cells and on serum proteins in the culture medium. All of the drugs tested, namely, BP, amoxicillin (AMX), ampicillin (AMP), cephalothin (CEP), cloxacillin (CLX), flucloxacillin (FLX), and phenoxymethylpenicillin (PMP) generated antigens in a concentration-dependent manner on cell and serum proteins, which could be detected by ELISA, although antigens generated by BP, CEP, FLX, or PMP in either cell- or serum-conjugated form were more readily detected than those generated by AMX, AMP, or CLX. Western blot analysis revealed that BP-derived antigens were generated relatively slowly on cell proteins (maximum binding was not yet reached after 8 h), compared to serum proteins (maximum binding within 1 h). BP, CEP, and PMP all generated similar distinctive patterns of immunostaining of electrophoresed cell or serum proteins which did not reflect the relative abundance of different proteins as revealed by Coomassie brilliant blue staining. FLX, CLX, AMP, and AMX did not generate antigens that could be detected on Western blots. In conclusion, we have shown that various beta-lactam antibiotics generate antigens on cell and serum proteins that can be detected and characterized immunochemically with polyclonal antiserum. Further application of these methods may offer potential for further identification of immunologically relevant cellular and serum antigens generated by these drugs.

  4. Quantitative Western ligand blotting reveals common patterns and differential features of IGFBP-fingerprints in domestic ruminant breeds and species.

    PubMed

    Wirthgen, Elisa; Höflich, Christine; Spitschak, Marion; Helmer, Carina; Brand, Bodo; Langbein, Jan; Metzger, Friedrich; Hoeflich, Andreas

    2016-02-01

    The insulin-like growth factor binding proteins (IGFBPs) are determinants of local IGF-effects and thus have an impact on growth and metabolism in vertebrate species. In farm animals, IGFBPs are associated with traits such as growth rate, body composition, milk production, or fertility. It may be assumed, that selective breeding and characteristic phenotypes of breeds are related to differential expression of IGFBPs. Therefore, the aim of the present study was to investigate the effects of selective breeding on blood IGFBP concentrations of farm animals. Breeds of the sheep, goat, and cattle species were investigated. IGFBP-3, -2, and -4 were analyzed with quantitative Western ligand blotting (qWLB), enabling comprehensive monitoring of intact IGFBPs with IGF-binding capacity. We show that in sera of all species and breeds investigated, IGFBP-3, -2, and -4 were simultaneously detectable by qWLB analysis. IGFBP-3 and the total amount of IGFBPs were significantly increased (P<0.05) in Cameroon sheep, if compared to 3 of 4 other sheep breeds, as well as in Dwarf goats versus Toggenburg and Boer goats (P<0.01). IGFBP-2 was elevated in Cameroon sheep and Boer goats, if compared to other breeds of these species (P<0.01), respectively. Holstein Friesian dairy cows had higher levels of IGFBP-4 (P<0.05), if compared to conventional crossbreeds of beef cattle. In Dwarf goats the ratio of IGFBP-3/IGFBP-2 was about 3-fold higher than in other goat breeds (P<0.001). The total IGFBP amount of Toggenburg goats was reduced (P<0.05), compared to the other goat breeds. In conclusion, our data indicate that common and specific features of IGFBP fingerprints are found in different ruminant species and breeds. Our findings may introduce quantitative Western ligand blotting as an attractive tool for biomarker development and molecular phenotyping in farm animal breeds. PMID:26597140

  5. Quantitative Western ligand blotting reveals common patterns and differential features of IGFBP-fingerprints in domestic ruminant breeds and species.

    PubMed

    Wirthgen, Elisa; Höflich, Christine; Spitschak, Marion; Helmer, Carina; Brand, Bodo; Langbein, Jan; Metzger, Friedrich; Hoeflich, Andreas

    2016-02-01

    The insulin-like growth factor binding proteins (IGFBPs) are determinants of local IGF-effects and thus have an impact on growth and metabolism in vertebrate species. In farm animals, IGFBPs are associated with traits such as growth rate, body composition, milk production, or fertility. It may be assumed, that selective breeding and characteristic phenotypes of breeds are related to differential expression of IGFBPs. Therefore, the aim of the present study was to investigate the effects of selective breeding on blood IGFBP concentrations of farm animals. Breeds of the sheep, goat, and cattle species were investigated. IGFBP-3, -2, and -4 were analyzed with quantitative Western ligand blotting (qWLB), enabling comprehensive monitoring of intact IGFBPs with IGF-binding capacity. We show that in sera of all species and breeds investigated, IGFBP-3, -2, and -4 were simultaneously detectable by qWLB analysis. IGFBP-3 and the total amount of IGFBPs were significantly increased (P<0.05) in Cameroon sheep, if compared to 3 of 4 other sheep breeds, as well as in Dwarf goats versus Toggenburg and Boer goats (P<0.01). IGFBP-2 was elevated in Cameroon sheep and Boer goats, if compared to other breeds of these species (P<0.01), respectively. Holstein Friesian dairy cows had higher levels of IGFBP-4 (P<0.05), if compared to conventional crossbreeds of beef cattle. In Dwarf goats the ratio of IGFBP-3/IGFBP-2 was about 3-fold higher than in other goat breeds (P<0.001). The total IGFBP amount of Toggenburg goats was reduced (P<0.05), compared to the other goat breeds. In conclusion, our data indicate that common and specific features of IGFBP fingerprints are found in different ruminant species and breeds. Our findings may introduce quantitative Western ligand blotting as an attractive tool for biomarker development and molecular phenotyping in farm animal breeds.

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

    PubMed

    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.

  7. Immunofluorescence, enzyme-linked immunosorbent assay, particle agglutination and western blot for the detection of antibody to human immunodeficiency virus type 1.

    PubMed

    Auwanit, W; Ayuthaya, P I; Balachandra, K; Jayavasu, C; Phanthumachinda, B; Ikuta, K; Yamanishi, K; Kanai, K

    1990-03-01

    Immunofluorescence assay (IFA) has been applied for detection of antibody to human immunodeficiency virus type 1 (HIV-1). To compare the IFA with an enzyme-linked immunosorbent assay (ELISA) and particle agglutination (PA), we examined the antibody response to HIV-1 in 475 sera from AIDS, PGL and ARC patients as well as several risk groups and healthy persons by three methods. The positive results by any methods were confirmed by western blot (WB). The results by all methods were well correlated on the sera from 45 asymptomatic male homosexuals and 70 female prostitutes. There were some false positive results by ELISA in the sera from prisoners and healthy persons. Four sera from drug abusers were positive only by PA and IFA and were negative by ELISA. All were WB-inconclusive. Particle agglutination and IFA results were compared with western blot analysis on 208 ELISA-positive sera. All IFA-strongly positive sera (84%) were positive by western blot. The sera with weakly positive, negative and inconclusive results by IFA (16%) were possibly any of positive, inconclusive or negative by western blot. By PA, 200 of 208 (97%) sera were PA-positive and 1% of these sera were WB-inconclusive while the PA-negative sera were either negative or inconclusive by western blot. These results suggested that PA is a simple and sensitive method for screening of HIV-1 antibody while IFA could be a primary confirmatory test and western blot would then be used for confirming any IFA-negative or inconclusive results.

  8. Identification of recombinant human EPO variants in greyhound plasma and urine by ELISA, LC-MS/MS and western blotting: a comparative study.

    PubMed

    Timms, Mark; Steel, Rohan; Vine, John

    2016-02-01

    The recombinant human erythropoietins epoetin alfa (Eprex®), darbepoetin (Aranesp®) and methoxy polyethylene glycol-epoetin beta (Mircera®) were administered to greyhounds for 7, 10 and 14 days respectively. Blood and urine samples were collected and analysed for erythropoietin by ELISA, LC-MS/MS and western blotting. Limits of confirmation in plasma for western blotting and LC-MS/MS methods ranged from a low of 2.5mIU/mL, and closely matched the sensitivity of ELISA screening. PMID:26290355

  9. Outbreak of larval Echinococcus multilocularis infection in Japanese monkey (Macaca fuscata) in a zoo, Hokkaido: western blotting patterns in the infected monkeys.

    PubMed

    Sato, Chiaki; Kawase, Shiro; Yano, Shoki; Nagano, Hideki; Fujimoto, Satoshi; Kobayashi, Nobuyuki; Miyahara, Kazuro; Yamada, Kazutaka; Sato, Motoyoshi; Kobayashi, Yoshiyasu

    2005-01-01

    A high prevalence of larval Echinococcus multilocularis (Em) infection was found in zoo primates in Hokkaido, Japan. In October 1997, a Japanese monkey (Macaca fuscata) died and histopathologically diagnosed as alveolar hydatidosis. Serum samples were collected from the remaining Japanese monkeys and examined for antibodies against Em by enzyme-linked immunosorbent assay and western blotting. Serological tests showed 12 more animals of the remaining 57 monkeys were possibly infected. Ultrasonography revealed that nine of these 12 animals had a cystic lesion in the liver. The band patterns of western blotting in the monkeys were very similar to those in human.

  10. 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.

    PubMed

    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.

  11. The Dot Blot ELISA.

    ERIC Educational Resources Information Center

    Gerbig, Donald G., Jr.; Fenk, Christopher J.; Goodhart, Amy S.

    2000-01-01

    Uses two laboratory techniques, Enzyme Linked Immunosorbent Assay (ELISA) and Western Blot, to demonstrate antibody-antigen binding concepts. Includes a list of required materials and directions for the procedure, and makes suggestions for classroom applications. (Contains 13 references.) (YDS)

  12. Identification of Novel Laminin- and Fibronectin-binding Proteins by Far-Western Blot: Capturing the Adhesins of Streptococcus suis Type 2

    PubMed Central

    Li, Quan; Liu, Hanze; Du, Dechao; Yu, Yanfei; Ma, Caifeng; Jiao, Fangfang; Yao, Huochun; Lu, Chengping; Zhang, Wei

    2015-01-01

    Bacterial cell wall (CW) and extracellular (EC) proteins are often involved in interactions with extracellular matrix (ECM) proteins such as laminin (LN) and fibronectin (FN), which play important roles in adhesion and invasion. In this study, an efficient method combining proteomic analysis and Far-Western blotting assays was developed to screen directly for bacterial surface proteins with LN- and FN-binding capacity. With this approach, fifteen potential LN-binding proteins and five potential FN-binding proteins were identified from Streptococcus suis serotype 2 (SS2) CW and EC proteins. Nine newly identified proteins, including oligopeptide-binding protein OppA precursor (OppA), elongation factor Tu (EF-Tu), enolase, lactate dehydrogenase (LDH), fructose-bisphosphate aldolase (FBA), 3-ketoacyl-ACP reductase (KAR), Gly ceraldehyde-3-phosphate dehydrogenase (GAPDH), Inosine 5′-monophosphate dehydrogenase (IMPDH), and amino acid ABC transporter permease (ABC) were cloned, expressed, purified and further confirmed by Far-Western blotting and ELISA. Five proteins (OppA, EF-Tu, enolase, LDH, and FBA) exhibited specifically binding activity to both human LN and human FN. Furthermore, seven important recombinant proteins were selected and identified to have the ability to bind Hep-2 cells by the indirect immunofluorescent assay. In addition, four recombinant proteins, and their corresponding polyclonal antibodies, were observed to decrease SS2 adhesion to Hep-2 cells, which indicates that these proteins contribute to the adherence of SS2 to host cell surface. Collectively, these results show that the approach described here represents a useful tool for investigating the host-pathogen interactions. PMID:26636044

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

    PubMed Central

    2011-01-01

    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 surveillance currently in

  14. Validity of interpretation criteria for standardized Western blots (immunoblots) for serodiagnosis of Lyme borreliosis based on sera collected throughout Europe.

    PubMed

    Hauser, U; Lehnert, G; Wilske, B

    1999-07-01

    Western blotting (WB; immunoblotting) is a widely used tool for the serodiagnosis of Lyme borreliosis (LB), but so far, no generally accepted criteria for performance and interpretation have been established in Europe. The current study was preceeded by a detailed analysis of WB with whole-cell lysates of three species of Borrelia burgdorferi sensu lato (U. Hauser, G. Lehnert, R. Lobentanzer, and B. Wilske, J. Clin. Microbiol. 35:1433-1444, 1997). In that study, interpretation criteria for a positive WB result were developed with the data for 330 serum samples (from patients with LB in different stages [n = 189] and from a control group [n = 141]) originating mostly from southern Germany. In the present work, the interpretation criteria for strains PKo (Borrelia afzelii) and PBi (Borrelia garinii) developed in the previous study were reevaluated with 224 serum samples (from patients with LB in different stages [n = 97] and from a control group [n = 127]) originating from throughout Europe that were provided by the European Union Concerted Action on Lyme Borreliosis (EUCALB). De novo criteria were developed on the basis of the reactivities of the EUCALB sera and were evaluated with the data for the samples from southern Germany. Comparison of all results led to the following recommendations: For WB for immunoglobulin G (IgG), at least two bands among p83/100, p58, p43, p39, p30, OspC, p21, p17, and p14 for PKo and at least one band among p83/100, p39, p30, OspC, p21, and p17b for PBi; for WB for IgM, at least one band among p39, OspC, and p17 or a strong p41 band for PKo and at least one band among p39 and OspC or a strong p41 band for PBi. WB with PKo was the most sensitive, and this strain is recommended for use in WB for the serodiagnosis of LB throughout Europe.

  15. Southern blotting.

    PubMed

    Brown, T

    2001-05-01

    Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This unit describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, and subsequent immobilization by UV irradiation (for nylon) or baking (for nitrocellulose). A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. A second alternate protocol describes a transfer method based on a different transfer-stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in the second alternate protocol is therefore more rapid and can result in more complete transfer. PMID:18432697

  16. Southern blotting.

    PubMed

    Brown, T

    2001-05-01

    Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support (the properties and advantages of the different types of membrane, transfer buffer, and transfer method are discussed in detail), resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This appendix describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, using a high-salt transfer buffer to promote binding of DNA to the membrane. With the high-salt buffer, the DNA becomes bound to the membrane during transfer but not permanently immobilized. Immobilization is achieved by UV irradiation (for nylon) or baking (for nitrocellulose). A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. The advantage of this combination is that no post-transfer immobilization step is required, as the positively charged membrane binds DNA irreversibly under alkaline transfer conditions. The method can also be used with neutral nylon membranes but less DNA will be retained. A second alternate protocol describes a transfer method based on a different transfer-stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane described in the first basic and alternate protocols has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in

  17. Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA*

    PubMed Central

    Schoenherr, Regine M.; Saul, Richard G.; Whiteaker, Jeffrey R.; Yan, Ping; Whiteley, Gordon R.; Paulovich, Amanda G.

    2015-01-01

    Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first

  18. A modified ELISA and western blot accurately determine anti-human immunodeficiency virus type 1 antibodies in oral fluids obtained with a special collecting device.

    PubMed

    Emmons, W W; Paparello, S F; Decker, C F; Sheffield, J M; Lowe-Bey, F H

    1995-06-01

    Serum and saliva from 195 known human immunodeficiency virus (HIV)-seropositive patients and 198 military personnel undergoing annual HIV serologic testing were evaluated in a prospective, blinded fashion for anti-HIV-1 antibodies. Oral specimens, collected with a device designed to concentrate oral mucosal transudate from whole saliva, were tested by a modified ELISA and by Western blot. Serum was tested in a standard manner. All 195 HIV-1-seropositive subjects had detectable anti-HIV-1 antibodies in their saliva by ELISA; 190 saliva samples were positive by Western blot and 5 were indeterminate. None of the 198 military personnel were positive by ELISA of serum or oral fluid. The sensitivity, specificity, and positive and negative predictive values for ELISA of saliva were each 100%. The serologic testing of oral mucosal transudate appears to be a simple, safe, sensitive, and specific method for detecting anti-HIV-1 antibodies.

  19. Western blot analyses of measles virus antibody in normal persons and in patients with multiple sclerosis, subacute sclerosing panencephalitis, or atypical measles.

    PubMed Central

    Hankins, R W; Black, F L

    1986-01-01

    A version of the Western blot was developed to detect serum antibodies against measles virus polypeptides. With this technique, a seroepidemiological survey of antibodies to the several measles virus proteins in diverse measles-related conditions was conducted. The sera were obtained from individuals with a recent or long-past history of natural measles, from persons with a history of immunization with live attenuated measles vaccine, and from patients with multiple sclerosis, subacute sclerosing panencephalitis, or atypical measles. The findings indicated that live attenuated measles vaccine elicits an antibody response qualitatively resembling that of a natural infection. In addition, multiple sclerosis patients made less antibody to the measles virus M protein than did individuals with a long-past history of natural measles. Thus, the immunological reaction of multiple sclerosis patients to measles virus is qualitatively, as well as quantitatively, different from that of normal persons. Finally, persons with subacute sclerosing panencephalitis and atypical measles mounted abnormally high antibody responses to measles virus polypeptides, in particular the P protein. PMID:3531224

  20. 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.

    PubMed

    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.

  1. Glycosylated VCAM-1 isoforms revealed in 2D western blots of HUVECs treated with tumoral soluble factors of breast cancer cells

    PubMed Central

    2009-01-01

    Background Several common aspects of endothelial phenotype, such as the expression of cell adhesion molecules, are shared between metastasis and inflammation. Here, we analyzed VCAM-1 variants as biological markers of these two types of endothelial cell activation. With the combination of 2-DE and western blot techniques and the aid of tunicamycin, we analyzed N-glycosylation variants of VCAM-1 in primary human endothelial cells stimulated with either TNF or tumoral soluble factors (TSF's) derived from the human breast cancer cell line ZR75.30. Results Treatments induced a pro-adhesive endothelial phenotype. 2D western blots analysis of cells subjected to both treatments revealed the expression of the two known VCAM-1 isoforms and of previously unknown isoforms. In particular TSFZR75.30 induced an isoform with a relative molecular mass (Mr) and isoelectric point (pI) of 75-77 kDa and 5.0, respectively. Conclusion The unknown isoforms of VCAM-1 that were found to be overexpressed after treatment with TSF's compared with TNF, could serve as biomarkers to discriminate between inflammation and metastasis. 2D western blots revealed three new VCAM-1 isoforms expressed in primary human endothelial cells in response to TSF stimulation. Each of these isoforms varies in Mr and pI and could be the result of differential glycosylation states. PMID:19930605

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

    PubMed

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

    2016-01-01

    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. PMID:27531616

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

    PubMed Central

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

    2016-01-01

    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. PMID:27531616

  4. The seroprevalence of Taenia solium cysticercosis among epileptic patients in León, Nicaragua, as evaluated by ELISA and western blotting.

    PubMed

    Bucardo, F; Meza-Lucas, A; Espinoza, F; García-Jerónimo, R C; García-Rodea, R; Correa, D

    2005-01-01

    The Taenia solium taeniasis/cysticercosis complex is an important public-health problem in several countries, where many epileptic seizures appear to be associated with neurocysticercosis. As few data on this problem in Nicaragua exist, the seroprevalence of antibodies reacting with antigens from T. solium cysticerci was investigated among 88 Nicaraguan epileptics (45 males and 43 females, aged 6-53 years). In questionnaire-based interviews, each adult subject and a caregiver of each child investigated were asked about potential risk factors for taeniasis/cysticercosis. When a serum sample from each subject was then checked for anti-cysticercus antibodies, 8.0% of the subjects were found seropositive by ELISA and 14.8% by western blotting. Five samples (all from individuals who had been epileptic for > 5 years) were positive in both tests. When the level of association between each potential risk factor and seropositivity (in ELISA or by blotting) was evaluated, the only statistically significant association detected was that between a positive ELISA and the subject living in a household where pigs were raised (odds ratio = 5.18; 95% confidence interval = 0.8-41.6; P = 0.05). The bands most frequently recognized in the western blots (of 50, 42-39, 24 and 14 kDa) were those previously reported. The results indicate that, in the city of Léon, cysticercosis may be endemic and the cause of a significant proportion of the epilepsy recorded.

  5. Evaluation of viral-lysate enzyme-linked immunosorbent assay kits for detecting human immunodeficiency virus (type 1) infections using human sera standardized by quantitative western blotting.

    PubMed

    Hardy, C T; Damrow, T A; Villareal, D B; Kenny, G E

    1992-06-01

    The first generation of proprietary reagents for detecting antibodies to the Human Immunodeficiency Virus Type 1 (HIV-1) by enzyme-linked immunosorbent assay (ELISA) used as antigen partially purified virus from cell culture lysates. These tests, which are still in use, may vary in their antibody measurement capabilities if different proportions of the viral polypeptides are present in the viral lysate mixtures. We determined the quantities of antibodies in the serum of persons infected with HIV-1 by dilution analysis using 3 ELISA kits: Abbott [A], Du Pont [D], Genetic Systems [G]. The proportionate antibody titres of each serum to p24gag and gp160env/120env were established by quantitative Western blotting. Serum antibody titres were high, frequently over 1:10,000, a result observed both by ELISA and Western blot. For Kit D, sera with high proportions of antibody to p24gag produced antibody titration curves with steep slopes whereas shallower slopes were found in sera with high proportions of antibody to gp160env. In contrast, Kit A gave steeper slopes with sera enriched for gp160env antibodies. Kit G gave results with slopes intermediate between Kits A and D. Serum antibody titres differed between kits depending upon the proportion and concentration of antibodies in a given serum to gp160env and p24gag. The findings that both the concentration and proportion of antibodies to specific viral polypeptides in human sera markedly affect the signal intensity produced by proprietary ELISAs suggest the need for several control sera which reflect the diversity of human serum responses. Standardization of human reference sera by quantitative Western blotting will assist in evaluation and quality control of ELISA tests.

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

    PubMed

    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.

  7. Western blot analysis of Amblyomma americanum-derived stage-specific and shared antigens using serum from guinea pigs expressing resistance.

    PubMed

    Brown, S J

    1988-04-01

    Serum from guinea pigs expressing resistance to larval, nymphal and adult Amblyomma americanum ticks was used in Western blot analyses to identify potential antigens from egg, larval and nymphal, and female salivary gland extract preparations. The results demonstrate multiple antigens unique to each life stage, as well as several shared proteins between the three life stages. However, it appears as if two particular proteins of 25 and 38 kDa may be more important than others, based upon their prevalence and intensity of recognition in this assay relative to other polypeptides. PMID:2455376

  8. 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.

    PubMed

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

    2016-01-01

    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. PMID:27479506

  9. Advanced laboratory techniques for diagnosing Toxoplasma gondii encephalitis in AIDS patients: significance of intrathecal production and comparison with PCR and ECL-western blotting.

    PubMed

    Contini, C; Fainardi, E; Cultrera, R; Canipari, R; Peyron, F; Delia, S; Paolino, E; Granieri, E

    1998-12-01

    The polymerase chain reaction (PCR) for detection of cerebral spinal fluid (CSF) Toxoplasma gondii DNA was combined with the study of intrathecal antibody synthesis by antibody specific index calculation (ASI) and the detection of specific oligoclonal IgG bands (OCB) by affinity mediated immunoblotting (AMI) in 11 AIDS patients with T. gondii encephalitis (TE) and in 20 control patients with or without neurological disorders. Enhanced chemiluminescence (ECL) western-blot technique was employed to evaluate the antigenic specificity of CSF-IgG towards individual T. gondii antigens. PCR was positive in all TE patients which displayed brain-derived or blood-derived specific OCB, even when comparative ASI failed. Four TE patients had a unique anti-T. gondii OCB restricted to the CSF and a strong antibody response toward the 29 kDa band by ECL western blot. This response could be an important marker to discriminate TE from other opportunistic central nervous system (CNS) infections in the course of AIDS. PMID:9916877

  10. 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

    PubMed Central

    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

    2015-01-01

    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

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

    PubMed

    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. PMID:26267107

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

    PubMed

    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.

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

    PubMed Central

    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. PMID:27494183

  14. IgG Western Blot for Confirmatory Diagnosis of Equivocal Cases of Toxoplasmosis by EIA-IgG and Fluorescent Antibody Test

    PubMed Central

    Saghrouni, Fatma; Yaacoub, Alia; Gaied Meksi, Sondoss; Ach, Hinda; Garma, Lamia; Fathallah, Akila; Ben Saïd, Moncef

    2013-01-01

    The performance values of available techniques used in serodiagnosis of toxoplasmosis are satisfactory but they raise problems of equivocal and discordant results for very low IgG titers. Recently marketed, LDBio-Toxo II IgG Western blot (IB) showed an excellent correlation with the dye test. We estimated the proportion of equivocal and discordant results between the enzyme immunoassay Platelia Toxo IgG (EIA-IgG) and fluorescent antibody test (FAT) and assessed the usefulness of the IB as a confirmatory test. Out of 2,136 sera collected from pregnant women, 1,644 (77.0%) tested unequivocally positive and 407 (19.0%) were negative in both EIA-IgG and FAT. The remaining 85 (4%) sera showed equivocal or discordant results. Among them, 73 (85.9%) were positive and 12 (14.1%) were negative in IB. Forty-one (89.1%) equivocal sera in EIA-IgG and 46 (86.8%) equivocal sera in FAT were positive in IB. Reducing the cut-off values of both screening techniques improved significantly their sensitivity in detecting very low IgG titers at the expense of their specificity. In conclusion, equivocal results in routine-used techniques and their discordance in determination of the immune status in pregnancy women were not uncommon. IB test appeard to be highly useful in these situations as a confirmatory technique. PMID:24039295

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

    PubMed

    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.

  16. Evaluation of the Bio-Rad Geenius HIV 1/2 Confirmation Assay as an Alternative to Western Blot in the Korean Population: A Multi-Center Study.

    PubMed

    Moon, Hee-Won; Huh, Hee Jin; Oh, Gwi Young; Lee, Sang Gon; Lee, Anna; Yun, Yeo-Min; Hur, Mina

    2015-01-01

    Recently updated recommendations for diagnosis of HIV infection suggest a new diagnostic algorithm including HIV-1/HIV-2 antibody differentiation immunoassay instead of western blot (WB) as a confirmatory testing. We evaluated Bio-Rad Geenius HIV1/2 confirmation assay as a simple and reliable alternative to WB in the Korean population with low HIV prevalence. The Geenius HIV1/2 was performed in a total of 192 serum specimens (140 reactive and 52 nonreactive specimens by ARCHITECT HIV Ag/Ab Combo assay) that were prospectively collected from five institutions. HIV-1 nucleic acid amplification test (NAT) was performed in negative or indeterminate specimens by Geenius HIV1/2 or WB. Among 140 reactive specimens by HIV Ag/Ab assay, 82 (58.6%) were positive for HIV-1 Ab by Geenius HIV1/2. Among 58 negative or indeterminate specimens by Geenius HIV1/2, four specimens (6.9%) were positive by HIV-1 NAT. The sensitivity and specificity of Geenius HIV1/2 were 95.3% and 100.0%, respectively. When we considered only WB, the sensitivity and specificity of Geenius HIV1/2 were 100.0% and 99.1%, respectively. Agreement between Geenius HIV1/2 and WB was excellent (weighted Kappa = 0.89). The Geenius HIV1/2 is simple and time-saving compared with WB. It has an excellent performance and can be a reliable alternative to WB. HIV-1 NAT should be performed in negative or indeterminate specimens by Geenius HIV1/2 to detect acute HIV infection as recommended in new HIV testing algorithms.

  17. Localization of α-synuclein in teleost central nervous system: immunohistochemical and Western blot evidence by 3D5 monoclonal antibody in the common carp, Cyprinus carpio.

    PubMed

    Vaccaro, Rosa; Toni, Mattia; Casini, Arianna; Vivacqua, Giorgio; Yu, Shun; D'este, Loredana; Cioni, Carla

    2015-05-01

    Alpha synuclein (α-syn) is a 140 amino acid vertebrate-specific protein, highly expressed in the human nervous system and abnormally accumulated in Parkinson's disease and other neurodegenerative disorders, known as synucleinopathies. The common occurrence of α-syn aggregates suggested a role for α-syn in these disorders, although its biological activity remains poorly understood. Given the high degree of sequence similarity between vertebrate α-syns, we investigated this proteins in the central nervous system (CNS) of the common carp, Cyprinus carpio, with the aim of comparing its anatomical and cellular distribution with that of mammalian α-syn. The distribution of α-syn was analyzed by semiquantitative western blot, immunohistochemistry, and immunofluorescence by a novel monoclonal antibody (3D5) against a fully conserved epitope between carp and human α-syn. The distribution of 3D5 immunoreactivity was also compared with that of choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and serotonin (5HT) by double immunolabelings. The results showed that a α-syn-like protein of about 17 kDa is expressed to different levels in several brain regions and in the spinal cord. Immunoreactive materials were localized in neuronal perikarya and varicose fibers but not in the nucleus. The present findings indicate that α-syn-like proteins may be expressed in a few subpopulations of catecholaminergic and serotoninergic neurons in the carp brain. However, evidence of cellular colocalization 3D5/TH or 3D5/5HT was rare. Differently, the same proteins appear to be coexpressed with ChAT by cholinergic neurons in several motor and reticular nuclei. These results sustain the functional conservation of the α-syn expression in cholinergic systems and suggest that α-syn modulates similar molecular pathways in phylogenetically distant vertebrates.

  18. Comparison of Enzyme-Linked Immunosorbent Assay, Western Blotting, Microagglutination, Indirect Immunofluorescence Assay, and Flow Cytometry for Serological Diagnosis of Tularemia

    PubMed Central

    Porsch-Özcürümez, Mustafa; Kischel, Nele; Priebe, Heidi; Splettstösser, Wolf; Finke, Ernst-Jürgen; Grunow, Roland

    2004-01-01

    The serodiagnostic efficiencies of five different approaches to detecting antibodies (immunoglobulins G, A, and M) developed in clinically proven infections with Francisella tularensis have been assessed. Fifty serum samples from patients suffering from tularemia during an outbreak in Sweden were compared with samples from 50 healthy blood donors (controls) by using an enzyme-linked immunosorbent assay (ELISA), microagglutination (MA), Western blotting (WB), an indirect immunofluorescence assay (IIFA), and flow cytometry (FC). ELISA, WB, and FC were based on the use of preparations of lipopolysaccharides (LPS) of the live vaccine strain of Francisella tularensis subsp. holarctica (ATCC 29684) as a capture antigen. Whole methanol-fixed bacteria were used for IIFA and MA. Optimized protocols yielded a diagnostic sensitivity and specificity of 100% for WB, MA, and FC, 98% for ELISA, and 93% for IIFA. A total of 6,632 serum samples from individuals between the ages of 18 and 79 years, representatively recruited from all regions of Germany, were screened to estimate and confirm the positive predictive value (PVpos) of the ELISA. Serum samples from 15 (0.226%) individuals tested positive for F. tularensis-specific antibodies by ELISA and confirmatory WB. The resulting prevalence-dependent PVpos of 10.2% and specificity of 98.1% were consistent with our findings for tularemia patients and controls. We conclude that the combined usage of a screening ELISA and a confirmatory WB based on LPS as a common antigen, as well as the MA, is a suitable serodiagnostic tool, while the quality of the IIFA is hampered by subjective variations of the results. FC is a promising new approach that might be improved further in terms of multiplex analyses or high-throughput applications. PMID:15539498

  19. Identification of Reference Proteins for Western Blot Analyses in Mouse Model Systems of 2,3,7,8-Tetrachlorodibenzo-P-Dioxin (TCDD) Toxicity

    PubMed Central

    Prokopec, Stephenie D.; Watson, John D.; Pohjanvirta, Raimo; Boutros, Paul C.

    2014-01-01

    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. PMID:25329058

  20. Identification of immunoreactive proteins of Chlamydia trachomatis by Western blot analysis of a two-dimensional electrophoresis map with patient sera.

    PubMed

    Sanchez-Campillo, M; Bini, L; Comanducci, M; Raggiaschi, R; Marzocchi, B; Pallini, V; Ratti, G

    1999-08-01

    Western blots of two-dimensional electrophoretic maps of proteins from Chlamydia trachomatis were probed with sera from 17 seropositive patients with genital inflammatory disease. Immunoblot patterns (comprising 28 to 2 spots, average 14.8) were different for each patient; however, antibodies against a spot-cluster due to the chlamydia-specific antigen outer membrane protein-2 (OMP2) were observed in all sera. The next most frequent group of antibodies (15/17; 88%) recognized the hsp60 GroEL-like protein, described as immunopathogenic in chlamydial infections. Reactivity to the major surface-exposed and variable antigen major outer membrane protein (MOMP) was observed at a relatively lower frequency (13/17; 76%). The hsp70 DnaK-like protein was also frequently recognized (11/17; 64.7%) in this patient group. Besides the above confirmatory findings, the study detected several new immunoreactive proteins, with frequencies ranging from 11/17 to 1/17. Some were characterized also by N-terminal amino acid sequencing and homology searches. Amongst these were a novel outer membrane protein (OmpB) and, interestingly, five conserved bacterial proteins: four (23%) sera reacted with the RNA polymerase alpha-subunit, five (29%) recognized the ribosomal protein S1, eight (47%) the protein elongation factor EF-Tu, seven (41%) a putative stress-induced protease of the HtrA family, and seven sera (41%) the ribosomal protein L7/L12. Homologs of the last two proteins were shown to confer protective immunity in other bacterial infections. The data show that immunological sensitization processes commonly thought to play a role in chlamydial pathogenicity may be sustained not only by the hsp60 GroEl-like protein, but also by other conserved bacterial antigens, some of which may be also considered as potential vaccine candidates. PMID:10493131

  1. 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?

    PubMed Central

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

    2015-01-01

    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: 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. PMID:26516904

  2. Detection of antibody to Toxocara vitulorum perieneteric fluid antigens (Pe) in the colostrum and serum of buffalo calves and cows by Western blotting.

    PubMed

    Ferreira, Fabiano P; Starke-Buzetti, Wilma A

    2005-04-20

    Toxocara vitulorum, a nematode parasite in the small intestine of cattle and water buffaloes, causes high morbidity and mortality of 1-3 months old buffalo calves. This research evaluated the specific perieneteric antigens (Pe) reactivity of anti-T. vitulorum-Pe antibody (Tv-Pe-Ab) in both immune sera and colostrum from buffalo cows immediately post-partum from buffalo cows. The presence of Tv-Pe-Ab in sera of buffalo newborn calves was also examined at 1 day before and after suckling the colostrum as well as in sera from naturally infected calves at the beginning and peak of the maximum infection and then again during the period of rejection and post-rejection of the parasite. Pe antigens were characterized for Tv-Pe-Ab by SDS-PAGE and Western blot (WB). The SDS-PAGE showed that Pe contained nine protein bands (11, 14, 31, 38, 58, 76, 88, 112 and 165 kDa). All Pe bands were recognized by Tv-Pe-Ab in sera and colostrum of buffalo cows. Only the serum antibodies of buffalo calves at 1 day of age after suckling the colostrum and during the beginning of T. vitulorum infection recognized Pe antigen's nine bands. In contrast, serum antibodies from 1-day-old buffalo calves, taken before suckling colostrum, did not react with any protein band. In suckling calves, which reached peak egg output, rejection and post-rejection stages of the infection, serum Tv-Pe-Ab reactivity with lower molecular weight protein bands (11-76 kDa) was lost and only reactivity with the Pe protein bands of higher molecular weight (88, 112 and 165 kDa) remained.

  3. Current state of HIV/AIDS in Taiwan and reappraisal of early diagnostic value of serial and comparative analyses of western blot patterns of HIV infection.

    PubMed

    Chen, Mao-Yuan; Chuang, Che-Yen

    2002-02-01

    Since Taiwan is not a member of the World Health Organization (WHO), the problems of human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) in Taiwan are not well known to the world. In this report we summarize the trend and current status of HIV/AIDS in Taiwan and also analyze Western blot (WB) patterns. The application of serial and comparative WB analyses is important in establishing an early diagnosis of neonatal HIV infection. These analyses will be useful as well in evaluating the clinical status of adults infected with HIV in developing countries. When there are "indeterminate" WBs in adults with recent HIV infection, it is imperative to perform a WB weekly to attain the earliest possible diagnosis and treatment. Usually anti-gag antibodies, most frequently the anti-p24, are followed by anti-env and anti-pol antibodies. When the set point is attained, WB studies may also be repeated if the clinical status changes or if the patients are receiving highly active antiretroviral therapy (HAART). In the advanced stages of HIV infection, usually the anti-gag antibodies will fade first and then disappear, this occurs next in the anti-pol and in the anti-env antibodies. During HAART, viral replication is usually controlled and there will thus be a partial restoration of the immunological function that will induce changes in the antigen-antibody ratio and, in the end, this will result in changes in the WB patterns. In the mid 1980s all the hepatitis B immunoglobulin (HBIG) used in Taiwan was anti-HIV positive. However, follow-up ELISA and WB studies of the HBIG injected newborns proved that HBIG was anti-HIV positive but that there was no replicable HIV in HBIG. Monthly WB tests of newborns and anti-HIV positive mothers were used to differentiate HIV infection from the passive placental transference by comparing their WB patterns. Comparative WB analyses was also done between blood donors and recipients, and also between husbands and wives

  4. Analysis of the serologic relationship among San Miguel sea lion virus and vesicular exanthema of swine virus isolates. Application of the western blot assay for detection of antibodies in swine sera to these virus types.

    PubMed

    Seal, B S; House, J A; Whetstone, C A; Neill, J D

    1995-04-01

    Caliciviruses are positive-sense single-stranded RNA viruses with a single capsid protein. The serotypes of the marine mammal calicivirus, San Miguel sea lion virus (SMSV), are antigenically related to vesicular exanthema of swine virus (VESV) and are potentially hazardous to swine. Western blot assays using purified SMSV serotypes 1 and 4 were used to further examine the serologic relationship among SMSV and VESV isolates. With the exception of SMSV 8 and SMSV 12, rabbit polyclonal antisera generated against all the available SMSV and VESV isolates reacted positively, as assessed by western blot, with purified capsid protein from SMSV 1 and SMSV 4. Consequently, the SMSV 8 and SMSV 12 virus isolates may not be members of the SMSV/VESV calicivirus group. Using antisera from pigs experimentally inoculated with SMSV and VESV as positive controls, a western blot assay for these virus types was utilized to check for the presence of antibodies to calciviruses in swine sera. Sera from colostrum-deprived gnotobiotic pigs were used as a negative control in all experiments. Examination of sera from domestic and feral swine collected in Iowa, California, and Florida was completed using this technique. The presence of antibodies to these virus types was not detected in any of the porcine sera tested. PMID:7619900

  5. Problem-Solving Test: Southwestern Blotting

    ERIC Educational Resources Information Center

    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,…

  6. Mutation detection by Southern blotting.

    PubMed

    Mellars, Gillian; Gomez, Keith

    2011-01-01

    Following the discovery of the structure of DNA in 1953, it became clear that scientists needed to be able to distinguish different DNA sequences. In 1975, Edward Southern published details of a new method for detecting DNA fragments based upon their specific sequence [corrected]. An indication of the importance of his work is that the technique was eponymously named after him and that subsequent methods based loosely on similar principles were named using a play on his surname (western and northern blot). The simplicity and effectiveness of the technique led to its universal acceptance as a standard method for identification of DNA sequences. In the modern laboratory where turn-around times assume ever greater importance, the process can seem relatively time-consuming. In some cases, this has led to its replacement by more rapid techniques such as long-range PCR. Nevertheless, more than 30 years after its invention, the Southern blot remains a cornerstone of molecular biology. PMID:20938846

  7. Collagen chains detected by western blotting using a /sup 125/I-labeled 45K fragment of fibronectin (45K FN)

    SciTech Connect

    Ristagno, R.; Heimer, R.; Fishman, A.P.; Sampson, P.M.

    1987-05-01

    The objective was to improve the sensitivity and specificity of detection of unlabeled collagen chains in biologic fluids. Chains of Types I,II,III,IV and XI (1..cap alpha..2..cap alpha..3..cap alpha..) collagen were separated by SDS PAGE. Their complete transfer to nitrocellulose was obtained by electrophoresis for 16 h at 150 mA with 10 mM Tris, 117 mM glycine, 100 mM cysteine, 0.1% SDS and 10% methanol. The 45K FN was prepared by chymotryptic digestion of fibronectin adsorbed to gelatin-Sepharose, followed by elution with 1.2 M urea, 1 M Tris-NaCl, pH 8.3 and iodination. When exposed to the nitrocellulose transblot at pH 9.5 and 4/sup 0/C, 45K FN did not react with IgG, fibrinogen, myosin, albumin or carbonic anhydrase. These proteins interfere in the assay under conditions of lower pH and higher temperature. The autoradiographs of the transblots were evaluated by densitometry and reflected results also obtained by dot blotting, that chains of collagen Types I,II,III were detectable at 4 ng and those of collagen Type IV at 12 ng. Generally, ..cap alpha..,BETA, and ..gamma.. chains were detectable. The 45K FN reacted equally with ..cap alpha..1(I) and ..cap alpha..2(I), but for Type XI the 1..cap alpha.. chain had considerably more reactivity than 2..cap alpha.. or 3..cap alpha... As the 45K FN was specific for collagens added to plasma, the authors method appears useful for qualitative and quantitative assays of unlabeled collagens in biologic fluids.

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

    PubMed

    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.

  9. Detection of proteins on blot transfer membranes.

    PubMed

    Sasse, Joachim; Gallagher, Sean R

    2008-11-01

    Staining of blot transfer membranes permits visualization of proteins and allows the extent of transfer to be monitored. In the protocols described in this unit, proteins are stained after electroblotting from one-dimensional or two-dimensional polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. Protocols are provided for the use of six general protein stains: Amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, and India ink. In addition, the fluorescent stains fluorescamine and IAEDANS, which covalently react with bound proteins, are described. Approximate detection limits for each nonfluorescent stain are indicated along with membrane compatibilities.

  10. Detection of proteins on blot membranes.

    PubMed

    Harper, S; Speicher, D W

    2001-05-01

    Staining of blot transfer membranes permits visualization of proteins and allows the extent of transfer to be monitored. In the protocols described in this unit, proteins are stained after electroblotting from one-dimensional or two-dimensional polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. Protocols are provided for the use of six general protein stains: amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, and India ink. In addition, the fluorescent stains fluorescamine and IAEDANS, which covalently react with bound proteins, are described. Approximate detection limits for each nonfluorescent stain are indicated along with membrane compatibilities. PMID:18429099

  11. Detection of proteins on blot transfer membranes.

    PubMed

    Sasse, Joachim; Gallagher, Sean R

    2008-11-01

    Staining of blot transfer membranes permits visualization of proteins and allows the extent of transfer to be monitored. In the protocols described in this unit, proteins are stained after electroblotting from one-dimensional or two-dimensional polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. Protocols are provided for the use of six general protein stains: Amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, and India ink. In addition, the fluorescent stains fluorescamine and IAEDANS, which covalently react with bound proteins, are described. Approximate detection limits for each nonfluorescent stain are indicated along with membrane compatibilities. PMID:19016450

  12. A new multi-host species indirect ELISA using protein A/G conjugate for detection of anti-Toxoplasma gondii IgG antibodies with comparison to ELISA-IgG, agglutination assay and Western blot.

    PubMed

    Al-Adhami, Batol H; Gajadhar, Alvin A

    2014-02-24

    Toxoplasma gondii is a zoonotic protozoan parasite which can cause significant disease and losses in livestock and wild animals. It is increasingly recognized as an important foodborne pathogen in a broad range of food animals and products. Effective control strategies require rapid, reliable and cost-effective detection methods for large scale surveys and diagnostic applications in a broad range of warm-blooded animals. To overcome one or more of these shortcomings in the currently available detection methods for T. gondii infection a non-species-specific protein A/G conjugate was used in the development of an indirect ELISA (ELISA-A/G) for the detection of IgG antibodies in serum samples obtained from experimentally infected pigs. The performance of the assay was evaluated using serum samples from pigs, cats, mice and seals with known positive or negative status for T. gondii infection. Results of the ELISA-A/G obtained with pig serum samples were compared with those generated by traditional ELISA using host specific IgG conjugate (ELISA-IgG), modified agglutination test (MAT) and Western blot analysis (WB). Using protein A/G conjugate, comparative analysis of results from 77 samples obtained from T. gondii infected pigs showed excellent agreement between the ELISA-A/G and in-house ELISA-IgG (0.917 κ). Similar agreements were also observed when these samples were tested by a commercial ELISA kit (0.816 κ), MAT (0.816 κ) and WB (0.79 κ). A total of 86 serum samples obtained from cats, mice and seals experimentally infected with T. gondii and tested by the ELISA-A/G as well as MAT for the presence of anti-Toxoplasma IgG antibodies yielded Kappa value of 1.0 for cats and mice and 0.79 for seals. These results show that the ELISA-A/G is a suitable method for serological detection of T. gondii infection in multiple host species and has the potential for testing samples from a broad range of domestic, wild, and aquatic mammalian host species. Simultaneous testing

  13. BLOT Ver. 1.65

    SciTech Connect

    MEYERS, RAY; GLICK, III, JOHN; FORSYTHE, CHRISTI; GILKEY, AMY; SJAARDEMA, GREGORY

    2009-03-24

    BLOT is a graphic program for post-processing finite element analyses output in the EXODUS II database format. It is command driven with free-format input and can drive graphics devices supported by the Sandia Virtual Device Interface. BLOT produces mesh plots of the analysis output variables including deformed mesh plots, line contours, filled (painted) contours, vector plots of two/three variables (velocity vectors), and symbol plots of scalar variables (discrete cracks). Features include pathlines of analysis variables drawn on the mesh, element selection by material, element birth and death, multiple views combining several displays on each plot, symmetry mirroring, and node and element numbering. X-Y plots of the analysis variables include time vs. variable plots or variable vs. variable plots, and distance vs. variable plots at selected time steps where distance is the accumulated distance between pairs of nodes or element centers. BLOT is written in as portable a form as possible. Fortran code is written in ANSI Standard FORTRAN-77. Machine-specific routines are limited in number and are grouped together to minimize the time required to adapt them to a new system. SEACAS codes have been ported to several Unix systems

  14. BLOT Ver. 1.65

    2009-03-24

    BLOT is a graphic program for post-processing finite element analyses output in the EXODUS II database format. It is command driven with free-format input and can drive graphics devices supported by the Sandia Virtual Device Interface. BLOT produces mesh plots of the analysis output variables including deformed mesh plots, line contours, filled (painted) contours, vector plots of two/three variables (velocity vectors), and symbol plots of scalar variables (discrete cracks). Features include pathlines of analysis variablesmore » drawn on the mesh, element selection by material, element birth and death, multiple views combining several displays on each plot, symmetry mirroring, and node and element numbering. X-Y plots of the analysis variables include time vs. variable plots or variable vs. variable plots, and distance vs. variable plots at selected time steps where distance is the accumulated distance between pairs of nodes or element centers. BLOT is written in as portable a form as possible. Fortran code is written in ANSI Standard FORTRAN-77. Machine-specific routines are limited in number and are grouped together to minimize the time required to adapt them to a new system. SEACAS codes have been ported to several Unix systems« less

  15. Detection of Blotted Proteins: Not All Blockers Are Created Equal.

    PubMed

    Kothari, Vishal; Mathews, Suresh T

    2015-01-01

    Western blotting is a standard analytical technique for detection of proteins. It is dependent on a number of components; from the specificity of the primary antibody to the reduction of competing biomolecules present in the assay. Blocking agents are a critical component for western blotting protocols as these diminish nonspecific binding by blocking off-target sites on the membrane. A variety of blocking agents are available and these are selected in an empirical manner, as no single blocker is compatible with every system. The best blocking agent and method for any particular assay will be an optimized but not absolute choice. Here, we describe characteristics of the most common blocking agents used in western blotting and discuss their advantages and disadvantages. PMID:26139251

  16. Effects of Roundup formulations, nutrient addition, and Western mosquitofish (Gambusia affinis) on aquatic communities.

    PubMed

    Geyer, Rebecca L; Smith, Geoffrey R; Rettig, Jessica E

    2016-06-01

    Aquatic communities can be affected by herbicides, nutrient addition, and non-native fish species. We conducted a mesocosm experiment to examine the direct and interactive effects of three stressors: (1) Roundup formulations (Roundup Weed and Grass Killer(®) and Roundup Poison Ivy and Tough Brush Killer Plus(®)), (2) nutrient addition, and (3) the presence of the non-native Western mosquitofish (Gambusia affinis), on experimental pond communities. Roundup formulations had the most widespread effects on the zooplankton community, but effects varied between formulations and among taxa. The only significant effect of nutrient addition was a lowering of Daphnia abundance in the nutrient addition treatments. The abundances of Daphnia, mid-sized cladocerans, and total zooplankton were lowered by mosquitofish, but no other taxa showed significant mosquitofish effects. We found several two-way and three-way interactions among the stressors, but these varied among zooplankton taxa. Chlorophyll a levels were higher with nutrient addition but were not significantly affected by Roundup formulation or mosquitofish. Our results suggest toxicity of Roundup formulations varies among taxa, and Roundup formulations differ in their toxicity to zooplankton, but with no cascading effects on primary producers. In addition, interactions among stressors affected the zooplankton community.

  17. Effects of Roundup formulations, nutrient addition, and Western mosquitofish (Gambusia affinis) on aquatic communities.

    PubMed

    Geyer, Rebecca L; Smith, Geoffrey R; Rettig, Jessica E

    2016-06-01

    Aquatic communities can be affected by herbicides, nutrient addition, and non-native fish species. We conducted a mesocosm experiment to examine the direct and interactive effects of three stressors: (1) Roundup formulations (Roundup Weed and Grass Killer(®) and Roundup Poison Ivy and Tough Brush Killer Plus(®)), (2) nutrient addition, and (3) the presence of the non-native Western mosquitofish (Gambusia affinis), on experimental pond communities. Roundup formulations had the most widespread effects on the zooplankton community, but effects varied between formulations and among taxa. The only significant effect of nutrient addition was a lowering of Daphnia abundance in the nutrient addition treatments. The abundances of Daphnia, mid-sized cladocerans, and total zooplankton were lowered by mosquitofish, but no other taxa showed significant mosquitofish effects. We found several two-way and three-way interactions among the stressors, but these varied among zooplankton taxa. Chlorophyll a levels were higher with nutrient addition but were not significantly affected by Roundup formulation or mosquitofish. Our results suggest toxicity of Roundup formulations varies among taxa, and Roundup formulations differ in their toxicity to zooplankton, but with no cascading effects on primary producers. In addition, interactions among stressors affected the zooplankton community. PMID:26944427

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

    ERIC Educational Resources Information Center

    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…

  19. Preliminary evaluation of feeder and lint slide moisture addition on ginning, fiber quality, and textile processing of western cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to evaluate the effects of moisture addition at the gin stand feeder conditioning hopper and/or the battery condenser slide on gin performance and Western cotton fiber quality and textile processing. The test treatments included no moisture addition, feeder hopper hum...

  20. A brief review of other notable protein blotting methods.

    PubMed

    Kurien, Biji T; Scofield, R Hal

    2009-01-01

    A plethora of methods have been used for transferring proteins from the gel to the membrane. These include centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low and high molecular weight proteins, blotting of Coomassie Brilliant Blue (CBB)-stained proteins from polyacrylamide gels to transparencies, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before MALDI tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter. PMID:19378075

  1. Other notable protein blotting methods: a brief review.

    PubMed

    Kurien, Biji T; Scofield, R Hal

    2015-01-01

    Proteins have been transferred from the gel to the membrane by a variety of methods. These include vacuum blotting, centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low- and high-molecular-weight proteins, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before matrix-assisted laser desorption/ionization tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter. PMID:26044032

  2. Mixed lubrication after rewetting of blotted pleural mesothelium.

    PubMed

    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.

  3. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  4. Streamlined Strategies to Better Visualize Southern Blotting

    ERIC Educational Resources Information Center

    Dean, Derek M.

    2012-01-01

    In this article, I describe an animated slideshow of Southern blotting that I have made freely available to other instructors. My hope is to provide a clear visualization of the logistics behind the technique so that instructors have a solid basis--as well as time freed up--to discuss its applications with students.

  5. BLOT II Ver.1.39

    2003-06-03

    BLOT II is a graphic program for post-processing finite element analyses output in the EXODUS II database format. It is command driven with free-format input and can drive graphics devices supported by the Sandia Virtual Device Interface. BLOT produces mesh plots of the analysis output variables including deformed mesh plots, line contours, filled (painted) contours, vector plots of two/three variables (velocity vectors), and symbol plots of scalar variables (discrete cracks). Features include pathlines of analysismore » variables drawn on the mesh, element selection by material, element birth and death, multiple views combining several displays on each plot, symmetry mirroring, and node and element numbering. X-Y plots of the analysis variables include time vs. variable plots or variable vs. variable plots, and distance vs. variable plots at selected time stips where distance is the accumulated distance between pairs of nodes or element centers.« less

  6. BLOT II Ver.1.39

    SciTech Connect

    2003-06-03

    BLOT II is a graphic program for post-processing finite element analyses output in the EXODUS II database format. It is command driven with free-format input and can drive graphics devices supported by the Sandia Virtual Device Interface. BLOT produces mesh plots of the analysis output variables including deformed mesh plots, line contours, filled (painted) contours, vector plots of two/three variables (velocity vectors), and symbol plots of scalar variables (discrete cracks). Features include pathlines of analysis variables drawn on the mesh, element selection by material, element birth and death, multiple views combining several displays on each plot, symmetry mirroring, and node and element numbering. X-Y plots of the analysis variables include time vs. variable plots or variable vs. variable plots, and distance vs. variable plots at selected time stips where distance is the accumulated distance between pairs of nodes or element centers.

  7. Blot-MS of Carbonylated Proteins: A Tool to Identify Oxidized Proteins.

    PubMed

    Ferreira, Rita; Domingues, Pedro; Amado, Francisco; Vitorino, Rui

    2016-01-01

    The efficiency of proteostasis regulation declines during aging and the failure of protein homeostasis is common in age-related diseases. Protein oxidation is a major contributor to the loss of proteome homeostasis, also called "proteostasis," precluding protein misfolding and aggregation. So, the identification of the molecular pathways impaired by protein oxidation will increase the understanding of proteostasis and the pathophysiological conditions related to the loss of proteostasis. Sample derivatization with dinitrophenyl hydrazine and western blot immunoassay detection of carbonylated proteins (commonly known as Oxyblot™) coupled to mass spectrometry (blot-MS) is an attractive methodological approach to identify proteins that are more prone to carbonylation, a typical oxidative modification of amino acid residues. The integration of blot-MS data of carbonylated proteins with bioinformatics tools allows the identification of the biological processes more affected by protein oxidation and that, eventually, result in the loss of proteostasis.In this chapter, we describe a blot-MS methodology to identify the proteins more prone to oxidation in biological samples, as cell and tissue extracts, and biofluids. Analysis of mitochondria isolated from cardiac tissue is provided as an example. Bioinformatic strategy to deal with data retrieved from blot-MS experiments are proposed for the identification of relevant biological processes modulated by oxidative stress stimuli. PMID:27613049

  8. Validation of a Dot-Blot quantitative technique for large scale analysis of beef tenderness biomarkers.

    PubMed

    Guillemin, N; Meunier, B; Jurie, C; Cassar-Malek, I; Hocquette, J-F; Leveziel, H; Picard, B

    2009-10-01

    Beef tenderness is a very complex and multifactorial sensorial meat quality trait, which depends partly on muscle characteristics. This tissue is very variable according to animal type (age, breed and sex) and rearing conditions. Consequently, beef tenderness exhibits a great variability. Different research programs have revealed several genes or proteins which could be good markers of beef tenderness. In order to validate the relation of these markers with beef tenderness on a large population of bovines, it is necessary to have a large-scale and trusty technique which can access different quantities of proteins related to tenderness. In this study we firstly compared Western-Blot and Dot-Blot. Secondly, we evaluated Dot-Blot technical and biological capabilities for the quantification of protein biomarkers. The results demonstrated that the Dot-Blot technique with fluorescence detection presents numerous interests. This technique allows a good reproducibility and permits the simultaneous analysis of a large number of samples. The Dot-Blot technique defined and validated in this study can be used for protein biomarkers analyses, notably to predict beef tenderness. Another major result of this study is that about 5 to 10 animals per group are required to detect large differences (>1.5) in biomarker expression between tender and tough beef, whereas much larger numbers of animals (10 to 30) are required to detect smaller differences (about 1.2 to 1.3) taking into account the biological variability of these markers.

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

    PubMed

    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. PMID:26924673

  10. Transcription Factor Proteomics: Identification by a Novel Gel Mobility Shift-Three-Dimensional Electrophoresis Method Coupled with Southwestern Blot and HPLC-Electrospray-Mass Spectrometry Analysis

    PubMed Central

    Jiang, Daifeng; Jia, Yinshan; Jarrett, Harry W.

    2011-01-01

    Transcription factor (TF) purification and identification is an important step in elucidating gene regulatory mechanisms. In this study, we present two new electrophoretic mobility shift assay (EMSA)-based multi-dimensional electrophoresis approaches to isolate and characterize TFs, using detection with either southwestern or western blotting and HPLC-nanoESI-MS/MS analysis for identification. These new techniques involve several major steps. First, EMSA is performed with agents that diminish non-specific DNA-binding and the DNA-protein complex is separated by native PAGE gel. The gel is then electrotransferred to PVDF membrane and visualized by autoradiography. Next, the DNA-protein complex, which has been transferred onto the blot, is extracted using a detergent-containing elution buffer. Following detergent removal, concentrated extract is separated by SDS-PAGE (EMSA-2DE), followed by in-gel trypsin digestion and HPLC-nanoESI-MS/MS analysis, or the concentrated extract is separated by two-dimensional gel electrophoresis EMSA-3DE), followed by southwestern or western blot analysis to localize DNA binding proteins on blot which are further identified by on-blot trypsin digestion and HPLC-nanoESI-MS/MS analysis. Finally, the identified DNA binding proteins are further validated by EMSA-immunoblotting or EMSA antibody supershift assay. This approach is used to purify and identify GFP-C/EBP fusion protein from bacterial crude extract, as well as purifying AP1 and CEBP DNA binding proteins from a human embryonic kidney cell line (HEK293) nuclear extract. AP1 components, c-Jun, Jun-D, c-Fos, CREB, ATF1 and ATF2 were successfully identified from 1.5 mg of nuclear extract (equivalent to 3 ×107 HEK293 cells) with AP1 binding activity of 750 fmol. In conclusion, this new strategy of combining EMSA with additional dimensions of electrophoresis and using southwestern blotting for detection proves to be a valuable approach in the identification of transcriptional complexes

  11. Improvement of antigen blotting in a tissue blot immunobinding assay for the detection of two chili pepper viruses.

    PubMed

    Han, Jung-Heon; Shin, Jun-Sung; Kim, Young Ho; Kim, Byung-Dong

    2007-11-01

    The tissue blot immunobinding assay (TBIA) is widely used for the detection and localization of plant viruses in various plant tissues. The basic experimental procedures of TBIA sampling and blotting were simplified using commercially available micropipette tips. This method was termed the ring-blot immunobinding assay (R-BIA), as the blot on the membrane forms a ring shape. The detection efficacy of R-BIA was tested for two chili pepper viruses, pepper mild mottle tobamovirus (PMMoV) and pepper mottle potyvirus (PepMoV), following the optimized serological procedures of TBIA (length of the incubation period and BSA concentration, and primary and secondary antibodies). Sensitivity of the R-BIA was about 1 ng/ml of purified PMMoV in pepper leaf sap from a healthy pepper plant. R-BIA also showed high specificity in the detection of PMMoV and PepMoV. Moreover, the modified sampling and blotting procedures were simpler and more reliable than other TBIA methods (such as whole-leaf blotting and crushed-leaf blotting), suggesting that the R-BIA may be used for medium- to large-scale detection of plant viruses in laboratories with minimal facilities.

  12. Multiplexed miRNA northern blots via hybridization chain reaction

    PubMed Central

    Schwarzkopf, Maayan; Pierce, Niles A.

    2016-01-01

    Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2′OMe-RNA probes. The HCR northern blot protocol takes ∼1.5 days independent of the number of target RNAs. PMID:27270083

  13. Multiplexed miRNA northern blots via hybridization chain reaction.

    PubMed

    Schwarzkopf, Maayan; Pierce, Niles A

    2016-09-01

    Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2'OMe-RNA probes. The HCR northern blot protocol takes ∼1.5 days independent of the number of target RNAs. PMID:27270083

  14. Identification of additional MAP kinases activated upon PAMP treatment

    PubMed Central

    Nitta, Yukino; Ding, Pingtao; Zhang, Yuelin

    2014-01-01

    Mitogen-activated protein (MAP) kinase cascades play important roles in plant immunity. Upon pathogen associated molecular pattern (PAMP) treatment, MPK3, MPK6 and MPK4 are quickly activated by upstream MKKs through phosphorylation. Western blot analysis using α-phospho-p44/42-ERK antibody suggests that additional MPKs with similar size as MPK4 are also activated upon PAMP perception. To identify these MAP kinases, 7 candidate MPKs with similar sizes as MPK4 were selected for further analysis. Transgenic plants expressing these MPKs with a ZZ-3xFLAG double tag of 17 kD were generated and analyzed by western blot. MPK1, MPK11 and MPK13 were found to be phosphorylated upon treatment with flg22. Our study revealed additional MAPKs being activated during PAMP-triggered immunity. PMID:25482788

  15. Evaluation of western shale-oil residue as an additive to petroleum asphalt for use as a pavement crack and joint sealant material

    SciTech Connect

    Harnsberger, P.M.; Wolf, J.M.; Robertson, R.E.

    1992-11-01

    The objective of this study was to perform a preliminary evaluation of using a distillation residue from Green River Formation (western) shale oil as an additive to a petroleum asphalt for use as a crack and joint filler material in portland cement concrete and asphaltic pavements. A commercially available rubberized asphalt crack and joint filler material was also tested for comparison. ASTM specification tests for sealant materials used in concrete and asphalt pavements were performed on the sealant materials. Portland cement concrete briquets prepared with an asphalt material sandwiched between two concrete wafers were tested in a stress-relaxation experiment to evaluate the relaxation and recovery properties of the sealant materials. The results show that the shale-oil modified petroleum asphalts and the neat petroleum asphalt do not pass the extension portion of the ASTM test; however, there is indication of improvement in the adhesive properties of the shale-oil modified asphalts. There is also evidence that the addition of shale-oil residue to the petroleum asphalt, especially at the 20% level, improves the relaxation and recovery properties compared with the petroleum asphalt.

  16. Detection of PrP**Sc in formalin-fixed tissues by western blot

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Formalin fixation is the most prevalent form of tissue preservative. As such, formalin fixed tissue represents an important source of archival material for study. Formalin fixation requires little environmental control and preserves the cellular architecture of a wide range of tissues, an importan...

  17. Detection of Traces of Ovalbumin and Casein in White and Red Wines by Quantitative Western Blotting.

    PubMed

    Meyer, Philippe; Zanetti, Simone

    2015-09-01

    Fining of wine with agents containing cow's milk or hen's egg white is a common and traditional procedure. In light of increasing food allergies all over the world, the presence of fining residues has been subject of intense debate. Switzerland does not make exception, and since 2009 the Federal Department of Home Affairs has modified its food regulations stating that the labels must show if traces of fining agents are present. Nevertheless, the application of this regulation is not based on an official analytical method. In this study we show that immunoblotting is an efficient technique to detect and quantify ovalbumin and casein residues in bottled wine. We showed that final filtration is an essential step to remove finings in red wine, and that overfining of white wine may result in fining residues in finished products. Finally, for the first time in Switzerland, 22 samples were taken by food safety inspectors and officially analyzed for the regional food control authority of the Canton of Vaud. These samples were allergen free, but a larger study is currently planned in collaboration with other regional authorities of Switzerland to complete these results and make a complete picture of the Swiss wine production.

  18. Western blotting of high and low molecular weight proteins using heat.

    PubMed

    Kurien, Biji T; Scofield, R Hal

    2015-01-01

    A method for the electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes following sodium dodecyl sulfate (SDS) polyacrylamide gel is described here. The transfer was performed with heated (70-75 °C) normal transfer buffer from which methanol had been omitted. Complete transfer of high and low molecular weight antigens (molecular weight protein standards, a purified protein, and proteins from a human tissue extract) could be carried out in 10 min for a 7 % (0.75 mm) SDS polyacrylamide gel. For 10 and 12.5 % gels (0.75 mm) the corresponding time was 15 min. A complete transfer could be carried out in 20 min for 7, 10, and 12.5 % gels (1.5 mm gels). The permeability of the gel is increased by heat, such that the proteins trapped in the polyacrylamide gel matrix can be easily transferred to the membrane. The heat mediated transfer method was compared with a conventional transfer protocol, under similar conditions. The conventional method transferred minimal low molecular weight proteins while retaining most of the high molecular weight proteins in the gel. In summary, this procedure is particularly useful for the transfer of high molecular weight proteins, very rapid, and avoids the use of methanol.

  19. Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins.

    PubMed

    Ramsey, Kathryn A; Rushton, Zachary L; Ehre, Camille

    2016-06-14

    Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

  20. Hydrogel Pore-Size Modulation for Enhanced Single-Cell Western Blotting.

    PubMed

    Duncombe, Todd A; Kang, Chi-Chih; Maity, Santanu; Ward, Toby M; Pegram, Mark D; Murthy, Niren; Herr, Amy E

    2016-01-13

    Pore-gradient microgel arrays enable thousands of parallel high-resolution single-cell protein electrophoresis separations for targets accross a wide molecular mass (25-289 kDa), yet within 1 mm separation distances. Dual crosslinked hydrogels facilitate gel-pore expansion after electrophoresis for efficient and uniform immunoprobing. The photopatterned, light-activated, and acid-expandable hydrogel underpins single-cell protein analysis, here for oncoprotein-related signaling in human breast biopsy.

  1. Analysis of casein using two-dimensional electrophoresis, western blot, and computer imaging.

    PubMed

    Goldfarb, M F

    2001-01-01

    Casein from milk of 20 mothers, 8 weeks postpartum, was analyzed using 2-dimensional electrophoresis. The casein micelle under 2-D denaturing conditions gives approximately 15 major spots. Beta-Casein, kappa-casein, para-kappa-casein, and casein peptides can be tentatively identified by molecular size, isoelectric point, and spot shape characteristics. All spots are not present in all samples. Two spots thought to represent different phosphorylated forms of beta-casein were selected for area and density measurements by means of the NIH-Image program. The larger area at approximately 26 kDa, isoelectric point 5.3, consisted of 1 to 3 closely linked spots, perhaps reflecting phenotype. The smaller spot 24kDa, isoelectric point 5.4, was always a single unit. Visual scanning of gels suggests a wide range of casein concentration. Computer comparison will allow a statistical analysis.

  2. Quantitative Northern Blot Analysis of Mammalian rRNA Processing.

    PubMed

    Wang, Minshi; Pestov, Dimitri G

    2016-01-01

    Assembly of eukaryotic ribosomes is an elaborate biosynthetic process that begins in the nucleolus and requires hundreds of cellular factors. Analysis of rRNA processing has been instrumental for studying the mechanisms of ribosome biogenesis and effects of stress conditions on the molecular milieu of the nucleolus. Here, we describe the quantitative analysis of the steady-state levels of rRNA precursors, applicable to studies in mammalian cells and other organisms. We include protocols for gel electrophoresis and northern blotting of rRNA precursors using procedures optimized for the large size of these RNAs. We also describe the ratio analysis of multiple precursors, a technique that facilitates the accurate assessment of changes in the efficiency of individual pre-rRNA processing steps. PMID:27576717

  3. The detection of DNA-binding proteins by protein blotting.

    PubMed Central

    Bowen, B; Steinberg, J; Laemmli, U K; Weintraub, H

    1980-01-01

    A method, called "protein blotting," for the detection of DNA-binding proteins is described. Proteins are separated on an SDA-polyacrylamide gel. The gel is sandwiched between 2 nitrocellulose filters and the proteins allowed to diffuse out of the gel and onto the filters. The proteins are tightly bound to each filter, producing a replica of the original gel pattern. The replica is used to detect DNA-binding proteins, RNA-binding proteins or histone-binding proteins by incubation of the filter with [32P]DNA, [125I]RNA, or [125I] histone. Evidence is also presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator. Strategies for the detection of specific protein-DNA interactions are discussed. Images PMID:6243775

  4. Transcription factor proteomics: identification by a novel gel mobility shift-three-dimensional electrophoresis method coupled with southwestern blot and high-performance liquid chromatography-electrospray-mass spectrometry analysis.

    PubMed

    Jiang, Daifeng; Jia, Yinshan; Jarrett, Harry W

    2011-09-28

    Transcription factor (TF) purification and identification is an important step in elucidating gene regulatory mechanisms. In this study, we present two new electrophoretic mobility shift assay (EMSA)-based multi-dimensional electrophoresis approaches to isolate and characterize TFs, using detection with either southwestern or western blotting and HPLC-nanoESI-MS/MS analysis for identification. These new techniques involve several major steps. First, EMSA is performed with agents that diminish non-specific DNA-binding and the DNA-protein complex is separated by native PAGE gel. The gel is then electrotransferred to PVDF membrane and visualized by autoradiography. Next, the DNA-protein complex, which has been transferred onto the blot, is extracted using a detergent-containing elution buffer. Following detergent removal, concentrated extract is separated by SDS-PAGE (EMSA-2DE), followed by in-gel trypsin digestion and HPLC-nanoESI-MS/MS analysis, or the concentrated extract is separated by two-dimensional gel electrophoresis (EMSA-3DE), followed by southwestern or western blot analysis to localize DNA binding proteins on blot which are further identified by on-blot trypsin digestion and HPLC-nanoESI-MS/MS analysis. Finally, the identified DNA binding proteins are further validated by EMSA-immunoblotting or EMSA antibody supershift assay. This approach is used to purify and identify GFP-C/EBP fusion protein from bacterial crude extract, as well as purifying AP1 and CEBP DNA binding proteins from a human embryonic kidney cell line (HEK293) nuclear extract. AP1 components, c-Jun, Jun-D, c-Fos, CREB, ATF1 and ATF2 were successfully identified from 1.5 mg of nuclear extract (equivalent to 3×10(7) HEK293 cells) with AP1 binding activity of 750 fmol. In conclusion, this new strategy of combining EMSA with additional dimensions of electrophoresis and using southwestern blotting for detection proves to be a valuable approach in the identification of transcriptional complexes

  5. Blotting Assisted by Heating and Solvent Extraction for DESI-MS Imaging

    NASA Astrophysics Data System (ADS)

    Cabral, Elaine C.; Mirabelli, Mario F.; Perez, Consuelo J.; Ifa, Demian R.

    2013-06-01

    Imprints of potato sprout ( Solanum tuberosum L.), gingko leaves (Gingko biloba L. ) and strawberries (Fragaria x ananassa Duch. ) were successfully imaged by desorption electrospray ionization mass spectrometry (DESI-MS) on TLC plates through blotting assisted by heating and/or solvent extraction. Ion images showing the distribution of significant compounds such as glycoalkaloid toxins in potato sprout, ginkgolic acids and flavonoids in ginkgo leaves, and sugars and anthocyanidin in strawberry were obtained. Practical implications of this work include analysis of a wide range of irregular or soft materials by different imprinting conditions without requiring the addition of matrices or use of specific kinds of surfaces.

  6. Blotting assisted by heating and solvent extraction for DESI-MS imaging.

    PubMed

    Cabral, Elaine C; Mirabelli, Mario F; Perez, Consuelo J; Ifa, Demian R

    2013-06-01

    Imprints of potato sprout (Solanum tuberosum L.), gingko leaves (Gingko biloba L.) and strawberries (Fragaria x ananassa Duch.) were successfully imaged by desorption electrospray ionization mass spectrometry (DESI-MS) on TLC plates through blotting assisted by heating and/or solvent extraction. Ion images showing the distribution of significant compounds such as glycoalkaloid toxins in potato sprout, ginkgolic acids and flavonoids in ginkgo leaves, and sugars and anthocyanidin in strawberry were obtained. Practical implications of this work include analysis of a wide range of irregular or soft materials by different imprinting conditions without requiring the addition of matrices or use of specific kinds of surfaces. PMID:23605686

  7. Blotting assisted by heating and solvent extraction for DESI-MS imaging.

    PubMed

    Cabral, Elaine C; Mirabelli, Mario F; Perez, Consuelo J; Ifa, Demian R

    2013-06-01

    Imprints of potato sprout (Solanum tuberosum L.), gingko leaves (Gingko biloba L.) and strawberries (Fragaria x ananassa Duch.) were successfully imaged by desorption electrospray ionization mass spectrometry (DESI-MS) on TLC plates through blotting assisted by heating and/or solvent extraction. Ion images showing the distribution of significant compounds such as glycoalkaloid toxins in potato sprout, ginkgolic acids and flavonoids in ginkgo leaves, and sugars and anthocyanidin in strawberry were obtained. Practical implications of this work include analysis of a wide range of irregular or soft materials by different imprinting conditions without requiring the addition of matrices or use of specific kinds of surfaces.

  8. Extraction of DNA from paraffin blocks for Southern blot analysis.

    PubMed

    Mies, C; Houldsworth, J; Chaganti, R S

    1991-02-01

    Tissues stored as paraffin blocks are a potential source of DNA for retrospective clinicogenetic analysis. To assess the feasibility of Southern blot analysis, DNA extracted from paraffin blocks was compared with DNA obtained from fresh-frozen controls of the same tissues. Sections 50-100 microns thick cut from paraffin blocks of 11 normal tissues, 18 lymphoid lesions, and 9 gastric carcinoma samples were deparaffinized and incubated at 45 degrees C for 48 to 72 h in a sodium dodecyl sulfate (SDS)/proteinase K solution. Following organic extraction, alcohol precipitation, restriction endonuclease digestion, and gel electrophoresis, DNA was transferred to nylon membranes. 32P-labelled DNA probes for the immunoglobulin heavy-chain locus and T-cell receptor beta-chain gene were hybridized to the normal tissue and lymphoid samples; the gastric cancers were probed for the HER-2/neu protooncogene. Intact DNA was obtained from the majority of formalin-fixed samples, yielding results qualitatively similar to those from fresh tissues. Degradation is the most significant problem in analyzing DNA extracted from paraffin blocks and compromises accurate quantitation. DNA analysis using paraffin-embedded tissue has potential clinical and research applications and may be a particularly useful way to study gene abnormalities in unusual tumors infrequently available as fresh specimens.

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

    PubMed Central

    Manis, Ashley E; Bowman, James R; Bowlin, Gary L; Simpson, David G

    2007-01-01

    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 < 110 mg/ml acetone deposited as 4–8 μm diameter beads; at 110 mg/ml-to-140 mg/ml starting concentrations, this polymer deposited as 100–4000 nm diameter fibers. Nylon formed fibers when electrospun from 60–140 mg/ml HFIP, fibers ranged from 120 nm-6000 nm in diameter. Electrospun nitrocellulose exhibited superior protein retention and increased sensitivity in slot blot experiments with respect to the parent nitrocellulose material. Western immunoblot experiments using fibronectin as a model protein demonstrated that electrospun nylon exhibits increased protein binding and increased dynamic range in the chemiluminescence detection of antigens than sheets of the parent starting material. Composites of electrospun nitrocellulose and electrospun nylon exhibit high protein binding activity and provide increased sensitivity for the immuno-detection of antigens. 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. PMID:18271978

  10. DMISA (dissociated membrane immunosorbent assay), a new ELISA technique performed with blotted samples.

    PubMed

    Guérin-Marchand, C; Batard, T; Brodard, V; Desvaux, F X; Sénéchal, H; David, B; Peltre, G

    1994-01-01

    This study describes the use of electrophoretically purified antigens blotted onto nitrocellulose, as solid phase antigens for enzyme-linked immunosorbent assays. This procedure is called DMISA, for dissociated membrane immunosorbent assay. The method is illustrated using immunoblotted antigens of Dactylis glomerata grass pollen extract. The band of interest was located on a print of nitrocellulose by light staining (India ink), then the corresponding strip of nitrocellulose was cut out. Immediately after its solubilization in ethyleneglycol monomethyl ether, the antigen coated nitrocellulose was precipitated by the addition of buffer. In this way the bulk of the antigen remained bound to the membrane. The resulting suspension was carefully washed, and used as a solid phase antigen in an enzyme-linked immunosorbent assay. Two different electrophoretic methods were used to separate the Dactylis glomerata antigens. We compared the results obtained with classical immunoblot and with DMISA, for IgG4 and IgE quantification using sera from patients allergic to D. glomerata and purified blotted antigens present at the nanogram level.

  11. Protein blot assays specific for the discrimination of the centromere autoantigen, CENP-A, from human cells.

    PubMed

    Billings, P B; Martinez, A; Haselby, J A; Hoch, S O

    1993-09-01

    The Western or protein blot has proven to be a valuable resource in detecting discrete, immunoreactive antigen targets associated with a variety of autoimmune diseases. As the roster of autoantigens has expanded, it has become increasingly common to tailor specific gel or blot conditions to a particular polypeptide antigen. Two such assays are reported here as applied to the fractionation and visualization of human centromere protein (CENP-A), a centromere autoantigen associated with the rheumatic disease, systemic sclerosis. The centromere antigens are effectively solubilized in the presence of 1 M MgCl2 to allow for further purification. CENP-A copurifies with the histone proteins, primarily H3 and H4. The two CENP-A-specific protein blot assays separate CENP-A from the histone proteins and enhance CENP-A immunoreactivity. The first assay is based on the use of acid-urea gels with a Triton X-100 concentration chosen to maximize separation of CENP-A from all the histones. The second assay is based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to differentiate two very basic proteins of similar molecular weight, namely CENP-A and histone H3. For each gel system, a selective choice of associated immunoblot parameters allows for the reproducible discrimination of the CENP-A antigen. PMID:8223400

  12. Lectin blot studies on proteins of Myxobolus cerebralis, the causative agent of whirling disease.

    PubMed

    Knaus, Martin; El-Matbouli, Mansour

    2005-07-18

    It is known that Myxobolus cerebralis antigens, both surficial and secreted, are key modulators for, or targets of, host immune system compounds. We undertook SDS-PAGE glycoprotein characterisation of M. cerebralis developmental stages isolated from infected rainbow trout and Western blot analyses using selected biotin-labelled plant lectins (GSA-I, PHA-E, SJA, GSA-II) and anti-triactinomyxon polyclonal antibodies. Glycoproteins were isolated with lectin-affinity chromatography, and prominent bands were characterised by matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI/MS). We identified glycoproteins of M. cerebralis myxospores that contained carbohydrate motifs reactive with Phaseolus vulgaris erythroagglutinin (proteins 20 to 209 kDa, PHA-E), Sophora japonica agglutinin (proteins 7 to 70 kDa, SJA), Griffonia simplicifolia Agglutinin I (proteins 10 to 209 kDa, GSA-I) and G. simplicifolia Agglutinin II (proteins 5 to 40 kDa, GSA-II). Mcgp33, a glycoprotein isolated by lectin-affinity chromatography, was reactive with SJA (about 33 kDa). Antiserum produced against M. cerebralis triactinomyxons was found to have differences in the antigenicity of isolated glycoproteins from both M. cerebralis myxospores and actinospores. We also demonstrated modified antigen expression, especially involving the glycoprotein Mcgp33, in different developmental stages of M. cerebralis. PMID:16119891

  13. PCR-Reverse Blot Hybridization Assay for Screening and Identification of Pathogens in Sepsis

    PubMed Central

    Choi, Yeonim; Wang, Hye-Young; Lee, Gyusang; Park, Soon-Deok; Jeon, Bo-Young; Uh, Young; Kim, Jong Bae

    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-specific and 13 species-specific probes; it uses additional probes for antibiotic resistance genes, i.e., the mecA gene of methicillin-resistant Staphylococcus aureus (MRSA) and the vanA and vanB genes of vancomycin-resistant enterococci (VRE). The REBA Sepsis-ID test successfully identified clinical isolates and blood culture samples as containing Gram-positive bacteria, Gram-negative bacteria, or fungi. The results matched those obtained with conventional microbiological methods. For the REBA Sepsis-ID test, of the 115 blood culture samples tested, 47 (40.8%) and 49 (42.6%) samples were identified to the species and genus levels, respectively, and the remaining 19 samples (16.5%), which included five Gram-positive rods, were identified as Gram-positive bacteria, Gram-negative bacteria, or fungi. The antibiotic resistances of the MRSA and VRE strains were identified using both conventional microbiological methods and the REBA Sepsis-ID test. In conclusion, the REBA Sepsis-ID test developed for this study is a fast and reliable test for the identification of Gram-positive bacteria, Gram-negative bacteria, fungi, and antibiotic resistance genes (including mecA for MRSA and the vanA and vanB genes for VRE) in bloodstream infections. PMID:23447637

  14. PCR and blot hybridization for rapid identification of Haloferax species.

    PubMed

    Asker, Dalal; Ohta, Yoshiyuki

    2002-05-01

    Based on the amplification of a 16S rDNA, a PCR assay for the identification of species of Haloferax to genus level was performed. Two variable regions of the 16S rDNA in Haloferax spp. were selected as genus-specific primers for the PCR assay and hybridization probe. Five genera of halophilic Archaea and Escherichia coli were examined as outside groups. Using this approach, all strains of Haloferax spp. were positive. In contrast, all species belonging to the most closely related genera, including Natrinema, Halorubrum, Halobacterium, and Haloarcula, were negative. In addition, the mass bloom of halophilic Archaea that develops in the El-Mallahet saltern of Alexandria City was positive using the same approach. This assay, which does not require pure cultures of microorganisms, is a specific and rapid method for identifying Haloferax spp. in hypersaline environments.

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

    PubMed

    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.

  16. Molecular combing compared to Southern blot for measuring D4Z4 contractions in FSHD.

    PubMed

    Vasale, Jessica; Boyar, Fatih; Jocson, Michael; Sulcova, Vladimira; Chan, Patricia; Liaquat, Khalida; Hoffman, Carol; Meservey, Marc; Chang, Isabell; Tsao, David; Hensley, Kerri; Liu, Yan; Owen, Renius; Braastad, Corey; Sun, Weimin; Walrafen, Pierre; Komatsu, Jun; Wang, Jia-Chi; Bensimon, Aaron; Anguiano, Arturo; Jaremko, Malgorzata; Wang, Zhenyuan; Batish, Sat; Strom, Charles; Higgins, Joseph

    2015-12-01

    We compare molecular combing to Southern blot in the analysis of the facioscapulohumeral muscular dystrophy type 1 locus (FSHD1) on chromosome 4q35-qter (chr 4q) in genomic DNA specimens sent to a clinical laboratory for FSHD testing. A de-identified set of 87 genomic DNA specimens determined by Southern blot as normal (n = 71), abnormal with D4Z4 macrosatellite repeat array contractions (n = 7), indeterminate (n = 6), borderline (n = 2), or mosaic (n = 1) was independently re-analyzed by molecular combing in a blinded fashion. The molecular combing results were identical to the Southern blot results in 75 (86%) of cases. All contractions (n = 7) and mosaics (n = 1) detected by Southern blot were confirmed by molecular combing. Of the 71 samples with normal Southern blot results, 67 (94%) had concordant molecular combing results. The four discrepancies were either mosaic (n = 2), rearranged (n = 1), or borderline by molecular combing (n = 1). All indeterminate Southern blot results (n = 6) were resolved by molecular combing as either normal (n = 4), borderline (n = 1), or rearranged (n = 1). The two borderline Southern blot results showed a D4Z4 contraction on the chr 4qA allele and a normal result by molecular combing. Molecular combing overcomes a number of technical limitations of Southern blot by providing direct visualization of D4Z4 macrosatellite repeat arrays on specific chr 4q and chr 10q alleles and more precise D4Z4 repeat sizing. This study suggests that molecular combing has superior analytical validity compared to Southern blot for determining D4Z4 contraction size, detecting mosaicism, and resolving borderline and indeterminate Southern blot results. Further studies are needed to establish the clinical validity and diagnostic accuracy of these findings in FSHD. PMID:26420234

  17. Analysis of common mitochondrial DNA mutations by allele-specific oligonucleotide and Southern blot hybridization.

    PubMed

    Tang, Sha; Halberg, Michelle C; Floyd, Kristen C; Wang, Jing

    2012-01-01

    Mitochondrial disorders are clinically and genetically heterogeneous. There are a set of recurrent point mutations in the mitochondrial DNA (mtDNA) that are responsible for common mitochondrial diseases, including MELAS (mitochondrial encephalopathy, lactic acidosis, stroke-like episodes), MERRF (myoclonic epilepsy and ragged red fibers), LHON (Leber's hereditary optic neuropathy), NARP (neuropathy, ataxia, retinitis pigmentosa), and Leigh syndrome. Most of the pathogenic mtDNA point mutations are present in the heteroplasmic state, meaning that the wild-type and mutant-containing mtDNA molecules are coexisting. Clinical heterogeneity may be due to the degree of mutant load (heteroplasmy) and distribution of heteroplasmic mutations in affected tissues. Additionally, Kearns-Sayre syndrome and Pearson syndrome are caused by large mtDNA deletions. In this chapter, we describe a multiplex PCR/allele-specific oligonucleotide (ASO) hybridization method for the screening of 13 common point mutations. This method allows the detection of low percentage of mutant heteroplasmy. In addition, a nonradioactive Southern blot hybridization protocol for the analysis of mtDNA large deletions is also described. PMID:22215554

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed

    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.

  20. Multiplexed microfluidic blotting of proteins and nucleic acids by parallel, serpentine microchannels.

    PubMed

    He, Sha; Zhang, Yi; Wang, Pei; Xu, Xingzhi; Zhu, Kui; Pan, Wenying; Liu, Wenwen; Cai, Kaiyong; Sun, Jiashu; Zhang, Wei; Jiang, Xingyu

    2015-01-01

    This work develops a high-throughput, high-efficiency and straightforward microfluidic blotting method for analyzing proteins and nucleic acids. Sample solutions containing antibodies (for protein detection) or hybridization probes (for nucleic acid detection) are introduced into the parallel, serpentine microchannels to specifically recognize the immobilized targets on the substrate, achieving the identification of multiple targets in multiple samples simultaneously. The loading control, molecular weight markers, and antigen/antibody titration are designed and integrated into the microfluidic chip, thus allowing for the quantification of proteins and nucleic acids. Importantly, we could easily distinguish the adjacent blotting bands inside parallel microchannels, which may be difficult to achieve in conventional blotting. The small dimensions of microfluidic channels also help to reduce the amount of probing molecules and to accelerate the biochemical reaction. Our microfluidic blotting could bypass the steps of blocking and washing, further reducing the operation time and complexity.

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

    PubMed

    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.

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

    PubMed

    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. PMID:27065589

  3. 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.

    PubMed

    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

  4. BLOT: A mesh and curve plot program for the output of a finite element analysis

    SciTech Connect

    Gilkey, A.P.; Glick, J.H.

    1989-06-01

    BLOT is a graphics program for post-processing of finite element analysis output that is presented in the EXODUS database format. It is command driven with free-format input and can drive any graphics device supported by the Sandia Virtual Device Interface. BLOT produces mesh plots with various representations of the analysis output variables. The major mesh plot capabilities are deformed mesh plots, line contours, banded contours, vector plots of two or three variables (e.g., velocity vectors), and symbol plots of scalar variables (e.g., temperature). Pathlines of analysis variables can also be drawn on the mesh. BLOT's features include element selection by material, element birth and death, multiple views for combining several displays on each plot, symmetry mirroring, and node and element numbering. BLOT can also produce X-Y curve plots of the analysis variables. BLOT generates time-versus-variable plots or variable-versus-variable plots. It also generates distance versus-variable plots at selected time steps where the distance is the accumulated distance between pairs of nodes or element centers. 14 refs.

  5. Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides

    PubMed Central

    Akiyama, Yasutoshi; Itoh, Kunihiko; Nankumo, Shinnosuke; Shima, Hisato; Kikuchi, Koichi; Takeuchi, Yoichi; Elkordy, Alaa; Suzuki, Takehiro; Niizuma, Kuniyasu; Ito, Sadayoshi; Tomioka, Yoshihisa; Abe, Takaaki

    2015-01-01

    The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism. PMID:26606401

  6. Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.

    PubMed

    Mishima, Eikan; Jinno, Daisuke; Akiyama, Yasutoshi; Itoh, Kunihiko; Nankumo, Shinnosuke; Shima, Hisato; Kikuchi, Koichi; Takeuchi, Yoichi; Elkordy, Alaa; Suzuki, Takehiro; Niizuma, Kuniyasu; Ito, Sadayoshi; Tomioka, Yoshihisa; Abe, Takaaki

    2015-01-01

    The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism. PMID:26606401

  7. Mammalian. cap alpha. -polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots

    SciTech Connect

    SenGupta, D.N.; Kumar, P.; Zmudzka, B.Z.; Coughlin, S.; Vishwanatha, J.K.; Robey, F.A.; Parrott, C.; Wilson, S.H.

    1987-02-10

    A new polyclonal antibody against the ..cap alpha..-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cell..cap alpha..-polymerase. The antibody neutralized ..cap alpha..-polymerase activity and was strong and specific for the ..cap alpha..-polymerase catalytic polypeptide (M/sub r/ 183,000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in lambdagt11. A positive phage was identified and plaque purified. This phage, designated lambdapol..cap alpha..1.2, also was found to be positive with an antibody against Drosophila ..cap alpha..-polymerase. The insert in lambdapol..cap alpha..1.2 (1183 base pairs) contained a poly(A) sequence at the 3' terminus and a short in-phase open reading frame at the 5' terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified ..cap alpha..-polymerase by enzyme-linked immunosorbent assay and was capable of immunoprecipitating ..cap alpha..-polymerase. This indicated the lambdapol..cap alpha..1.2 insert encoded an ..cap alpha..-polymerase epitope and suggested that the cDNA corresponded to an ..cap alpha..-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(A+) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding ..cap alpha..-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(A+) RNA revealed that this mRNA is approx.5.4 kilobases.

  8. Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis.

    PubMed

    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.

  9. Evaluation of a Treponema pallidum western immunoblot assay as a confirmatory test for syphilis.

    PubMed Central

    Byrne, R E; Laska, S; Bell, M; Larson, D; Phillips, J; Todd, J

    1992-01-01

    Tests for the detection of antibodies to Treponema pallidum are recommended for the confirmation of reactive nontreponemal test results and the accurate diagnosis of syphilis. The present-day use of Western blot (immunoblot) technology for the diagnosis of retroviruses prompted the development and evaluation of a Western blot assay with whole-cell T. pallidum as the antigen. The assay detected antibodies in syphilitic serum or plasma from dilutions of specimens incubated overnight with test strips. A test was considered positive when at least three of four major antigens having molecular masses of 15.5, 17, 44.5, and 47 kDa were detected. The Western blot assay had 93.8% sensitivity and 100% specificity for clinically defined samples. The Western blot assay was compared with double-staining fluorescent treponemal antibody absorption [FTA-ABS (DS)], which had a sensitivity and a specificity of 91.7 and 92.0%, respectively. Dilution series studies of syphilis-positive specimens indicated that the Western blot assay has an endpoint of reactivity at least 3 to 4 serial dilutions greater than that for FTA-ABS (DS). Overall, the greater than 95% agreement between the Western blot assay and FTA-ABS (DS) for clinically defined specimens indicates that the sensitivity of the Western blot assay is equal to or greater than that of FTA-ABS (DS). The Western blot assay demonstrated no false-positive or equivocal reactivities for nonsyphilitic specimens, including normal specimens (both plasma and serum), biological false-positives, and specimens with elevated gamma globulin or antinuclear antibody. We conclude that the high sensitivity and specificity of the T. pallidum Western blot assay, together with its simplicity and objectivity, make it a good confirmatory test for syphilis. Images PMID:1734042

  10. Western Samoa.

    PubMed

    1985-12-01

    This discussion of Western Samoa, which lies 2575 km northeast of Auckland, New Zealand, focuses on the following: geography; the people; history; government; political conditions; the economy; foreign relations; and relations the US. The population of Western Samoa, as of 1985, totals 163,000 with an annual growth rate of 0.9%. The infant mortality rate is 13/1000; life expectancy is 65 years. The main islands are formed ranges of extinct volcanoes. Volcanic activity last occurred in 1911. More than 2000 years age, waves of Polynesians migrated from Southeast Asia to the Samoan Islands. Samoans are the 2nd largest Polynesian group, after the Maoris of New Zealand, and speak a Polynesian dialect. Samoans have tended to retain their traditional ways despite exposure to European influence for more than 150 years. Most Samoans live within the traditional social system based on an extended family group, headed by a chief. Western Samoans are Christian. Education is free but not compulsory. In 1967, 95% of the children of primary school age attended school. From 1947 to 1961, a series of constitutional advances, assisted by visits from UN missions, brought Western Samoa from dependent status to self-government and finally to independence. The 1960 constitution is based on the British pattern of parliamentary democracy, modified to take Samoan customs into account. The present head of state holds his position for life. Future heads of state will be elected by the Legislative Assembly for 5-year terms. The Parliament consists of the Legislative Assembly and the head of state. The Supreme Court is the superior court of record and has full jurisdiction in civil, criminal, and constitutional matters. The "matai" of chief system still dominates the politics of Western Samoa, although several political parties have been formed and seem to be taking root. The "matai" system is a predominantly conservative force but does provide for change. Western Samoa is predominantly

  11. Detection of human cytomegalovirus by slot-blot hybridization assay employing oligo-primed /sup 32/P-labelled probe

    SciTech Connect

    Agha, S.A.; Coleman, J.C.; Selwyn, S.; Mahmound, L.A.; Abd-Elaal, A.M.; Archard, L.C.

    1988-12-01

    A /sup 32/P-labelled Hind III-0 DNA fragment (nine Kilobases; Kb) from human cytomegalovirus AD-169 (HCMV) was used in slot-blot hybridization assay for the detection of HCMV in clinical samples. The results obtained with DNA hybridization assay (DNA HA) were compared with virus isolation using conventional tube cell culture (CTC) and centrifugation vial culture (CVC), immunofluorescence (IF), and complement fixation test (CFT). Of 15 CTC-positive samples, 13 were positive with DNA HA (sensitivity 86.7%). Also, 14 additional samples were DNA HA-positive but CTC-negative. CVC and/or IF confirmed the diagnosis in nine of 14; the remaining five samples were from three patients who showed fourfold rising antibody titre by CFT. Although DNA HA using /sup 32/P-labelled probes is relatively cumbersome and expensive, it is a valuable test for quantitation of viral shedding in patients with HCMV infections who may benefit from antiviral therapy.

  12. The squash blot technique and the detection of Leishmania major in Phlebotomus papatasi in Tunisia.

    PubMed

    Esseghir, S; Ftaïti, A; Ready, P D; Khadraoui, B; Zaafouri, B; Dellagi, K; Ben Ismaïl, R

    1993-01-01

    This study describes the preliminary applications of the squash blot technique in Tunisia, to detect Leishmania major in naturally infected Phlebotomus papatasi. 309 P. papatasi among 364 female sandflies squashed on to nylon Gene Screen DNA transfer membranes, were identified using the 3.2 kb ribosomal P. papatasi specific DNA probe described by Ready et al. A second hybridization using the Taq1 DNA probe described by Smith et al. (1989) allowed the detection and identification of the parasite in 15 (4.9%) of these P. papatasi specimens. The dissection of P. papatasi females during the same period and from the same biotopes showed an infection rate of 7.9% (9 positives among 113 dissected). The t proportion comparison test indicated that there is no significant statistical difference between the dissection and the squash blot technique for the estimation of infection rates of P. papatasi.

  13. Mapping Point Mutations in the Drosophila Rosy Locus Using Denaturing Gradient Gel Blots

    PubMed Central

    Gray, M.; Charpentier, A.; Walsh, K.; Wu, P.; Bender, W.

    1991-01-01

    Mutations within the rosy locus of Drosophila were mapped using blots of genomic DNA fragments separated on denaturing gradient gels. DNA sequence differences between otherwise identical small rosy DNA fragments were detected among the mutants as mobility shifts on the blots. Mutations were mapped to within a few hundred base pairs of rosy sequence in 100 of 130 mutants tested--a 77% detection rate. The sequence changes in 43 rosy mutations are presented; all but six of these were single base changes. Thirty-four of 36 sequenced mutations induced by the alkylating agents N-ethyl-N-nitrosourea and ethyl methanesulfonate were transitions. All of the mutations mapped in the rosy transcription unit. Twenty-three of the 43 sequenced mutations change the predicted rosy gene polypeptide sequence; the remainder would interrupt protein translation (17), or disrupt mRNA processing (3). PMID:1901817

  14. A Dual Color Southern Blot to Visualize Two Genomes or Genic Regions Simultaneously

    PubMed Central

    Zavala, Anamaria G.; Kulkarni, Amit S.; Fortunato, Elizabeth A.

    2014-01-01

    This report describes the development of a novel dual color Southern protocol to visualize two distinct genomes or genic regions simultaneously on a single Southern blot. The blot is developed with IRDye-conjugated antibody (Ab) and streptavidin that recognize Digoxigenin- (Dig) or biotin-labeled probes, respectively and visualized on an infrared imager. This protocol was validated by visualizing viral and host genomes of human cytomegalovirus (HCMV)-infected human fibroblasts. This technique utilizes extremely sensitive fluorescent imaging, allowing the detection of nanogram quantities of DNA, as opposed to microgram quantities needed in Southerns using radioactively labeled probes, and eliminates the inherent loss in signal after stripping and reprobing a Southern blot. The probes are labeled with non-radioactive Dig and biotin and can be stored for extended periods of time. This protocol will aid in studies of any system with two genomes, such as cells infected with numerous types of microorganisms (virus/parasites/bacteria), or studies of mitochondrial and nuclear DNA within the same cells. PMID:24389128

  15. Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis.

    PubMed

    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. PMID:27329815

  16. RNA Colony Blot Hybridization Method for Enumeration of Culturable Vibrio cholerae and Vibrio mimicus Bacteria▿

    PubMed Central

    Grim, Christopher J.; Zo, Young-Gun; Hasan, Nur A.; Ali, Afsar; Chowdhury, Wasimul B.; Islam, Atiqul; Rashid, Mohammed H.; Alam, Munirul; Morris, J. Glenn; Huq, Anwar; Colwell, Rita R.

    2009-01-01

    A species-specific RNA colony blot hybridization protocol was developed for enumeration of culturable Vibrio cholerae and Vibrio mimicus bacteria in environmental water samples. Bacterial colonies on selective or nonselective plates were lysed by sodium dodecyl sulfate, and the lysates were immobilized on nylon membranes. A fluorescently labeled oligonucleotide probe targeting a phylogenetic signature sequence of 16S rRNA of V. cholerae and V. mimicus was hybridized to rRNA molecules immobilized on the nylon colony lift blots. The protocol produced strong positive signals for all colonies of the 15 diverse V. cholerae-V. mimicus strains tested, indicating 100% sensitivity of the probe for the targeted species. For visible colonies of 10 nontarget species, the specificity of the probe was calculated to be 90% because of a weak positive signal produced by Grimontia (Vibrio) hollisae, a marine bacterium. When both the sensitivity and specificity of the assay were evaluated using lake water samples amended with a bioluminescent V. cholerae strain, no false-negative or false-positive results were found, indicating 100% sensitivity and specificity for culturable bacterial populations in freshwater samples when G. hollisae was not present. When the protocol was applied to laboratory microcosms containing V. cholerae attached to live copepods, copepods were found to carry approximately 10,000 to 50,000 CFU of V. cholerae per copepod. The protocol was also used to analyze pond water samples collected in an area of cholera endemicity in Bangladesh over a 9-month period. Water samples collected from six ponds demonstrated a peak in abundance of total culturable V. cholerae bacteria 1 to 2 months prior to observed increases in pathogenic V. cholerae and in clinical cases recorded by the area health clinic. The method provides a highly specific and sensitive tool for monitoring the dynamics of V. cholerae in the environment. The RNA blot hybridization protocol can also be

  17. Analysis of sperm antigens by sodium dodecyl sulfate gel/protein blot radioimmunobinding method

    SciTech Connect

    Lee, C.Y.G.; Huang, Y.S.; Hu, P.C.; Gomel, V.; Menge, A.C.

    1982-06-01

    A radioimmunobinding method based on the blotting of renatured proteins from sodium dodecyl sulfate gels on to nitrocellulose filter papers was developed to analyze the sperm antigens that elicit serum anti-sperm antibodies. In rabbits, serum anti-sperm antibodies were raised by immunization with homologous epididymal spermatozoa mixed with complete Freund's adjuvant. The raised antisera from either male or female rabbits were shown to react with three major sperm protein bands on sodium dodecyl sulfate gels with the corresponding molecular weights of about 70,000 +/- 5000, 14,000, and 13,000, respectively. In humans, the monoclonal antibodies against human sperm were raised by a hybridoma technique. Out of six independent hybrid cell lines that were generated, three of them were shown to secrete immunoglobulins that react with the same two protein bands on sodium dodecyl sulfate gels, which have the approximate molecular weight of 10,000. The same procedure was also used to analyze human serum samples that were shown to contain anti-sperm antibodies by the known techniques. Unique sperm antigens that elicit anti-sperm antibodies in humans were identified and correlated. The results of this study suggest that sodium dodecyl sulfate gel/protein blot radioimmunobinding method may be a sensitive and useful tool for the study of sperm antigens that elicit autoimmune responses and their association with human infertility.

  18. A sensitive non-radioactive northern blot method to detect small RNAs.

    PubMed

    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. PMID:20081203

  19. Selective analysis of lipids by thin-layer chromatography blot matrix-assisted laser desorption/ionization imaging mass spectrometry.

    PubMed

    Zaima, Nobuhiro; Goto-Inoue, Naoko; Adachi, Kohsuke; Setou, Mitsutoshi

    2011-01-01

    Thin-layer chromatography (TLC) is an essential method for food composition analyses such as lipid nutrition analysis. TLC can be used to obtain information about the lipid composition of foods; however, it cannot be used for analyses at the molecular level. Recently we developed a new method that combines matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) with TLC-blotting (TLC-Blot-MALDI-IMS). The combination of MALDI-IMS and TLC blotting enabled detailed and sensitive analyses of lipids. In this study, we applied TLC-Blot-MALDI-IMS for analysis of major phospholipids extracted from bluefin tuna. We showed that TLC-Blot-MALDI-IMS analysis could visualize and identify major phospholipids such as phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin.

  20. A bead-based western for high-throughput cellular signal transduction analyses

    PubMed Central

    Treindl, Fridolin; Ruprecht, Benjamin; Beiter, Yvonne; Schultz, Silke; Döttinger, Anette; Staebler, Annette; Joos, Thomas O.; Kling, Simon; Poetz, Oliver; Fehm, Tanja; Neubauer, Hans; Kuster, Bernhard; Templin, Markus F.

    2016-01-01

    Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. By combining gel-based protein separation with immobilization on microspheres, hundreds of replicas of the initial blot are created, thus enabling the comprehensive analysis of limited material, such as cells collected by laser capture microdissection, and extending traditional western blotting to reach proteomic scales. The combination of molecular weight resolution, sensitivity and signal linearity on an automated platform enables the rapid quantification of hundreds of specific proteins and protein modifications in complex samples. This high-throughput western blot approach allowed us to identify and characterize alterations in cellular signal transduction that occur during the development of resistance to the kinase inhibitor Lapatinib, revealing major changes in the activation state of Ephrin-mediated signalling and a central role for p53-controlled processes. PMID:27659302

  1. Restoration of antibody binding to blotted meningococcal outer membrane proteins using various detergents.

    PubMed

    Wedege, E; Bryn, K; Frøholm, L O

    1988-10-01

    Restoration of IgG antibody binding to heat-denatured meningococcal outer membrane proteins has been studied on immunoblots with a series of 14 detergents. Nitrocellulose strips with the blotted proteins were incubated with the detergents and sera from human volunteers vaccinated with meningococcal membrane proteins. Zwitterionic and ionic detergents, containing substituted quarternary ammonium or amino groups with a minimum of 10 C atoms in the alkyl chain, restored the antigenicity of the serotype-specific class 2 porin protein. The concentrations of the Zwittergent detergents necessary for activation decreased with increasing alkyl chain length of the homologues. Only zwitterionic detergents renatured the class 1 protein. Both proteins were weakly antigenic in the presence of the nonionic detergents Triton X-100 and Tween 20. Meningococcal lipopolysaccharide restored antibody binding to the porin, but not to the class 1 protein. Similar concentrations of lipopolysaccharides from two other gram-negative bacteria had no effect.

  2. Rotavirus detection by dot blot hybridization assay using a non-radioactive synthetic oligodeoxynucleotide probe.

    PubMed Central

    Fernández, J.; Sandino, A.; Yudelevich, A.; Avendaño, L. F.; Venegas, A.; Hinrichsen, V.; Spencer, E.

    1992-01-01

    A synthetic oligodeoxynucleotide of 40 nucleotides corresponding to nucleotides 33-72 of the gene coding for the viral protein VP7 of rotavirus, was used as a nucleic acid probe to develop a non-radioactive hybridization method for rotavirus detection. The probe was labelled at the 3' end with biotin-7-dATP. The sensitivity and specificity of the dot blot hybridization assay for rotavirus detection was evaluated with 303 stool specimens. The results indicate that the hybridization assay has a higher sensitivity than both PAGE and EIA. Among the rotavirus strains tested 37 different electropherotypes were found. The results suggest that rotavirus diagnosis by dot hybridization using a non-radioactive probe may become routine laboratory procedure because it is simple, highly specific and very sensitive. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:1312480

  3. Reexamination of alcohol dehydrogenase structural mutants in Drosophila using protein blotting

    SciTech Connect

    Hollocher, H.; Place, A.R.

    1987-06-01

    Using protein blotting and an immuno-overlay procedure, the authors have reexamined the cross-reacting material produced by ADH null-activity mutants generated with ethyl methanesulfonate (EMS). Of the 13 mutants, 11 have an immunodetectable polypeptide of wild-type size. The native and urea denatured isoelectric points (pI) establish that 7 of 13 of the mutations have no effect on protein charge. The electrophoretic mobilities of each variant on increasing percent acrylamide gels (Ferguson analysis), reveal that 9 of the 11 immunodetectable mutations have retained the ability form dimers under native conditions. None of the inactive mutant proteins has the ability to form the adduct-bound isozyme. The authors have found no correlation between protein pI and i vivo stability. The observed frequencies of specific charge class alterations do not dispute the propensity of G:A transitions previously found for EMS mutagenesis.

  4. Food additives

    PubMed Central

    Spencer, Michael

    1974-01-01

    Food additives are discussed from the food technology point of view. The reasons for their use are summarized: (1) to protect food from chemical and microbiological attack; (2) to even out seasonal supplies; (3) to improve their eating quality; (4) to improve their nutritional value. The various types of food additives are considered, e.g. colours, flavours, emulsifiers, bread and flour additives, preservatives, and nutritional additives. The paper concludes with consideration of those circumstances in which the use of additives is (a) justified and (b) unjustified. PMID:4467857

  5. Identification of Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans with novel markers and using a dot blot platform coupled with automatic data analysis.

    PubMed

    Albuquerque, Pedro; Caridade, Cristina M R; Marcal, Andre R S; Cruz, Joana; Cruz, Leonor; Santos, Catarina L; Mendes, Marta V; Tavares, Fernando

    2011-08-15

    Phytosanitary regulations and the provision of plant health certificates still rely mainly on long and laborious culture-based methods of diagnosis, which are frequently inconclusive. DNA-based methods of detection can circumvent many of the limitations of currently used screening methods, allowing a fast and accurate monitoring of samples. The genus Xanthomonas includes 13 phytopathogenic quarantine organisms for which improved methods of diagnosis are needed. In this work, we propose 21 new Xanthomonas-specific molecular markers, within loci coding for Xanthomonas-specific protein domains, useful for DNA-based methods of identification of xanthomonads. The specificity of these markers was assessed by a dot blot hybridization array using 23 non-Xanthomonas species, mostly soil dwelling and/or phytopathogens for the same host plants. In addition, the validation of these markers on 15 Xanthomonas spp. suggested species-specific hybridization patterns, which allowed discrimination among the different Xanthomonas species. Having in mind that DNA-based methods of diagnosis are particularly hampered for unsequenced species, namely, Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans, for which comparative genomics tools to search for DNA signatures are not yet applicable, emphasis was given to the selection of informative markers able to identify X. fragariae, X. axonopodis pv. phaseoli, and X. fuscans subsp. fuscans strains. In order to avoid inconsistencies due to operator-dependent interpretation of dot blot data, an image-processing algorithm was developed to analyze automatically the dot blot patterns. Ultimately, the proposed markers and the dot blot platform, coupled with automatic data analyses, have the potential to foster a thorough monitoring of phytopathogenic xanthomonads. PMID:21705524

  6. Detection and typing of human papillomavirus using the Vira Type "in situ" kit: comparison with a conventional dot blot technique.

    PubMed Central

    Faulkner-Jones, B E; Bellomarino, V M; Borg, A J; Orzeszko, K; Garland, S M

    1990-01-01

    A new commercial kit (Vira Type "in situ", Life Technologies, Inc., Molecular Diagnostics Division, Guithersburg, Maryland, USA) for the detection of human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33 and 35 in routinely processed human anogenital tissue was compared with a conventional dot blot assay for HPV 6, 11, 16 and 18. Both systems use double-stranded genomic DNA probes for the detection of type specific HPV DNA. The probes used on the dot blots were labelled with 32P and visualised autoradiographically. The Vira Type probes were labelled with biotin and visualised using a streptavidin-alkaline phosphatase conjugate with NBT-BCIP substrate. Biopsy specimens from the cervix, vagina, and vulva of 46 women were processed by both methods and compared. The histological diagnoses ranged from benign changes, to dysplasia, and invasive carcinoma. Overall, 50% of biopsy specimens were positive for HPV DNA by dot blot hybridisation; only 39% were positive by Vira Type in situ hybridisation. Three of the specimens positive by the Vira Type "in situ" kit showed no cross hybridisation and were the same HPV type as the dot blot. A further 13 showed hybridisation, but the showed cross hybridisation, but the to the dot blot results. One biopsy specimen was positive for different HPV types by the two tests and one was positive by Vira Type and negative by dot blot. Six biopsy specimens were negative by Vira Type but positive by dot blot. It is concluded that the Vira Type "in situ" kit has a similar specificity but lower sensitivity than the dot blot hybridisation method for the detection of HPV DNA. Images PMID:2175755

  7. [Measurement of antiganglioside autoantibodies by immunodot-blot assay: clinical importance in peripheral neuropathies].

    PubMed

    Caudie, C; Vial, C; Petiot, P; Bancel, J; Later, R; Gonnaud, P M

    1999-01-01

    We retrospectively evaluated measurement data and clinical relevance of autoantibodies to gangliosides in peripheral neuropathies (PN). The IgG and IgM antiganglioside autoantibodies were determined by our own immunodot-blot assay on membrane and by enzyme-linked immunosorbent assay (Elisa) in sera of 1,342 patients with peripheral neuropathies. Anti-GM1 and anti-GD1b autoantibodies formed a part of the normal autoantibody repertoire and were common place in 12% of normal subjects and in 14% of disease control groups. Polyclonal IgM antiganglioside autoantibodies were detected in chronic PN, polyclonal IgG antiganglioside autoantibodies were detected in acute PN. Polyclonal IgM anti-GM1 and anti-GD1b autoantibodies were detected in 35 patients out of 48 with treatable multifocal motor neuropathy with persistent conduction blocks. These autoantibodies well discriminated between suspected motor peripheral neuropathies and motor neuron diseases (sensitivity 73%, specificity 83%, positive predictive value 60%, negative predictive value 91%). Monoclonal IgM autoantibodies reacted strongly with gangliosides in 15 patients out of 77 with M-IgM neuropathy (19%). M-IgM autoantibodies differed in their fine specificities with different principal target antigens as demonstrated with cross-reactivity. Such findings provide further evidence for a relationship between neurological syndromes and antiganglioside antibody profiles and also suggest that different gangliosides could be principal target antigens such as GM1, GD1b, GT1b, GD1a or GM2. Polyclonal IgG anti-GM1 and anti-GD1b autoantibodies were detected in 21 patients out of 22 with acute motor axonal Guillain-Barré syndrome with antecedent of infection by Campylobacter jejuni, polyclonal IgG anti-GQ1b autoantibodies in 9 patients out of 10 with Miller-Fisher syndrome. Detection of antiganglioside autoantibodies by immunodot-blot assay which is simple and quick in testing a large panel of gangliosides has become very

  8. Rapid detection of intestinal pathogens in fecal samples by an improved reverse dot blot method

    PubMed Central

    Xing, Jian-Ming; Zhang, Su; Du, Ying; Bi, Dan; Yao, Li-Hui

    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. PMID:19469006

  9. Retrospective Study of Hemoparasites in Cattle in Southern Italy by Reverse Line Blot Hybridization

    PubMed Central

    CECI, Luigi; IARUSSI, Fabrizio; GRECO, Beatrice; LACINIO, Rosanna; FORNELLI, Stefania; CARELLI, Grazia

    2014-01-01

    ABSTRACT Tick-borne diseases are widespread in tropical and temperate regions and are responsible for important economic losses in those areas. In order to assess the presence and prevalence of various pathogens in southern Italy, we retrospectively analyzed cattle blood samples collected for a previous study in 2000 using reverse line blot (RLB) hybridization. The study had been carried out in three regions of southern Italy on 1,500 randomly selected and apparently healthy adult cattle. RLB showed that 43.7% of the cattle were positive for nine different species of hemoparasites with either a single infection or a mixed infection. Theileria buffeli was the most common species found, being present in 27.3% of the animals, followed by Anaplasma marginale in 18.1%, Anaplasma centrale in 13.8%, Babesia bigemina and Anaplasma bovis in 4.2%, Anaplasma phagocytophilum in 1.7%, Babesia bovis in 1.6%, Babesia major in 0.2% and Babesia divergens in 0.1%. Complete blood counts showed different degrees of anemia in 363 animals (24.2%) and of these, 169 were RLB-positive for at least one pathogen. Among the ticks that were collected from the cattle, the following species were identified: Rhipicephalus bursa, Ixodes ricinus, Hyalomma marginatum, Boophilus annulatus, Dermacentor marginatus and Haemaphysalis (sulcata, parva, inermis and punctata). The results obtained confirmed the spread of endemic tick-borne pathogens in the regions studied. PMID:24614604

  10. Pairwise detection of site-specific receptor phosphorylations using single-molecule blotting

    PubMed Central

    Kim, Kyung Lock; Kim, Daehyung; Lee, Seongsil; Kim, Su-Jeong; Noh, Jung Eun; Kim, Joung-Hun; Chae, Young Chan; Lee, Jong-Bong; Ryu, Sung Ho

    2016-01-01

    Post-translational modifications (PTMs) of receptor tyrosine kinases (RTKs) at the plasma membrane (PM) determine the signal transduction efficacy alone and in combination. However, current approaches to identify PTMs provide ensemble results, inherently overlooking combinatorial PTMs in a single polypeptide molecule. Here, we describe a single-molecule blotting (SiMBlot) assay that combines biotinylation of cell surface receptors with single-molecule fluorescence microscopy. This method enables quantitative measurement of the phosphorylation status of individual membrane receptor molecules and colocalization analysis of multiple immunofluorescence signals to directly visualize pairwise site-specific phosphorylation patterns at the single-molecule level. Strikingly, application of SiMBlot to study ligand-dependent epidermal growth factor receptor (EGFR) phosphorylation, which is widely thought to be multi-phosphorylated, reveals that EGFR on cell membranes is hardly multi-phosphorylated, unlike in vitro autophosphorylated EGFR. Therefore, we expect SiMBlot to aid understanding of vast combinatorial PTM patterns, which are concealed in ensemble methods, and to broaden knowledge of RTK signaling. PMID:27009355

  11. Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose.

    PubMed

    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.

  12. Reverse line blot macroarray for simultaneous detection and characterization of four biological warfare agents.

    PubMed

    Vanlalhmuaka; Thavachelvam, Kulanthaivel; Tuteja, Urmil; Sarika, Kumari; Nagendra, Suryanarayana; Kumar, Subodh

    2013-03-01

    The need for a rapid detection and characterization of biowarfare (BW) agents cannot be over emphasized. With diverse array of potential BW pathogen available presently, rapid identification of the pathogen is crucial, so that specific therapy and control measures can be initiated. We have developed a multiplex polymerase chain reaction based reverse line blot macroarray to simultaneously detect four pathogens of BW importance viz. Bacillus anthracis, Yersinia pestis, Brucella melitensis and Burkholderia pseudomallei. The multiplex PCR utilizes 14 pairs of primers targeting 18 specific markers. These markers include genes which are genus specific, species-specific chromosomal sequences and virulence markers of plasmid origin. The assay was evaluated on various human, environment and animal isolates. The assay w successful in simultaneous detection and characterization of isolates of the four pathogens on as a single platform with sensitivity ranging from 0.3 pg to 0.3 ng of genomic DNA. The assay was able to detect 5 × 10(2) cfu/ml for B. anthracis, 8 × 10(2) cfu/ml for Yersinia sp., 1.4 × 10(2) cfu/ml for B. melitensis and 4 × 10(2) cfu/ml for B. pseudomallei.

  13. Development of a dot blot assay using gene probes for the detection of enteroviruses in water

    SciTech Connect

    Margolin, A.B.

    1986-01-01

    Enteric viruses are viruses which replicate in the intestinal tract of man and animals. One mode of transmission for enteric viruses is the fecal-oral route. Drinking water which has been contaminated with sewage or sewage effluent has been implicated as a means for the spread of enteric viruses. Current methods for the detection of enteric viruses in water requires the use of animal cell culture. This technique has several drawbacks. More rapid techniques, such as fluorescent antibody or radioimmunoassay do not have the needed sensitivity to detect the low levels of virus found in contaminated water. An alternative technique for the detection of viruses in water was sought. Recent advances in recombinant DNA technology now makes it possible to detect viruses without the use of cell culture or antibodies. Gene probes that hybridize to the RNA of poliovirus and hepatitis A virus were tested for their ability to detect different enteric viruses. The probes were labeled with /sup 32/P dCTP and /sup 32/P dATP to a specific activity greater then 1.0 x 10/sup 9/ cpm/ug DNA. One infectious unit of poliovirus and hepatitis A virus was detected using labeled cDNA probes. Upon comparison, the dot blot assay was as sensitive as tissue culture for the detection of poliovirus in beef extract, secondary effluent, and tap water. Environmental samples, such as secondary effluent, reclaimed wastewater and unchlorinated drinking water were also assayed for poliovirus and hepatitis A virus with the use of gene probes. The results presented here offer an alternative method for screening water samples for the presence of enteric viruses.

  14. Validity of the Enzyme-linked Immunoelectrotransfer Blot (EITB) for naturally acquired porcine cysticercosis.

    PubMed

    Jayashi, César M; Gonzalez, Armando E; Castillo Neyra, Ricardo; Rodríguez, Silvia; García, Hector H; Lightowlers, Marshall W

    2014-01-17

    The Enzyme-linked Immunoelectrotransfer Blot (EITB) has been used widely as a screening test for Taenia solium cysticercosis in swine. However, the relation between seropositivity and infection in pig populations from endemic areas has not been well defined. The aim of this study is to relate EITB seropositivity with infection and infection burden, analyse the trade-off between sensitivity and specificity with various cut-off points for the EITB assay, and finally describe the serology changes in a cohort of rural pigs raised under natural conditions. A group of 107 pigs that were used as controls during a vaccination field trial in Peru was our study population. The prevalence of porcine cysticercosis determined by necropsy examination was 16.82% (18/107) in these animals. Using EITB reactivity to ≥ 1 band as a cut-off point for the assay, the sensitivity was 88.89% (65.29-98.62, 95% CI) and the specificity was 48.31% (37.59-59.16, 95% CI). Comparing other cut-off points, involving up to as many as 7 reactive bands, a reactivity of ≥ 3 bands provided the best trade-offs in sensitivity and specificity. Using this cut-off point for the assay, the sensitivity was 77.77% (52.36-93.59, 95% CI) and the specificity was 76.40% (66.22-84.76, 95% CI). A significant association was found between cyst counts over 100 cysts and reactivity to ≥ 3 bands in the EITB assay (Fisher's exact test, p<0.05). The results of this study suggest that the use of the EITB assay to study porcine cysticercosis may require setting different cut-offs under field and experimental conditions, and depending upon the objective of the screening process.

  15. Application of reverse dot blot hybridization to simultaneous detection and identification of harmful algae.

    PubMed

    Chen, Guo Fu; Zhang, Chun Yun; Wang, Yuan Yuan; Chen, Wen

    2015-07-01

    Warning and monitoring projects of harmful algal blooms require simple and rapid methods for simultaneous and accurate detection and identification of causative algae present in the environmental samples. Here, reverse dot blot hybridization (RDBH) was employed to simultaneously detect several harmful algae by using five representative bloom-forming microalgae along the Chinese coast. A set of specific probes for RDBH were developed by PCR, cloning, and sequencing of the internal transcribed spacer (ITS), alignment analysis, and probe design. Each probe was oligo (dT)-tailed and spotted onto positively charged nylon membrane to make up a low-density oligonucleotide array. Universal primers designed within the conserved regions were used to amplify the ITS sequences by using genomic DNA of target as templates. The digoxigenin (Dig)-labeled PCR products were denatured and then hybridized to the oligonucleotide array. The array produced a unique hybridization pattern for each target species differentiating them from each other. The preparations of oligonucleotide array and hybridization conditions were optimized. The developed RDBH demonstrated a detection limit up to 10 cells. The detection performance of RDBH was relatively stable and not affected by non-target species and the fixation time of target species over at least 30 days. The RDBH could recover all the target species from the simulated field samples and target species confirmed by the subsequent microscopy examination in the environmental samples. These results indicate that RDBH can be a new technical platform for parallel discrimination of harmful algae and is promising for environmental monitoring of these microorganisms.

  16. Application of reverse dot blot hybridization to simultaneous detection and identification of harmful algae.

    PubMed

    Chen, Guo Fu; Zhang, Chun Yun; Wang, Yuan Yuan; Chen, Wen

    2015-07-01

    Warning and monitoring projects of harmful algal blooms require simple and rapid methods for simultaneous and accurate detection and identification of causative algae present in the environmental samples. Here, reverse dot blot hybridization (RDBH) was employed to simultaneously detect several harmful algae by using five representative bloom-forming microalgae along the Chinese coast. A set of specific probes for RDBH were developed by PCR, cloning, and sequencing of the internal transcribed spacer (ITS), alignment analysis, and probe design. Each probe was oligo (dT)-tailed and spotted onto positively charged nylon membrane to make up a low-density oligonucleotide array. Universal primers designed within the conserved regions were used to amplify the ITS sequences by using genomic DNA of target as templates. The digoxigenin (Dig)-labeled PCR products were denatured and then hybridized to the oligonucleotide array. The array produced a unique hybridization pattern for each target species differentiating them from each other. The preparations of oligonucleotide array and hybridization conditions were optimized. The developed RDBH demonstrated a detection limit up to 10 cells. The detection performance of RDBH was relatively stable and not affected by non-target species and the fixation time of target species over at least 30 days. The RDBH could recover all the target species from the simulated field samples and target species confirmed by the subsequent microscopy examination in the environmental samples. These results indicate that RDBH can be a new technical platform for parallel discrimination of harmful algae and is promising for environmental monitoring of these microorganisms. PMID:25731086

  17. Performance of commercial reverse line blot assays for human papillomavirus genotyping.

    PubMed

    Steinau, Martin; Onyekwuluje, Juanita M; Scarbrough, Mariela Z; Unger, Elizabeth R; Dillner, Joakim; Zhou, Tiequn

    2012-05-01

    The performance of three line blot assays (LBAs), the Linear Array HPV genotyping assay (LA) (Roche Diagnostics), INNO-LiPA HPV Genotyping Extra (LiPA) (Innogenetics), and the reverse hybridization assay (RH) (Qiagen), was evaluated using quantitated whole genomic human papillomavirus (HPV) plasmids (types 6, 11, 16, 18, 31, 33, 35, 39, 51, 52, 56, 58, 59, and 68b) as well as epidemiologic samples. In a plasmid titration series, LiPA and RH did not detect 50 international units (IU) of HPV type 18 (HPV18) in the presence of 5 × 10(4) IU or more of HPV16. HPV DNA (1 to 6 types) in the plasmid challenges at 50 IU or genome equivalents (GE) were identified with an accuracy of 99.9% by LA, 97.3% by LiPA, and 95.4% by RH, with positive reproducibility of 99.8% (kappa = 0.992), 88.2% (kappa = 0.928), and 88.1% (kappa = 0.926), respectively. Two instances of mistyping occurred with LiPA. Of the 120 epidemiologic samples, 76 were positive for high-risk types by LA, 90 by LiPA, and 69 by RH, with a positive reproducibility of 87.3% (kappa = 0.925), 83.9% (kappa = 0.899), and 90.2% (kappa = 0.942), respectively. Although the assays had good concordance in the clinical samples, the greater accuracy and specificity in the plasmid panel suggest that LA has an advantage for internationally comparable genotyping studies.

  18. Induction of a protective antibody response to FMDV in mice following oral immunization with transgenic Stylosanthes spp. as a feedstuff additive.

    PubMed

    Wang, Dong Mei; Zhu, Jian Bo; Peng, Ming; Zhou, Peng

    2008-12-01

    The expression of antigens in transgenic plants has increasingly been used as an alternative to the classical methodologies for the development of experimental vaccines, and it remains one of the real challenges in this field to use transgenic plant-based vaccines effectively as feedstuff additives. We report herein the development of a new oral immunization system for foot and mouth disease with the structural protein VP1 of the foot and mouth disease virus (FMDV) produced in transgenic Stylosanthes guianensis cv. Reyan II. The transgenic plantlets were identified by polymerase chain reaction (PCR), Southern blotting, and northern blotting; and the production of VP1 protein in transgenic plants was confirmed and quantified by western blotting and enzyme-linked immunosorbent assays (ELISA). Six transformed lines were obtained, and the level of the expressed protein was 0.1-0.5% total soluble protein (TSP). Mice that were orally immunized using studded feedstuff mixed with desiccated powder of the transgenic plants developed a virus-specific immune response to the structural VP1 and intact FMDV particles. To our knowledge, this is the first report of transgenic plants expressing the antigen protein of FMDV as feedstuff additives that has demonstrated the induction of a protective systemic antibody response in animals. These results support the feasibility of producing edible vaccines from transgenic forage plants, and provide proof of the possibility of using plant-based vaccines as feedstuff additives. PMID:18651235

  19. Detection of Babesia and Theileria species infection in cattle from Portugal using a reverse line blotting method.

    PubMed

    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.

  20. Phosphazene additives

    DOEpatents

    Harrup, Mason K; Rollins, Harry W

    2013-11-26

    An additive comprising a phosphazene compound that has at least two reactive functional groups and at least one capping functional group bonded to phosphorus atoms of the phosphazene compound. One of the at least two reactive functional groups is configured to react with cellulose and the other of the at least two reactive functional groups is configured to react with a resin, such as an amine resin of a polycarboxylic acid resin. The at least one capping functional group is selected from the group consisting of a short chain ether group, an alkoxy group, or an aryloxy group. Also disclosed are an additive-resin admixture, a method of treating a wood product, and a wood product.

  1. Potlining Additives

    SciTech Connect

    Rudolf Keller

    2004-08-10

    In this project, a concept to improve the performance of aluminum production cells by introducing potlining additives was examined and tested. Boron oxide was added to cathode blocks, and titanium was dissolved in the metal pool; this resulted in the formation of titanium diboride and caused the molten aluminum to wet the carbonaceous cathode surface. Such wetting reportedly leads to operational improvements and extended cell life. In addition, boron oxide suppresses cyanide formation. This final report presents and discusses the results of this project. Substantial economic benefits for the practical implementation of the technology are projected, especially for modern cells with graphitized blocks. For example, with an energy savings of about 5% and an increase in pot life from 1500 to 2500 days, a cost savings of $ 0.023 per pound of aluminum produced is projected for a 200 kA pot.

  2. Detection of enterotoxigenic Clostridium perfringens in spices used in Mexico by dot blotting using a DNA probe.

    PubMed

    Rodríguez-Romo, L A; Heredia, N L; Labbé, R G; García-Alvarado, J S

    1998-02-01

    Several reports on the microbiology of spices and herbs indicate the presence of Clostridium perfringens, a spore-forming foodborne pathogen responsible for gastrointestinal disease. In the present study, a total of 380 samples of spices and herbs (cumin seed, black pepper, oregano, garlic powder, and bay leaves) widely used in Mexico were analyzed for the presence of C. perfringens, and the enterotoxigenicity of the isolates was determined by a dot-blot technique using an enterotoxin degoxigenin-labeled DNA probe. C. perfringens counts varied from <100 to 433 CFU/g in garlic powder, from <100 to 200 CFU/g in black pepper, from <100 to 433 CFU/g in cumin seed, from <100 to 340 CFU/g in oregano, and from < 100 to 450 CFU/g in bay leaves. The dot-blot technique detected the enterotoxin gene in 8 (4.25%) of 188 confirmed isolates of C. perfringens. dot-blot.

  3. HPTLC Plate Blotting for Liquid Microjunction Surface Sampling Probe Mass Spectrometric Analysis of Analytes Separated on a Wettable Phase Plate

    SciTech Connect

    Walworth, Matthew J; Stankovich, Joseph J; Van Berkel, Gary J; Schulz, Michael; Minarik, susanne

    2012-01-01

    A blotting method that transfers analytes separated on wettable HPTLC plates to a hydrophobic reversed-phase C8 HPLTC plate suitable for analysis with a liquid microjunction surface sampling probe electrospray ionization mass spectrometry system was described and demonstrated. The simple blotting procedure transfers the analyte from the wettable plate to the topmost surface of a rigidly backed, easy-to-mount hydrophobic substrate that already has been proven viable for analysis by this sampling probe/mass spectrometry system. The utility of the approach was demonstrated by the analysis of a four-component peptide mixture originally separated on a ProteoChrom HPTLC cellulose sheet and then blotted to the reversed phase HPTLC plate.

  4. Enzyme-linked immunoelectrotransfer blot analysis of a cryptosporidiosis outbreak on a United States Coast Guard cutter.

    PubMed

    Moss, D M; Bennett, S N; Arrowood, M J; Wahlquist, S P; Lammie, P J

    1998-01-01

    Symptoms consistent with an outbreak of cryptosporidiosis (diarrhea, vomiting, nausea, and abdominal cramps) occurred on a U.S. Coast Guard cutter within 0-18 days after the cutter filled its tanks with Milwaukee, Wisconsin city water in March 1993. At three-weeks postdocking (PD), the suspected water was removed, and serum samples and stool specimens were collected from 47 of the 58 crew members, as well as questionnaire data on their water consumption and symptoms aboard the cutter. At 10-weeks PD and/or at 28-weeks PD, additional serum specimens were collected. Intensitometric data from enzyme-linked immunoelectrotransfer blot (EITB) were obtained on IgA responses to a 17-kD antigen group, IgM responses to a 27-kD antigen group, and IgG responses to 27-, 17-, and 15-kD antigen groups extracted from oocysts. In addition, IgG responses to crude oocyst antigens were obtained by ELISA. Based on reported symptoms, EITB results, and stool examination, the crew members were classified as confirmed (10), probable (10), suspected (22), and noncases (16). Of the 10 confirmed cases (all symptomatic) and the 10 probable cases (eight symptomatic) whose stools were positive and negative, respectively, for Cryptosporidium oocysts by microscopy, all showed changes in EITB intensities to the antigen groups and were considered EITB positive. The remaining 38 crew members, 22 suspected cases (all symptomatic), and 16 noncases (all asymptomatic), if tested, had negative stool examinations and were considered EITB negative. Of the 10 confirmed cases, only four showed a significant change in IgG responses (P < 0.05) between three-weeks PD and follow-up serum specimens by ELISA. Crew members considered confirmed cases consumed significantly more water (P < or = 0.005) aboard the cutter than noncases. Crew members considered EITB positive consumed more water (P < or = 0.04) than crew members considered EITB negative while there was no significant difference in water consumption (P > or

  5. Use of sera from humans and dolphins with lacaziosis and sera from experimentally infected mice for Western Blot analyses of Lacazia loboi antigens.

    PubMed

    Mendoza, Leonel; Belone, Andréa F F; Vilela, Raquel; Rehtanz, Manuela; Bossart, Gregory D; Reif, John S; Fair, Patricia A; Durden, Wendy N; St Leger, Judy; Travassos, Luiz R; Rosa, Patricia S

    2008-01-01

    Antibodies in the sera of patients with lacaziosis recognized an approximately 193-kDa antigen and other Lacazia loboi antigens. Paracoccidioides brasiliensis gp43 antigen was detected by all evaluated sera, but they failed to detect a protein with the same molecular mass in L. loboi extracts. This study is the first to examine the humoral response to L. loboi antigens by using multiple host sera.

  6. A Western Blot-based Investigation of the Yeast Secretory Pathway Designed for an Intermediate-Level Undergraduate Cell Biology Laboratory

    ERIC Educational Resources Information Center

    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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. Identification of species in tribe Brassiceae by dot-blot hybridization using species-specific ITS1 probes.

    PubMed

    Tonosaki, K; Nishio, Takeshi

    2010-10-01

    Simple, reliable methods for identification of species are required for management of many species and lines in a plant gene bank. Species-specific probes were designed from published sequences of the ITS1 region in rDNA of 16 species in Brassica and its related genera, and used as probes for dot-blot hybridization with plant genomic DNA. All the probes detected species-specific signals at dot-blots of genomic DNAs of the 16 species in Brassica, Diplotaxis, Eruca, and Raphanus. Signals of the Brassica digenomic species in the U's triangle, i.e., B. napus, B. juncea, and B. carinata, were detected by the probes of their parental monogenomic species, i.e., B. rapa, B. nigra, and B. oleracea. The probe for B. oleracea showed signals of B. balearica, B. cretica, B. incana, B. insularis, and B. macrocarpa, which have the C genome as B. oleracea. Eruca vesicaria DNA was detected by the probe for E. sativa, which has been classified as a subspecies of E. vescaria. DNA of leaf tissue extracted by an alkaline solution and seed DNA prepared by the NaI method can be used directly for dot-blotting. Misidentification of species was revealed in 20 accessions in the Tohoku University Brassica Seed Bank. These results indicate dot-blot hybridization to be a simple and efficient technique for identification of plant species in a gene bank.

  9. Detection of genetically modified microorganisms in soil using the most-probable-number method with multiplex PCR and DNA dot blot.

    PubMed

    Yeom, Jinki; Lee, Yunho; Noh, Jaemin; Jung, Jaejoon; Park, Jungsoon; Seo, Hyoju; Kim, Jisun; Han, Jiwon; Jeon, Che Ok; Kim, Taesung; Park, Woojun

    2011-10-01

    The principal objective of this study was to detect genetically modified microorganisms (GMMs) that might be accidentally released into the environment from laboratories. Two methods [plate counting and most-probable-number (MPN)] coupled with either multiplex PCR or DNA dot blots were compared using genetically modified Escherichia coli, Pseudomonas putida, and Acinetobacter oleivorans harboring an antibiotic-resistance gene with additional gfp and lacZ genes as markers. Alignments of sequences collected from databases using the Perl scripting language (Perl API) and from denaturing gradient gel electrophoresis analysis revealed that the gfp, lacZ and antibiotic-resistance genes (kanamycin, tetracycline, and ampicillin) in GMMs differed from the counterpart genes in many sequenced genomes and in soil DNA. Thus, specific multiplex PCR primer sets for detection of plasmid-based gfp and lacZ antibiotic-resistance genes could be generated. In the plate counting method, many antibiotic-resistant bacteria from a soil microcosm grew as colonies on antibiotic-containing agar plates. The multiplex PCR verification of randomly selected antibiotic-resistant colonies with specific primers proved ineffective. The MPN-multiplex PCR method and antibiotic-resistant phenotype could be successfully used to detect GMMs, although this method is quite laborious. The MPN-DNA dot blot method screened more cells at a time in a microtiter plate containing the corresponding antibiotics, and was shown to be a more efficient method for the detection of GMMs in soil using specific probes in terms of labor and accuracy. PMID:21810467

  10. Snapshot blotting: transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to grids for electron microscopy.

    PubMed Central

    Jett, S D; Bear, D G

    1994-01-01

    We present a technique, "snapshot blotting," for the electrophoretic transfer of nucleic acids and nucleoprotein complexes in gel electrophoresis bands onto highly stable carbon film-coated grids for imaging by electron microscopy. The method permits structural analysis of macromolecular species that have been resolved by a gel mobility-shift assay. To demonstrate the efficiency and integrity of the transfer process for a multiprotein-DNA assembly, we have imaged various species of a prokaryotic transcription complex, using the cleavage-defective EcoRI(Q111) protein as an orientation marker and as a blockade of transcription elongation. Snapshot blotting should be of great utility in the structural characterization of nucleic acids and protein-nucleic acid interactions. Images PMID:8041711

  11. Differentiation of Enterococcus faecium from Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains by PCR and dot-blot hybridisation.

    PubMed

    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.

  12. Assessment by Southern blot analysis of UV-induced damage and repair in human immunoglobulin genes.

    PubMed

    Bianchi, M S; Bianchi, N O; de la Chapelle, A

    1990-09-01

    Irradiation of DNA with UV light induces pyrimidine dimers and (6-4) photoproducts. The presence of one of these photolesions in the restriction site of a given endonuclease inhibits DNA cleavage and induces the formation of fragments by incomplete DNA digestion which appear as additional, facultative bands in Southern hybridization autoradiograms. The number and size of these fragments show a positive correlation with the UV dose. The response to UV light of immunoglobulin light-chain constant kappa and heavy-chain constant mu genes was analyzed with 2 specific probes. Constant kappa and mu genes when irradiated as part of the chromatin of living lymphocytes showed a UV sensitivity similar to that of naked DNA. The same genes from granulocytes had 50-60 times lower UV sensitivity. When cells were allowed to repair photolesions for 24 h the facultative bands from granulocytes disappeared indicating that these cells were able to remove photolesions from constant kappa and mu genes. Facultative bands from lymphocytes showed a smaller decrease of density after 24 h repair. This suggests that lymphocytes are less efficient than granulocytes in removing UV damage from constant kappa and mu genes.

  13. Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique.

    PubMed

    Bhaskara, Srividya

    2016-01-14

    Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA. Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

  14. Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique.

    PubMed

    Bhaskara, Srividya

    2016-01-01

    Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA. Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples. PMID:26863264

  15. Detection of Rickettsia in Rhipicephalus sanguineus ticks and Ctenocephalides felis fleas from southeastern Tunisia by reverse line blot assay.

    PubMed

    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.

  16. Interspecies sharing of two distinct nonstructural protein 1 alleles among human and animal rotaviruses as revealed by dot blot hybridization.

    PubMed

    Fujiwara, Y; Nakagomi, O

    1997-10-01

    The distribution of the nonstructural protein 1 (NSP1) alleles from human strain AU-1 and canine strain K9 among rotaviruses of human, feline, canine, bovine, and simian origin was studied by a dot blot hybridization assay. Human and feline strains belonging to the AU-1 genogroup had the same NSP1 allele, while canine and feline strains belonging to the canine-feline genogroup shared another NSP1 allele. This canine-feline NSP1 allele had a significant level of homology with the NSP1 of rhesus rotavirus strain MMU18006. PMID:9316942

  17. RT-PCR and Northern blot analysis in search for a putative Paramecium beta-adrenergic receptor.

    PubMed

    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.

  18. What Is Western Civilization?

    ERIC Educational Resources Information Center

    Birken, Lawrence

    1992-01-01

    Discusses opposing tendencies in the interpretation of Western Civilization. Describes the expanded definition that includes Byzantine and Islamic cultures as heirs of the Greco-Roman cultures. Suggests that a limited definition of Western culture will facilitate a problems approach, emphasize diversity among cultures, and integrate the classical…

  19. [THE SCREENING OF DIAGNOSTIC POTENTIAL OF NATIVE PROTEIN FRACTIONS OF MYCOBACTERIUM TUBERCULOSIS USING TECHNIQUE OF IMMUNE BLOTTING].

    PubMed

    Tsibulkin, A P; Khaertinova, I M; Urazov, N G; Khaertinov, K S

    2016-02-01

    The technique of immune blotting was used to analyze surface proteins obtained from cells M Tuberculosis exposed to partialmode of delipidization. At that, there were applied serums of patients with tuberculosis of lungs; HIV agents and patients with concomitant infection tuberculosis-AIDS and also HIV-negative patients without clinical signs of disease of lungs and with chronic diseases of lungs of other etiology The fractions oflow-molecular antigens with molecular mass 6.5-30 kilodaltons became diagnostically significant. To this fraction of antigens reacted serums of all patients with tuberculosis of lungs and serums of 91% of patients with concomitant tuberculosis-AIDS infection. The antigens of protein fractions with high (70-100 kilodaltons) and interim (30-69 kilodaltons) molecular mass became diagnostically insignificant. PMID:27455562

  20. Amplification with molecular beacon primers and reverse line blotting for the detection and typing of human papillomaviruses.

    PubMed

    Jordens, J Z; Lanham, S; Pickett, M A; Amarasekara, S; Abeywickrema, I; Watt, P J

    2000-09-01

    A novel method for the detection and typing of human papillomavirus (HPV) was developed using molecular beacon primers. The method is based on the use of HPV-specific primers containing a hairpin loop structure in which fluorescent donor and quencher groups are held in close proximity such that fluorescence is quenched. Amplification of the target sequence results in the opening of the loop and the resulting fluorescence can be detected on a sequence detector system (SDS) 7700 (Applied Biosystems), as used for TaqMan assays. Fluorescent amplicons were identified on the SDS 7700 and then typed by a single hybridisation with specific probes immobilised in lines on a nylon membrane and detected on a fluorescent scanner. This novel beacon primer method compared well with conventional PCR for cervical scrape specimens. The combination of the beacon primer method and reverse line blotting should enable large-scale population studies of HPV infection.

  1. Imprinting mutations in Angelman syndrome detected by Southern blotting using a probe containing exon {alpha} of SNRPN

    SciTech Connect

    Beuten, J.; Sutcliffe, J.S.; Nakao, M.

    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.

  2. Identification of Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans with Novel Markers and Using a Dot Blot Platform Coupled with Automatic Data Analysis ▿ †

    PubMed Central

    Albuquerque, Pedro; Caridade, Cristina M. R.; Marcal, Andre R. S.; Cruz, Joana; Cruz, Leonor; Santos, Catarina L.; Mendes, Marta V.; Tavares, Fernando

    2011-01-01

    Phytosanitary regulations and the provision of plant health certificates still rely mainly on long and laborious culture-based methods of diagnosis, which are frequently inconclusive. DNA-based methods of detection can circumvent many of the limitations of currently used screening methods, allowing a fast and accurate monitoring of samples. The genus Xanthomonas includes 13 phytopathogenic quarantine organisms for which improved methods of diagnosis are needed. In this work, we propose 21 new Xanthomonas-specific molecular markers, within loci coding for Xanthomonas-specific protein domains, useful for DNA-based methods of identification of xanthomonads. The specificity of these markers was assessed by a dot blot hybridization array using 23 non-Xanthomonas species, mostly soil dwelling and/or phytopathogens for the same host plants. In addition, the validation of these markers on 15 Xanthomonas spp. suggested species-specific hybridization patterns, which allowed discrimination among the different Xanthomonas species. Having in mind that DNA-based methods of diagnosis are particularly hampered for unsequenced species, namely, Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans, for which comparative genomics tools to search for DNA signatures are not yet applicable, emphasis was given to the selection of informative markers able to identify X. fragariae, X. axonopodis pv. phaseoli, and X. fuscans subsp. fuscans strains. In order to avoid inconsistencies due to operator-dependent interpretation of dot blot data, an image-processing algorithm was developed to analyze automatically the dot blot patterns. Ultimately, the proposed markers and the dot blot platform, coupled with automatic data analyses, have the potential to foster a thorough monitoring of phytopathogenic xanthomonads. PMID:21705524

  3. Detection of antibodies against avian antigens in bronchoalveolar lavage from patients with pigeon breeder's disease: usefulness of enzyme-linked immunosorbent assay and enzyme immunotransfer blotting.

    PubMed

    Sandoval, J; Bañales, J L; Cortés, J J; Mendoza, F; Selman, M; Reyes, P A

    1990-01-01

    The study reported here evaluated the usefulness of the enzyme-linked immunosorbent assay (ELISA) in the detection of antibodies against pigeon antigens in the serum and bronchoalveolar lavage (BAL) of patients with clinical, radiological, and functional evidence of interstitial lung disease (ILD) with and without pigeon breeder's disease (PBD). The results were compared with those obtained by the simultaneous use of counterimmunoelectrophoresis (CIE) in the same patients. In PBD, ELISA detected antibodies against pigeon's sera in both serum and BAL in 100% of patients, while CIE failed to detect the antibodies in the serum of one patient and in most of the samples of BAL. In addition, we used enzyme immunotransfer blotting to determine the number of epitopes in pigeon serum recognized by antibodies present in serum and BAL. There was a heterogeneous response in both fluids, but the reaction pattern demonstrated that patient's sera recognize to-25 different pigeon epitopes. We conclude that ELISA is a highly sensitive and specific method for the detection of antibodies against pigeon antigens in the serum and BAL of patients with PBD and that the host response involves a great number of avian antigens.

  4. Violence the Western way.

    PubMed

    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.

  5. Paula Fox's "Western Wind."

    ERIC Educational Resources Information Center

    Corsaro, Julie

    1997-01-01

    Summarizes the novel, "Western Wind," by Paula Fox, lists 12 discussion questions, describes six activities, and contains an annotated bibliography of seven other novels related to birth, death, and families. (DGM)

  6. Western Disturbances: A review

    NASA Astrophysics Data System (ADS)

    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.

  7. Simultaneous direct identification of genital microorganisms in voided urine using multiplex PCR-based reverse line blot assays.

    PubMed

    McKechnie, Michelle L; Kong, Fanrong; Gilbert, Gwendolyn L

    2013-01-01

    Our aim was to develop and evaluate sensitive methods that would allow simultaneous direct identification of multiple potential pathogens in clinical specimens for diagnosis and epidemiological studies, using a multiplex PCR-based reverse line blot assay. We have previously developed assays suitable for detection of bacterial respiratory and systemic pathogens. In this chapter we describe, in detail, a method developed to identify 14 genital microorganisms, for use in epidemiological studies of genital infection or colonization, using first voided urine specimens. The 14 urogenital pathogens or putative pathogens studied were Trichomonas vaginalis, Streptococcus pneumoniae, Neisseria gonorrhoeae, N. meningitidis, Chlamydia trachomatis, Ureaplasma parvum, U. urealyticum, Mycoplasma hominis, M. genitalium, Gardnerella vaginalis, Haemophilus influenzae, herpes simplex virus 1 and 2, and adenovirus. Two species-specific primer pairs and probes were designed for each target. The method was validated using a reference strain or a well-characterized clinical isolate of each target organism. In a clinical study among men attending sexual health clinics in Sydney, we used the assay to compare rates of detection of the 14 organisms in men with urethritis with those in asymptomatic controls and found the method to be sensitive, specific, convenient, and relatively inexpensive.

  8. A Southern Blot Assay for Detection of Hepatitis B Virus Covalently Closed Circular DNA from Cell Cultures

    PubMed Central

    Cai, Dawei; Nie, Hui; Yan, Ran; Guo, Ju-Tao; Block, Timothy M.; Guo, Haitao

    2016-01-01

    Chronic hepatitis B remains a substantial public health burden affecting approximately 350 million people worldwide, causing cirrhosis and liver cancer, and about 1 million people die each year from hepatitis B and its complications. Hepatitis B is caused by hepatitis B virus (HBV) infection. As an essential component of the viral life cycle, HBV covalently closed circular DNA (cccDNA) is synthesized and maintained at low copy numbers in the nucleus of infected hepatocytes, and serves as the transcription template for all viral RNAs. Therefore, cccDNA is responsible for the establishment of viral infection and persistence. The presence and longevity of cccDNA may also explain the limitations of current antiviral therapy for hepatitis B. Thus, understanding the mechanisms underlying cccDNA formation and regulation is critical in understanding the HBV pathogenesis and finding a cure for hepatitis B. Here we describe a protocol for HBV cccDNA extraction and detection in detail. The procedure includes two major steps: (1) HBV cccDNA extraction by Hirt protein-free DNA extraction method and (2) HBV cccDNA detection by Southern blot analysis. The method is straightforward and reliable for cccDNA assay with cell culture samples, and it is useful for both HBV molecular biology and antiviral research. PMID:23821267

  9. [Establishment of a method for HLA-DRB genotyping in cord blood by reverse dot-blot hybridization technique].

    PubMed

    Huang, Yi-Ning; Liao, Can; Tang, Xue-Wei; Li, Yan; Xie, Xing-Mei; Zeng, Rui-Ping

    2002-04-01

    The HLA system was discovered by virtue of the fact that it was polymorphic. The impetus for its discovery was the search for polymorphic antigens to match for transplantation, by analogy with the human red cell blood groups. The most usually DNA method of HLA typing is sequence specific oligonucleotides (SSO) and PCR sequence specific primers (SSP). SSO technique is perfectly suited for analyzing large number of samples, it is not suitable for individual or small numbers. The SSP method is ideal for typing individual samples, but it is costly and requires high capacity thermal cycles for larger numbers of samples. To set up a simple, quick, cheap and high resolution DNA method, were collected sixty-three cord blood samples from Guangzhou Cord Blood Bank, got DNA from blood by the traditional guanidine hydrochloride distillation method. Each sample was simultaneously typed by SSOP, PCR-SSP and reverse dot-blot hybridization (RDB) methods. All of typed is success. The results of three DNA methods are consistent each other. 60 HLA-DRB1 alleles could be accurately distinguished with the RDB method. Our results show that RDB method is a simple, quick, cheap and high resolution method for HLA-DRB types. It can be used in any HLA typing.

  10. A Modified Quantum Dot-Based Dot Blot Assay for Rapid Detection of Fish Pathogen Vibrio anguillarum.

    PubMed

    Zhang, Yang; Xiao, Jingfan; Wang, Qiyao; Zhang, Yuanxing

    2016-08-28

    Vibrio anguillarum, a devastating pathogen causing vibriosis among marine fish, is prevailing in worldwide fishery industries and accounts for grievous economic losses. Therefore, a rapid on-site detection and diagnostic technique for this pathogen is in urgent need. In this study, two mouse monoclonal antibodies (MAbs) against V. anguillarum, 6B3-C5 and 8G3-B5, were generated by using hybridoma technology and their isotypes were characterized. MAb 6B3-C5 was chosen as the detector antibody and conjugated with quantum dots. Based on MAb 6B3- C5 labeled with quantum dots, a modified dot blot assay was developed for the on-site determination of V. anguillarum. It was found that the method had no cross-reactivity with other than V. anguillarum bacteria. The detection limit (LOD) for V. anguillarum was 1 × 10(3) CFU/ml in cultured bacterial suspension samples, which was a 100-fold higher sensitivity than the reported colloidal gold immunochromatographic test strip. When V. anguillarum was mixed with turbot tissue homogenates, the LOD was 1 × 10(3) CFU/ml, suggesting that tissue homogenates did not influence the detection capabilities. Preenrichment with the tissue homogenates for 12 h could raise the LOD up to 1 × 10(2) CFU/ml, confirming the reliability of the method.

  11. Development of rapid, sensitive and non-radioactive tissue-blot diagnostic method for the detection of citrus greening.

    PubMed

    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.

  12. Simultaneous direct identification of genital microorganisms in voided urine using multiplex PCR-based reverse line blot assays.

    PubMed

    McKechnie, Michelle L; Kong, Fanrong; Gilbert, Gwendolyn L

    2013-01-01

    Our aim was to develop and evaluate sensitive methods that would allow simultaneous direct identification of multiple potential pathogens in clinical specimens for diagnosis and epidemiological studies, using a multiplex PCR-based reverse line blot assay. We have previously developed assays suitable for detection of bacterial respiratory and systemic pathogens. In this chapter we describe, in detail, a method developed to identify 14 genital microorganisms, for use in epidemiological studies of genital infection or colonization, using first voided urine specimens. The 14 urogenital pathogens or putative pathogens studied were Trichomonas vaginalis, Streptococcus pneumoniae, Neisseria gonorrhoeae, N. meningitidis, Chlamydia trachomatis, Ureaplasma parvum, U. urealyticum, Mycoplasma hominis, M. genitalium, Gardnerella vaginalis, Haemophilus influenzae, herpes simplex virus 1 and 2, and adenovirus. Two species-specific primer pairs and probes were designed for each target. The method was validated using a reference strain or a well-characterized clinical isolate of each target organism. In a clinical study among men attending sexual health clinics in Sydney, we used the assay to compare rates of detection of the 14 organisms in men with urethritis with those in asymptomatic controls and found the method to be sensitive, specific, convenient, and relatively inexpensive. PMID:23104293

  13. A Modified Quantum Dot-Based Dot Blot Assay for Rapid Detection of Fish Pathogen Vibrio anguillarum.

    PubMed

    Zhang, Yang; Xiao, Jingfan; Wang, Qiyao; Zhang, Yuanxing

    2016-08-28

    Vibrio anguillarum, a devastating pathogen causing vibriosis among marine fish, is prevailing in worldwide fishery industries and accounts for grievous economic losses. Therefore, a rapid on-site detection and diagnostic technique for this pathogen is in urgent need. In this study, two mouse monoclonal antibodies (MAbs) against V. anguillarum, 6B3-C5 and 8G3-B5, were generated by using hybridoma technology and their isotypes were characterized. MAb 6B3-C5 was chosen as the detector antibody and conjugated with quantum dots. Based on MAb 6B3- C5 labeled with quantum dots, a modified dot blot assay was developed for the on-site determination of V. anguillarum. It was found that the method had no cross-reactivity with other than V. anguillarum bacteria. The detection limit (LOD) for V. anguillarum was 1 × 10(3) CFU/ml in cultured bacterial suspension samples, which was a 100-fold higher sensitivity than the reported colloidal gold immunochromatographic test strip. When V. anguillarum was mixed with turbot tissue homogenates, the LOD was 1 × 10(3) CFU/ml, suggesting that tissue homogenates did not influence the detection capabilities. Preenrichment with the tissue homogenates for 12 h could raise the LOD up to 1 × 10(2) CFU/ml, confirming the reliability of the method. PMID:27116991

  14. A southern blot assay for detection of hepatitis B virus covalently closed circular DNA from cell cultures.

    PubMed

    Cai, Dawei; Nie, Hui; Yan, Ran; Guo, Ju-Tao; Block, Timothy M; Guo, Haitao

    2013-01-01

    Chronic hepatitis B remains a substantial public health burden affecting approximately 350 million people worldwide, causing cirrhosis and liver cancer, and about 1 million people die each year from hepatitis B and its complications. Hepatitis B is caused by hepatitis B virus (HBV) infection. As an essential component of the viral life cycle, HBV covalently closed circular DNA (cccDNA) is synthesized and maintained at low copy numbers in the nucleus of infected hepatocytes, and serves as the transcription template for all viral RNAs. Therefore, cccDNA is responsible for the establishment of viral infection and persistence. The presence and longevity of cccDNA may also explain the limitations of current antiviral therapy for hepatitis B. Thus, understanding the mechanisms underlying cccDNA formation and regulation is critical in understanding the HBV pathogenesis and finding a cure for hepatitis B. Here we describe a protocol for HBV cccDNA extraction and detection in detail. The procedure includes two major steps: (1) HBV cccDNA extraction by Hirt protein-free DNA extraction method and (2) HBV cccDNA detection by Southern blot analysis. The method is straightforward and reliable for cccDNA assay with cell culture samples, and it is useful for both HBV molecular biology and antiviral research. PMID:23821267

  15. [The beginning of western medical education].

    PubMed

    Kee, C D

    1992-01-01

    Our country had quite an advanced system of medical education during the era of the Koryo Kingdom, and during the Choson Dynasty, the Kyong Guk Dae Jon, in which a systematized medical education was clearly described, was compiled in the era of King Sejong. However, the educational system was not for Western medicine. Western medicine was first introduced to our country in the 9th year of King Injo (1631) when Chong Du Won, Yi Yong Jun, etc. returned from Yon Gyong (Beiuin) with Chik Bang Oe Gi. Knowledge of Western medicine was disseminated by Shil Hak (practical learning) scholars who read a translation in Chinese characters, of Chik Bang Oe Gi. Yi Ik (Song Ho), Yi Gyu Gyong (O ju), Choe Han Gi (Hye Gang), Chong Yak Yong (Ta San), etc., read books of Western medicine and introduced in writing the excellent theory of Western medicine. In addition, Yu Hyong Won (Pan Gye), Pak Ji Won (Yon Am), Pak Je Ga (Cho Jong), etc., showed much interest in Western medicine, but no writings by them about western medicine can be found. With the establishment of a treaty of amity with Japan in the 13th year of King Kojong (1876), followed by the succession of amity treaties with Western powers, foreigners including medical doctors were permitted to flow into this country. At that time, doctors Horace N. Allen, W. B. Scranton, John W. Heron, Rosetta Sherwood (Rosetta S. Hall), etc., came to Korea and inaugurated hospitals, where they taught Western medicine to Korean students. Dr. Horace N. Allen, with the permission of king Kojong, established Che Jung Won in April 1885, and in March 1886, he began at the hospital to provide education of Western medicine to Korean students who were recrutied by the Korean Government. However, the education was not conduted on a regular basis, only training them for work as assistants. This is considered to be the pioneer case of Western medical education in this country. Before that time, Japanese medical doctors came to Korea, but there are no

  16. Paraffin-embedded tissue blot as a sensitive method for discrimination between classical scrapie and experimental bovine spongiform encephalopathy in sheep.

    PubMed

    Webb, Paul R; Denyer, Margaret; Gough, Julie; Spiropoulos, John; Simmons, Marion M; Spencer, Yvonne I

    2011-05-01

    The paraffin-embedded tissue (PET) blot was modified for use as a tool to differentiate between classical scrapie and experimental bovine spongiform encephalopathy (BSE) in sheep. Medulla (obex) from 21 cases of classical scrapie and 6 cases of experimental ovine BSE were used to develop the method such that it can be used as a tool to differentiate between BSE and scrapie in the same way that differential immunohistochemistry (IHC) has been used previously. The differential PET blot successfully differentiated between all of the scrapie and ovine BSE cases. Differentiation was permitted more easily with PET blot than by differential IHC, with accurate observations possible at the macroscopic level. At the microscopic level, sensitivity was such that discrimination by the differential PET blot could be made with more confidence than with differential IHC in cases where the immunohistochemical differences were subtle. The differential PET blot makes use of harsh epitope demasking conditions, and, because of the differences in the way prion protein is processed in different prion diseases, it can serve as a new, highly sensitive method to discriminate between classical scrapie and experimental BSE in sheep.

  17. Rings dominate western Gulf

    NASA Astrophysics Data System (ADS)

    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.

  18. 75 FR 33269 - Procurement List; Additions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-11

    ... Street, Boise, ID. NPA: Western Idaho Training Company, Caldwell, ID Contracting Activity: Dept of Trans... . SUPPLEMENTARY INFORMATION: Additions On 4/9/2010 (75 FR 18164-18165), the Committee for Purchase From People Who... contractor for the commodity or service. Comments were received from a commercial company that claims to...

  19. Western Aeronautical Test Range

    NASA Technical Reports Server (NTRS)

    Sakahara, Robert D.

    2008-01-01

    This viewgraph presentation reviews the work of the Western Aeronautical Test Range (WATR). NASA's Western Aeronautical Test Range is a network of facilities used to support aeronautical research, science missions, exploration system concepts, and space operations. The WATR resides at NASA's Dryden Flight Research Center located at Edwards Air Force Base, California. The WATR is a part of NASA's Corporate Management of Aeronautical Facilities and funded by the Strategic Capability Asset Program (SCAP). Maps show the general location of the WATR area that is used for aeronautical testing and evaluation. The products, services and facilities of WATR are discussed,

  20. Comparison of chemical assay, bioassay, enzyme-linked immunosorbent assay, and dot blot hybridization for detection of aerobactin in members of the family Enterobacteriaceae.

    PubMed Central

    Le Roy, D; Bouchet, A; Saulnier, P; Pecquet, S; Andremont, A

    1993-01-01

    In order to determine the best strategy for detection of aerobactin in members of the family Enterobacteriaceae, we compared the results of three phenotypic assays, including a chemical assay, a cross-feeding bioassay, and an enzyme-linked immunosorbent assay (ELISA), with the results of a dot blot hybridization assay using a specific probe for the aerobactin genes. The sensitivity and specificity of the ELISA were better than those of the chemical and cross-feeding assays, but the results of dot blot hybridization were the most reproducible. However, none of the Serratia and Enterobacter cloacae strains which produced aerobactin hybridized with the probe. We concluded that the best strategy for aerobactin detection is a two-step procedure that combines screening by dot blot hybridization with an ELISA for negative strains. PMID:8481015

  1. Development of Double Eastern Blotting for Major Licorice Components, Glycyrrhizin and Liquiritin for Chemical Quality Control of Licorice Using anti-Glycyrrhizin and anti-Liquiritin Monoclonal Antibodies.

    PubMed

    Fujii, Shunsuke; Morinaga, Osamu; Uto, Takuhiro; Nomura, Shuichi; Shoyama, Yukihiro

    2016-02-10

    Licorice is utilized in various food industries around the world for seasoning agents, confectioneries, drinks, and functional foods. Glycyrrhizin (GL) and liquiritin (Liq) are major quality control chemical markers of licorice that have multifunctional bioactivities. Chemical quality control of licorice is important because its component profiles change depending environmental factors (climate, soil condition, and water deficit) and differences between species. Double eastern blotting using anti-GL and anti-Liq monoclonal antibodies was developed for more convenient, rapid, and specific quality control analysis of GL and Liq, respectively. Moreover, double eastern blotting was applied to investigate the immunohistochemical distributions of GL and Liq in the root of fresh licorice; the localization of both components was then clarified visually. This double eastern blotting technique for GL and Liq may serve as a powerful approach for visually determining the chemical quality of licorice.

  2. Development of Double Eastern Blotting for Major Licorice Components, Glycyrrhizin and Liquiritin for Chemical Quality Control of Licorice Using anti-Glycyrrhizin and anti-Liquiritin Monoclonal Antibodies.

    PubMed

    Fujii, Shunsuke; Morinaga, Osamu; Uto, Takuhiro; Nomura, Shuichi; Shoyama, Yukihiro

    2016-02-10

    Licorice is utilized in various food industries around the world for seasoning agents, confectioneries, drinks, and functional foods. Glycyrrhizin (GL) and liquiritin (Liq) are major quality control chemical markers of licorice that have multifunctional bioactivities. Chemical quality control of licorice is important because its component profiles change depending environmental factors (climate, soil condition, and water deficit) and differences between species. Double eastern blotting using anti-GL and anti-Liq monoclonal antibodies was developed for more convenient, rapid, and specific quality control analysis of GL and Liq, respectively. Moreover, double eastern blotting was applied to investigate the immunohistochemical distributions of GL and Liq in the root of fresh licorice; the localization of both components was then clarified visually. This double eastern blotting technique for GL and Liq may serve as a powerful approach for visually determining the chemical quality of licorice. PMID:26765784

  3. Molecular diagnosis of Theileria and Babesia species infecting cattle in Northern Spain using reverse line blot macroarrays

    PubMed Central

    García-Sanmartín, Josune; Nagore, Daniel; García-Pérez, Ana L; Juste, Ramón A; Hurtado, Ana

    2006-01-01

    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 tests like RLB that clearly

  4. Identification of a 17-kilodalton Fasciola hepatica immunodiagnostic antigen by the enzyme-linked immunoelectrotransfer blot technique.

    PubMed Central

    Hillyer, G V; Soler de Galanes, M

    1988-01-01

    Sera obtained from human patients, calves, sheep, and rabbits infected with Fasciola hepatica were tested by the Falcon assay screening test enzyme-linked immunosorbent assay (FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) techniques with Fasciola hepatica excretory-secretory antigens in order to evaluate their immunodiagnostic potential. The study included sera from 13 patients infected with F. hepatica or a history suggesting fascioliasis, 5 patients infected and treated with bithionol or praziquantel (3 were cured with bithionol), 10 patients infected with Schistosoma mansoni, 6 infected with Trichinella spiralis, and 13 controls and sera from calves, sheep, and rabbits with a primary F. hepatica infection. By FAST-ELISA with F. hepatica excretory-secretory antigens, the serum samples from fascioliasis patients gave the highest absorbance values, and the schistosomiasis patient sera gave intermediate values compared with a normal human serum control. Also by FAST-ELISA, the values for serum from patients with fascioliasis decreased steadily after cure, reaching normal levels 20 to 47 weeks postcure. In contrast, the serum from two patients who had been treated but were not yet cured had high levels of antibodies for up to 3 years of infection. By EITB, the serum samples from humans, rabbits, cattle, and sheep with fascioliasis recognized two antigenic polypeptides of 17 and 63 kilodaltons (kDa) in the form of sharp bands. For humans, this recognition lasted for at least 3 years of infection. Sera from individuals with schistosomiasis mansoni or trichinosis or from normal controls did not recognize the 17-kDa F. hepatica antigenic polypeptide. However, serum from one human with S. mansoni and one with T. spiralis infection has slight bands in the 63-kDa region, suggesting cross-reactivity. Reactivity to the 17-kDa polypeptide was absent in fascioliasis patients at 1 year postcure. Reactivity to the 63-kDa polypeptide was significantly

  5. 5. Photocopy of old exterior photo showing Western Saving Fund ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. Photocopy of old exterior photo showing Western Saving Fund Society before the addition of ca. 1910 building. Original photo, late 19th century, is at the Philadelphia Free Library, Philadelphia Collection, Print and Picture Department. - Western Saving Fund Society of Philadelphia, 1000-1008 Walnut Street, Philadelphia, Philadelphia County, PA

  6. Rethinking the "Western Tradition"

    ERIC Educational Resources Information Center

    Enslin, Penny; Horsthemke, Kai

    2015-01-01

    In recent years, the "Western tradition" has increasingly come under attack in anti-colonialist and postmodernist discourses. It is not difficult to sympathise with the concerns that underlie advocacy of historically marginalised traditions, and the West undoubtedly has a lot to answer for. Nonetheless, while arguing a qualified yes to…

  7. Typhoon Odessa, Western Pacific

    NASA Technical Reports Server (NTRS)

    1985-01-01

    This near vertical and detailed view of the eye of Typhoon Odessa was photographed somewhere in the Western Pacific, exact location unknown. The presence of a tight gyre and well formed eye, bordered by a cloud berm in the center, are all indicators of a very powerful and devastating typhoon with very high internal cyclonic wind speeds.

  8. NASA 2007 Western States Fire Missions (WSFM)

    NASA Technical Reports Server (NTRS)

    Posada, Herman A.

    2008-01-01

    This presentation describes the objectives of the 2007 Western States Fire Mission (WSFM), which included demonstrating capabilities of the Ikhana unmanned aerial system (UAS) to overfly and collect sensor data on widespread fires throughout the Western United States, demonstrating long-endurance (20+ hours) mission capabilities, and delivering real-time imagery within 10 minutes of acquisition. Additionally, the operations concept, operational zones, and landing sites are highlighted. Provisions of the certificate of authorization are also addressed. Imagery obtained from the WSFM are included.

  9. Western Hemisphere Knowledge Partnerships

    NASA Astrophysics Data System (ADS)

    Malone, T. F.

    2001-05-01

    Society in general, and geophysicists in particular, are challenged by problems and opportunities in the prospects for an additional three billion people on finite planet Earth by 2050 in a global economy four to six times larger than it is at present. A problem was identified by the Pilot Assessment of Global Ecosystems (PAGE): "If we choose to continue our current patterns of use, we face almost certain decline in the ability of ecosystems to yield their broad spectrum of benefits - from clean water to stable climate, fuel wood to food crops, timber to wildlife habitat." This is the issue of environmental sustainability. Another problem is the widening gap in wealth and health between affluent nations and impoverished countries. Every day each of the more than a billion people in the industrial nations produces goods and services worth nearly 60 dollars to meet their basic needs and "wants." This figure increases by about 85 cents annually. Every day each of the 600 million people in the least developed countries produces goods and services worth about 75 cents to meet their basic needs and limited wants. That number grows by less that a penny a day annually. This is the issue of economic prosperity and equity. By harnessing revolutionary technologies in communications to distribute expanding knowledge in the physical, chemical, and geophysical sciences and exploding knowledge in the biological and health sciences, a new vision for world society is brought within reach in The Knowledge Age. It is a society in which all of the basic human needs and an equitable share of human wants can be met while maintaining healthy, attractive, and biologically productive ecosystems. This society is environmentally sustainable, economically prosperous and equitable, and therefore likely to be politically stable. The time has arrived to fashion a strategy to pursue that vision. A knowledge-based and human-centered strategy will involve the discovery, integration, dissemination

  10. Detail, external parabolic antenna (later addition). Note how waveguide was ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Detail, external parabolic antenna (later addition). Note how waveguide was cut to remove active portion of antenna. - Western Union Telegraph Company, Jennerstown Relay, Laurel Summit Road off U.S. 30, Laughlintown, Westmoreland County, PA

  11. Western Kentucky thrives

    SciTech Connect

    Buchsbaum, L.

    2005-10-01

    Independents and big boys struggle to keep up with increasing demand and a lack of experienced workers in the Illinois Basin. This is the second of a two part series reviewing the coal mining industry in the Illinois Basin which also includes Indiana and Western Kentucky. It includes a classification/correction to Part 1 of the article published in the September 2005 issue (see Coal Abstracts Entry data/number Dec 2005 00204). 4 photos.

  12. Western Gas Sands Subprogram

    SciTech Connect

    Not Available

    1983-12-01

    The Western Gas Sands Subprogram (WGSS) is a multidisciplinary research effort within the US Department of Energy program on Unconventional Gas Recovery. The subprogram, managed by DOE's Morgantown Energy Technology Center, is directed towards the development of tight (very low permeability) lenticular gas sands in the western United States. The purpose of the subprogram is to demonstrate the feasibility of economically producing natural gas from low-permeability reservoirs. The subprogram has two broad goals: (1) to reduce the uncertainty of the reservoir production potential and (2) to improve the extraction technology. With input from the gas industry, universities, and geologic and engineering consulting firms, the WGSS was broadened to include more fundamental research and development. Consequently, for the last five years it has focused on improving diagnostic instrumentation, geophysical and engineering interpretation, and stimulation techniques. Integrated geologic studies of the three priority basins containing tight sands and selected by DOE as research targets have also been pursued as part of this new effort. To date, the following tentative conclusions have evolved: Permeability of the tight gas sands can be as much as three to four orders of magnitude lower than conventional gas deposits. Nineteen western geologic basins and trends containing significant amounts of tight gas have been identified. Gas resources in the priority geologic basins are Piceance Basin, 49 tcf., Uinta Basin, 20 tcf., and Greater Green River Basin, 136 tcf. The presence of natural micro-fractures within the production zone of a reservoir and the effective propped length of hydraulically-induced fractures are the critical parameters for successful development of tight sand resources. 8 figures.

  13. 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.

    PubMed

    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.

  14. 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.

    PubMed

    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%.

  15. Clinical Application of a Dot Blot Test for Diagnosis of Enteric Fever Due to Salmonella enterica Serovar Typhi in Patients with Typhoid Fever from Colombia and Peru

    PubMed Central

    Cardona-Castro, Nora; Gotuzzo, Eduardo; Rodriguez, Monica; Guerra, Humberto

    2000-01-01

    Clinical application of a dot blot test to detect immunoglobulin G (IgG) (88% sensitivity and specificity) and IgM (12.1% sensitivity and 97% specificity) against flagellar antigen from Salmonella enterica serovar Typhi was performed in Peruvian and Colombian patients with typhoid fever. This test can be used as a good predictor of serovar Typhi infection in regions lacking laboratory facilities and in field studies. PMID:10702512

  16. Use of an AC electric field in galvanotactic on/off switching of the motion of a microstructure blotted by Serratia marcescens

    NASA Astrophysics Data System (ADS)

    Tran, Trung-Hieu; Hyung Kim, Dal; Kim, Jihoon; Jun Kim, Min; Byun, Doyoung

    2011-08-01

    In this study, we manipulated the swimming direction of bacteria and controlled the switching off movement by using dc and ac galvanotaxis. The microstructures blotted by Serratia marcescens could be spontaneously manipulated and switched off at the desired position. The optimum ac frequency for switching off the microstructural motion was 7 Hz. We built a mathematical model to analyze and understand the oscillating motion of microstructure.

  17. Western Regional Remote Sensing Conference Proceedings, 1979

    NASA Technical Reports Server (NTRS)

    1980-01-01

    Remote sensing users from the 14 western states explained their diverse applications of LANDSAT data, discussed operational goals, and exchanged problems and solutions. In addition, conference participants stressed the need for increased cooperation among state and local governments, private industry, and universities to aid NASA's objective of transferring to user agencies the ability to operationally use remote sensing technology for resource and environmental quality management.

  18. [Food additives and healthiness].

    PubMed

    Heinonen, Marina

    2014-01-01

    Additives are used for improving food structure or preventing its spoilage, for example. Many substances used as additives are also naturally present in food. The safety of additives is evaluated according to commonly agreed principles. If high concentrations of an additive cause adverse health effects for humans, a limit of acceptable daily intake (ADI) is set for it. An additive is a risk only when ADI is exceeded. The healthiness of food is measured on the basis of nutrient density and scientifically proven effects.

  19. Lightning in Western Patagonia

    NASA Astrophysics Data System (ADS)

    Garreaud, René D.; Gabriela Nicora, M.; Bürgesser, Rodrigo E.; Ávila, Eldo E.

    2014-04-01

    On the basis of 8 years (2005-2012) of stroke data from the World Wide Lightning Location Network we describe the spatial distribution and temporal variability of lightning activity over Western Patagonia. This region extends from ~40°S to 55°S along the west coast of South America, is limited to the east by the austral Andes, and features a hyper-humid, maritime climate. Stroke density exhibits a sharp maximum along the coast of southern Chile. Although precipitation there is largely produced by cold nimbostratus, days with more than one stroke occur up to a third of the time somewhere along the coastal strip. Disperse strokes are also observed off southern Chile. In contrast, strokes are virtually nonexistent over the austral Andes—where precipitation is maximum—and farther east over the dry lowlands of Argentina. Atmospheric reanalysis and satellite imagery are used to characterize the synoptic environment of lightning-producing storms, exemplified by a case study and generalized by a compositing analysis. Lightning activity tends to occur when Western Patagonia is immersed in a pool of cold air behind a front that has reached the coast at ~40°S. Under these circumstances, midlevel cooling occurs before and is more prominent than near-surface cooling, leading to a weakly unstable postfrontal condition. Forced uplift of the strong westerlies impinging on the coastal mountains can trigger convection and produces significant lightning activity in this zone. Farther offshore, large-scale ascent near the cyclone's center may lift near-surface air parcels, fostering shallow convection and dispersing lightning activity.

  20. Production of Hybrids between Western Gray Wolves and Western Coyotes

    PubMed Central

    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. PMID:24586418

  1. Production of hybrids between western gray wolves and western coyotes

    USGS Publications Warehouse

    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.

  2. Production of hybrids between western gray wolves and western coyotes.

    PubMed

    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. Polyimide processing additives

    NASA Technical Reports Server (NTRS)

    Fletcher, James C. (Inventor); Pratt, J. Richard (Inventor); St.clair, Terry L. (Inventor); Stoakley, Diane M. (Inventor); Burks, Harold D. (Inventor)

    1992-01-01

    A process for preparing polyimides having enhanced melt flow properties is described. The process consists of heating a mixture of a high molecular weight poly-(amic acid) or polyimide with a low molecular weight amic acid or imide additive in the range of 0.05 to 15 percent by weight of additive. The polyimide powders so obtained show improved processability, as evidenced by lower melt viscosity by capillary rheometry. Likewise, films prepared from mixtures of polymers with additives show improved processability with earlier onset of stretching by TMA.

  4. Polyimide processing additives

    NASA Technical Reports Server (NTRS)

    Pratt, J. Richard (Inventor); St.clair, Terry L. (Inventor); Stoakley, Diane M. (Inventor); Burks, Harold D. (Inventor)

    1993-01-01

    A process for preparing polyimides having enhanced melt flow properties is described. The process consists of heating a mixture of a high molecular weight poly-(amic acid) or polyimide with a low molecular weight amic acid or imide additive in the range of 0.05 to 15 percent by weight of the additive. The polyimide powders so obtained show improved processability, as evidenced by lower melt viscosity by capillary rheometry. Likewise, films prepared from mixtures of polymers with additives show improved processability with earlier onset of stretching by TMA.

  5. Comparing the Southern blot method and polymerase chain reaction product analysis for chimeric RCCX detection in CYP21A2 deficiency.

    PubMed

    Lee, Hsien-Hsiung; Lee, Yann-Jinn; Chao, Mei-Chyn

    2010-04-15

    The 3.2-kb TaqI-produced fragment of the CYP21A1P pseudogene and the 3.7-kb TaqI-produced fragment of the functional CYP21A2 gene exist on chromosome 6p21.3. We used the polymerase chain reaction (PCR) product and Southern blot method with TaqI endonuclease digestion to identify a chimeric RCCX module in two unrelated patients with congenital adrenal hyperplasia (CAH). After TaqI cleavage, the PCR product analysis revealed that patient 1 with the chimeric CYP21A1P/CYP21A2 gene in one allele and IVS2-12A/C>G in combination with the 707-714del mutation in the other allele produced a configuration of 3.2- and 2.4-kb fragments. Patient 2, who carried IVS2-12A/C>G in combination with the 707-714del mutation in one allele and the chimeric TNXA/TNXB gene in the other allele, presented with 3.2- and 2.3-kb fragments. However, Southern blot analysis showed that patients 1 and 2 produced 3.2-, 2.4-, and 2.5-kb fragments. We conclude that the chimeric CYP21A1P/CYP21A2 gene, IVS2-12A/C>G in combination with the 707-714del mutation, and the chimeric TNXA/TNXB gene cannot be distinguished by the Southern blot method. Conversely, the chimeric TNXA/TNXB gene was identified in the PCR product analysis due to the appearance of the 2.37-kb fragment, which indicates the occurrence of the chimeric TNXA/TNXB formation extending to the boundary of TNXA in the RCCX region. PMID:19961824

  6. Quantitative Expression Analysis in Brassica napus by Northern Blot Analysis and Reverse Transcription-Quantitative PCR in a Complex Experimental Setting

    PubMed Central

    Rumlow, Annekathrin; Keunen, Els; Klein, Jan; Pallmann, Philip; Riemenschneider, Anja; Cuypers, Ann

    2016-01-01

    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. PMID:27685087

  7. Upolu Island, Western Samoa

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Island nations in the South Pacific Ocean experience natural disasters associated with typhoons, and with their proximity to the Pacific Ocean's 'Ring of Fire.' This radar image shows the western end of the island of Upolu in the nation of Western Samoa. Disaster managers use digital elevation models (DEMs) generated from radar data to assist in research toward disaster mitigation and management. Geologists also use DEM data of volcanic features, such as the circular craters in this image, to study eruption rates and volumes, and volcanic landform evolution.

    Black areas near the top of the image are areas where steep topography causes holes in the data; these holes can be filled in by collecting data at other look directions. Color represents topography and intensity represents across-section of the radar backscatter. Since rough areas return more of the incident signal, they appear brighter on the image than relatively smooth areas, such as the ocean surface at the top of the image.

    This image was acquired by the AIRborne Synthetic Aperture (AIRSAR) radar instrument aboard a DC-8 aircraft operated out of NASA's Dryden Flight Research Center. AIRSAR collects fully polarimetric data at three wavelengths; C-band (0.057 meter), L-band (0.25 meter) and P-band (0.68 meter). AIRSAR also collects cross-track and along track interferometric data that results in topographic measurements and motion detection, respectively.

    This image was collected during the Pacific Rim mission, a three-month mission from July to October 2000 that collected data at over 200 sites in eighteen countries and territories around the Pacific Rim. AIRSAR is managed by NASA's Jet Propulsion Laboratory, Pasadena, CA, for NASA's Earth Science Enterprise,Washington, D.C.

    Size: 10 km (6.2 miles) x 10 km (6.2 miles) Location: 14.02 deg. North lat., 171.52 deg. West Orientation: North at top Date Acquired: August 10, 2000

  8. Upolu Island, Western Samoa

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Island nations in the South Pacific Ocean experience natural disasters associated with typhoons, and with their proximity to the Pacific Ocean's 'Ring of Fire.' This radar image shows most of the northern coast of the island of Upolu in the nation of Western Samoa. Disaster managers use digital elevation models (DEMs) generated from radar data to assist in research toward disaster mitigation and management. Geologists also use DEM data of volcanic features, such as the line of circular craters in this image, to study eruption rates and volumes, and volcanic landform evolution. The capital of Western Samoa, Apia, is in the lower left of the image.

    Angular black areas in the image are areas where steep topography causes holes in the data; these holes can be filled in by collecting data at other look directions. Color represents topography and intensity represents across-section of the radar backscatter. Since rough areas return more of the incident signal, they appear brighter on the image than relatively smooth areas, such as the ocean surface , along the left side of the image.

    This image was acquired by the AIRborne Synthetic Aperture (AIRSAR) radar instrument aboard a DC-8 aircraft operated out of NASA's Dryden Flight Research Center. AIRSAR collects fully polarimetric data at three wavelengths; C-band (0.057 meter), L-band (0.25 meter) and P-band (0.68 meter). AIRSAR also collects cross-track and along track interferometric data that results in topographic measurements and motion detection, respectively.

    This image was collected during the Pacific Rim mission, a three-month mission from July to October 2000 that collected data at over 200 sites in eighteen countries and territories around the Pacific Rim. AIRSAR is managed by NASA's Jet Propulsion Laboratory, Pasadena, CA, for NASA's Earth Science Enterprise,Washington, D.C.

    Size: 10 km (6.2 miles) x 63 km (37.3 miles) Location: 14.16 deg. North lat., 171.75 deg. West Orientation: North towards

  9. Additional Types of Neuropathy

    MedlinePlus

    ... A A Listen En Español Additional Types of Neuropathy Charcot's Joint Charcot's Joint, also called neuropathic arthropathy, ... can stop bone destruction and aid healing. Cranial Neuropathy Cranial neuropathy affects the 12 pairs of nerves ...

  10. Food Additives and Hyperkinesis

    ERIC Educational Resources Information Center

    Wender, Ester H.

    1977-01-01

    The hypothesis that food additives are causally associated with hyperkinesis and learning disabilities in children is reviewed, and available data are summarized. Available from: American Medical Association 535 North Dearborn Street Chicago, Illinois 60610. (JG)

  11. Smog control fuel additives

    SciTech Connect

    Lundby, W.

    1993-06-29

    A method is described of controlling, reducing or eliminating, ozone and related smog resulting from photochemical reactions between ozone and automotive or industrial gases comprising the addition of iodine or compounds of iodine to hydrocarbon-base fuels prior to or during combustion in an amount of about 1 part iodine per 240 to 10,000,000 parts fuel, by weight, to be accomplished by: (a) the addition of these inhibitors during or after the refining or manufacturing process of liquid fuels; (b) the production of these inhibitors for addition into fuel tanks, such as automotive or industrial tanks; or (c) the addition of these inhibitors into combustion chambers of equipment utilizing solid fuels for the purpose of reducing ozone.

  12. 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.

    PubMed

    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.

  13. 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.

    PubMed

    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. PMID:25864766

  14. Investigation of the effects of experimental autolysis on the detection of abnormal prion protein in lymphoid and central nervous system tissues from elk and sheep using the Western blotting method

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic wasting disease CWD is a transmissible spongiform encephalopathy of cervid ruminants, including white tailed deer, mule deer, black tailed deer, moose, and elk. The disease is related to the scrapie of sheep. In both diseases, diagnosis is typically made by detection of the disease associa...

  15. Western Aeronautical Test Range

    NASA Technical Reports Server (NTRS)

    Sakahara, Robert D.

    2008-01-01

    NASA's Western Aeronautical Test Range (WATR) is a network of facilities used to support aeronautical research, science missions, exploration system concepts, and space operations. The WATR resides at NASA's Dryden Flight Research Center located at Edwards Air Force Base, California. The WATR is a part of NASA's Corporate Management of Aeronautical Facilities and funded by the Strategic Capability Asset Program (SCAP). It is managed by the Aeronautics Test Program (ATP) of the Aeronautics Research Mission Directorate (ARMD) to provide the right facility at the right time. NASA is a tenant on Edwards Air Force Base and has an agreement with the Air Force Flight Test Center to use the land and airspace controlled by the Department of Defense (DoD). The topics include: 1) The WATR supports a variety of vehicles; 2) Dryden shares airspace with the AFFTC; 3) Restricted airspace, corridors, and special use areas are available for experimental aircraft; 4) WATR Products and Services; 5) WATR Support Configuration; 6) Telemetry Tracking; 7) Time Space Positioning; 8) Video; 9) Voice Communication; 10) Mobile Operations Facilities; 11) Data Processing; 12) Mission Control Center; 13) Real-Time Data Analysis; and 14) Range Safety.

  16. Moon - Western Hemisphere

    NASA Technical Reports Server (NTRS)

    1990-01-01

    This image of the western hemisphere of the Moon was taken through a green filter by the Galileo spacecraft at 9:35 a.m. PST Dec. 9 at a range of about 350,000 miles. In the center is the Orientale Basin, 600 miles in diameter, formed about 3.8 billion years ago by the impact of an asteroid-size body. Orientale's dark center is a small mare. To the right is the lunar nearside with the great, dark Oceanus Procellarum above and the small, circular, dark Mare Humorum below. Maria are broad plains formed mostly over 3 billion years ago as vast basaltic lava flows. To the left is the lunar far side with fewer maria but, at lower left, the South-Pole-Aitken basin, about 1200 miles in diameter, which resembles Orientale but is much older and more weathered and battered by cratering. The intervening cratered highlands of both sides, as well as the maria, are dotted with bright, young craters. This image was 'reprojected' so as to center the Orientale Basin, and was filtered to enhance the visibility of small features. The digital image processing was done by DLR, the German Aerospace Research Establishment near Munich, an international collaborator in the Galileo mission.

  17. Additive Manufacturing Infrared Inspection

    NASA Technical Reports Server (NTRS)

    Gaddy, Darrell

    2014-01-01

    Additive manufacturing is a rapid prototyping technology that allows parts to be built in a series of thin layers from plastic, ceramics, and metallics. Metallic additive manufacturing is an emerging form of rapid prototyping that allows complex structures to be built using various metallic powders. Significant time and cost savings have also been observed using the metallic additive manufacturing compared with traditional techniques. Development of the metallic additive manufacturing technology has advanced significantly over the last decade, although many of the techniques to inspect parts made from these processes have not advanced significantly or have limitations. Several external geometry inspection techniques exist such as Coordinate Measurement Machines (CMM), Laser Scanners, Structured Light Scanning Systems, or even traditional calipers and gages. All of the aforementioned techniques are limited to external geometry and contours or must use a contact probe to inspect limited internal dimensions. This presentation will document the development of a process for real-time dimensional inspection technique and digital quality record of the additive manufacturing process using Infrared camera imaging and processing techniques.

  18. Effects of maternally exposed coloring food additives on receptor expressions related to learning and memory in rats.

    PubMed

    Ceyhan, Betul Mermi; Gultekin, Fatih; Doguc, Duygu Kumbul; Kulac, Esin

    2013-06-01

    Exposure to artificial food colors and additives (AFCAs) has been implicated in the induction and severity of some childhood behavioral and learning disabilities. N-methyl-D-aspartate receptors (NMDARs) and nicotinic acetylcholine receptors (nACHRs) are thought to be effective in the learning and memory-generating process. In this study, we investigated the effects of intrauterine exposure to AFCAs on subunit concentrations of NMDARs and nAChRs isoforms in rats. We administered a mixture of AFCAs (Eritrosin, Ponceau 4R, Allura Red AC, Sunset Yellow FCF, Tartrazin, Amaranth, Brilliant Blue, Azorubin and Indigotin) to female rats before and during gestation. The concentration of NR2A and NR2B subunits and nAChR α7, α4β2 isoforms in their offspring's hippocampi were measured by Western Blotting. Expressions of NR2B and nAChR β2 were significantly increased (17% and 6.70%, respectively), whereas expression of nAChR α4 was significantly decreased (5.67%) in male experimental group compared to the male control group (p<0.05). In the female experimental group, AFCAs caused a 14% decrease in NR2B expression when compared to the female control group (p<0.05). Our results indicate that exposure to AFCAs during the fetal period may lead to alterations in expressions of NMDARs and nAChRs in adulthood. These alterations were different between male and female genders. PMID:23429044

  19. Detection of IgM antibodies against a recombinant nucleocapsid protein of canine distemper virus in dog sera using a dot-blot assay.

    PubMed

    Barben, G; Stettler, M; Jaggy, A; Vandevelde, M; Zurbriggen, A

    1999-03-01

    A dot-blot assay for the detection of IgM antibodies (ABs) against canine distemper virus (CDV) in canine serum is described. The diagnostic potential of this technique was evaluated by analysing sera from three test groups: (i) specific pathogen-free (SPF) beagle dogs experimentally infected with virulent CDV; (ii) SPF dogs immunized with a combined vaccine containing CDV, and (iii) SPF dogs immunized with a CDV-free vaccine. As antigen for the dot-blot assay we used the recombinant nucleocapsid protein (N protein) of the virulent A75/17 CDV strain. All 12 dogs of group 1, infected with virulent CDV, showed detectable CDV-specific IgM levels in their serum. All dogs of group 2 were also positive for anti-CDV IgM after the first immunization with the CDV-containing vaccine. The four dogs immunized with a CDV-free vaccine (group iii) remained negative throughout the course of the experiment. From these results, we conclude that the IgM detection test, which requires only a single serum sample, is a useful method for diagnosing current or recent CDV infection in CDV-infected or CDV-immunized dogs under experimental conditions. PMID:10216448

  20. A Comparison of Antibacterial Activity of Selected Thyme (Thymus) Species by Means of the Dot Blot Test with Direct Bioautographic Detection.

    PubMed

    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.

  1. Polymerase chain reaction and Southern blot-based analysis of the C9orf72 hexanucleotide repeat in different motor neuron diseases.

    PubMed

    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 neuron diseases affecting primarily the first or the second motor neuron and hereditary spastic paraplegia). Whereas most studies on C9orf72 repeat expansions published so far rely on a polymerase chain reaction-based screening, we applied both polymerase chain reaction-based techniques and Southern blotting. Furthermore, we determined the sensitivity and specificity of Southern blotting of the C9orf72 hexanucleotide repeat in DNA derived from lymphoblastoid cell lines. C9orf72 repeat expansions were found in 27.1% out of 166 familial ALS patients, only once in 68 sporadic ALS patients, and not in 61 hereditary spastic paraplegia patients or 52 patients with motor neuron diseases affecting clinically primarily either the first or the second motor neuron. We found hints for a correlation between C9orf72 repeat length and the age of onset. Somatic instability of the C9orf72 repeat was observed in lymphoblastoid cell lines compared with DNA derived from whole blood from the same patient and therefore caution is warranted for repeat length determination in immortalized cell lines.

  2. A Comparison of Antibacterial Activity of Selected Thyme (Thymus) Species by Means of the Dot Blot Test with Direct Bioautographic Detection.

    PubMed

    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. PMID:26268965

  3. Analysis of the insulin receptor gene in noninsulin-dependent diabetes mellitus by denaturing gradient gel blots: A clinical research center study

    SciTech Connect

    Magre, J.; Goldfine, A.B.; Warram, J.H.

    1995-06-01

    We have used a new technique of denaturing gradient gel blotting to determine the prevalence of alterations in the intracellular domain of the insulin receptor in normal individuals and subjects with non-insulin-dependent diabetes mellitus (NIDDM). This method detects DNA sequence differences as restriction fragment melting polymorphisms (RFMP) and is sensitive to changes in sequence at both restriction sites and within the fragments themselves. Using restriction digests with AluI, HaeIII, HinfI, RsaI, Sau3A, and Sau96, 12 RFMPs were found to localize to the region of the {beta}-subunit of the insulin receptor gene. Using exon-specific probes, these RFMPs could be localized to specific regions surrounding individual exons, including exons, 14, 15, 16, 18, 20, and 22. In general, linkage disequilibrium between polymorphisms was inversely related to their distance in the gene structure, although there was a {open_quotes}hot spot{close_quotes} for recombination between exons 19 and 20. No difference in melting temperatures or allele frequency was observed between NIDDM patients and controls. These data indicate that the region of the insulin receptor gene coding for the intracellular portion of the {beta}-subunit is highly polymorphic and that polymorphisms surrounding specific exons can be identified by denaturing gradient gel blotting, but there is no evidence that variation at this locus contributes to NIDDM susceptibility in most individuals. 36 refs., 3 figs., 3 tabs.

  4. Phenylethynyl Containing Reactive Additives

    NASA Technical Reports Server (NTRS)

    Connell, John W. (Inventor); Smith, Joseph G., Jr. (Inventor); Hergenrother, Paul M. (Inventor)

    2002-01-01

    Phenylethynyl containing reactive additives were prepared from aromatic diamine, containing phenylethvnvl groups and various ratios of phthalic anhydride and 4-phenylethynviphthalic anhydride in glacial acetic acid to form the imide in one step or in N-methyl-2-pvrrolidinone to form the amide acid intermediate. The reactive additives were mixed in various amounts (10% to 90%) with oligomers containing either terminal or pendent phenylethynyl groups (or both) to reduce the melt viscosity and thereby enhance processability. Upon thermal cure, the additives react and become chemically incorporated into the matrix and effect an increase in crosslink density relative to that of the host resin. This resultant increase in crosslink density has advantageous consequences on the cured resin properties such as higher glass transition temperature and higher modulus as compared to that of the host resin.

  5. Western gas sands

    SciTech Connect

    Not Available

    1985-03-01

    The purpose of this research is to demonstrate the feasibility of economically producing natural gas from low-permeability reservoirs. Two broad research goals have been defined: (1) reducing the uncertainty of the reservoir production potential, and (2) improving the extraction technology. These goals are being pursued by conducting research and encouraging industrial efforts in developing the necessary technology, including: (1) providing fundamental research into the nature of tight, lenticular gas sands and the technologies for diagnosing and developing them: (2) developing and verifying the technology for effective gas production; and (3) promoting the transfer of research products and technology advances to the gas industry in usable forms. The focus of the research for the last several years has been improving diagnostic instrumentation for reservoir and stimulation performance evaluation, geophysical and engineering interpretation, and stimulation techniques. Integrated geologic studies of three basins containing tight lenticular sands, which were selected by DOE as priority research targets, have also been pursued as part of this new effort. To date, the following tentative conclusions have been formed: Permeability of the tight gas sands can be as much as three to four orders of magnitude lower than that of conventional gas deposits. Nineteen western geologic basins and trends containing significant volumes of tight gas have been identified. Gas resources in the priority geologic basins have been estimated - Piceance Basin 49 Tcf.; Greater Green River Basin, 136 Tcf.; Uinta Basin, 20 Tcf. Presence of natural micro-fractures within a reservoir and the effective propped length of hydraulically induced fratures are the critical parameters for successful development of tight sand resources. Stimulation technology at the present time is insufficient to efficiently recover gas from lenticular tight reservoirs. 8 figs., 3 tabs.

  6. Multifunctional fuel additives

    SciTech Connect

    Baillargeon, D.J.; Cardis, A.B.; Heck, D.B.

    1991-03-26

    This paper discusses a composition comprising a major amount of a liquid hydrocarbyl fuel and a minor low-temperature flow properties improving amount of an additive product of the reaction of a suitable diol and product of a benzophenone tetracarboxylic dianhydride and a long-chain hydrocarbyl aminoalcohol.

  7. Biobased lubricant additives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fully biobased lubricants are those formulated using all biobased ingredients, i.e. biobased base oils and biobased additives. Such formulations provide the maximum environmental, safety, and economic benefits expected from a biobased product. Currently, there are a number of biobased base oils that...

  8. Vinyl capped addition polyimides

    NASA Technical Reports Server (NTRS)

    Vannucci, Raymond D. (Inventor); Malarik, Diane C. (Inventor); Delvigs, Peter (Inventor)

    1991-01-01

    Polyimide resins (PMR) are generally useful where high strength and temperature capabilities are required (at temperatures up to about 700 F). Polyimide resins are particularly useful in applications such as jet engine compressor components, for example, blades, vanes, air seals, air splitters, and engine casing parts. Aromatic vinyl capped addition polyimides are obtained by reacting a diamine, an ester of tetracarboxylic acid, and an aromatic vinyl compound. Low void materials with improved oxidative stability when exposed to 700 F air may be fabricated as fiber reinforced high molecular weight capped polyimide composites. The aromatic vinyl capped polyimides are provided with a more aromatic nature and are more thermally stable than highly aliphatic, norbornenyl-type end-capped polyimides employed in PMR resins. The substitution of aromatic vinyl end-caps for norbornenyl end-caps in addition polyimides results in polymers with improved oxidative stability.

  9. Tackifier for addition polyimides

    NASA Technical Reports Server (NTRS)

    Butler, J. M.; St.clair, T. L.

    1980-01-01

    A modification to the addition polyimide, LaRC-160, was prepared to improve tack and drape and increase prepeg out-time. The essentially solventless, high viscosity laminating resin is synthesized from low cost liquid monomers. The modified version takes advantage of a reactive, liquid plasticizer which is used in place of solvent and helps solve a major problem of maintaining good prepeg tack and drape, or the ability of the prepeg to adhere to adjacent plies and conform to a desired shape during the lay up process. This alternate solventless approach allows both longer life of the polymer prepeg and the processing of low void laminates. This approach appears to be applicable to all addition polyimide systems.

  10. Traditional Chinese Medicine and Western Psychopharmacology: Building Bridges

    PubMed Central

    Shorter, Edward; Segesser, Kathryn

    2013-01-01

    This paper demonstrates that in the treatment of psychiatric disorders, there are striking similarities between the mechanisms of psychoactive agents used in Traditional Chinese Medicine (TCM) and those of western psychopharmacology. While western researchers search for new treatments and novel mechanisms of action, investigators in Asia are analyzing traditional remedies in order to understand the mechanisms responsible for their effectiveness. A review of contemporary pharmacologic studies of agents used in TCM for psychiatric indications reveals that virtually all of the active principles of drug action established in 20th century psychopharmacology were encountered empirically in Chinese herbal medicine over the past 2000 years. Building bridges between these two traditions may thus be of benefit to both cultures. In addition to providing western patients with a wider selection of treatment options, the effort may help Asian clinicians and researchers avoid some of the errors that have troubled their western counterparts. PMID:23418138

  11. Traditional Chinese medicine and Western psychopharmacology: building bridges.

    PubMed

    Shorter, Edward; Segesser, Kathryn

    2013-12-01

    This paper demonstrates that in the treatment of psychiatric disorders, there are striking similarities between the mechanisms of psychoactive agents used in Traditional Chinese Medicine (TCM) and those of western psychopharmacology. While western researchers search for new treatments and novel mechanisms of action, investigators in Asia are analyzing traditional remedies in order to understand the mechanisms responsible for their effectiveness. A review of contemporary pharmacologic studies of agents used in TCM for psychiatric indications reveals that virtually all of the active principles of drug action established in 20th century psychopharmacology were encountered empirically in Chinese herbal medicine over the past 2000 years. Building bridges between these two traditions may thus be of benefit to both cultures. In addition to providing western patients with a wider selection of treatment options, the effort may help Asian clinicians and researchers avoid some of the errors that have troubled their western counterparts.

  12. A simple, inexpensive, robust and sensitive dot-blot assay for equal detection of the nonstructural-1 glycoprotein of all dengue virus serotypes

    PubMed Central

    2013-01-01

    Background Detection of dengue virus (DENV) soluble/excreted (s/e) form of the nonstructural-1 (NS1) glycoprotein in patient acute-phase sera is ideal for diagnosis. The commercially-available detection assays are, however, too expensive for routine use and have low specificity, particularly for the s/e NS1 glycoprotein of DENV-2 and DENV-4, which are important causes of lethal human disease worldwide. Methods Mouse monoclonal antibodies (MAbs) were generated and screened against s/e NS1 glycoprotein purified from each DENV serotype to obtain those that reacted equally with each serotype, but not with yellow fever virus (YFV) s/e NS1 glycoprotein or human serum proteins. One MAb, MAb 2C4.6, was further tested against these DENV glycoproteins in human sera using simple, peroxidase-labelled secondary antibody/substrate-developed dot-blot assays. Results Optimal quenching of endogenous human serum peroxidases was attained using 3% H2O2 in H20 for 5 min. MAb 2C4.6 showed an acceptable detection sensitivity of < 32 ng/ml for the s/e NS1 glycoprotein of each DENV serotype but did not cross-react with the YFV s/e NS1 glycoprotein or human serum proteins. By contrast, the LX1 epitope-specific MAb, 3D1.4, showed similar detection sensitivity against only the DENV-1 NS1 glycoprotein, consistent with results from commercial DENV s/e NS1 glycoprotein detection assays. DENV s/e NS1 glycoproteins were stable in human sera after drying on the nitrocellulose membranes and storage for one month at ambient temperature (28°C) before being processed. The total assay time was reduced to 3 h without any loss of detection sensitivity. This dot-blot format was ideal for the circulating immune complex disruption step, which is required for increased DENV s/e NS1 glycoprotein detection. Conclusions This is the first study to determine the detection sensitivity of MAbs against known concentrations of s/e NS1 glycoprotein from each DENV serotype. The preparation of patient serum samples for

  13. Electrophilic addition of astatine

    SciTech Connect

    Norseev, Yu.V.; Vasaros, L.; Nhan, D.D.; Huan, N.K.

    1988-03-01

    It has been shown for the first time that astatine is capable of undergoing addition reactions to unsaturated hydrocarbons. A new compound of astatine, viz., ethylene astatohydrin, has been obtained, and its retention numbers of squalane, Apiezon, and tricresyl phosphate have been found. The influence of various factors on the formation of ethylene astatohydrin has been studied. It has been concluded on the basis of the results obtained that the univalent cations of astatine in an acidic medium is protonated hypoastatous acid.

  14. Functional Generalized Additive Models.

    PubMed

    McLean, Mathew W; Hooker, Giles; Staicu, Ana-Maria; Scheipl, Fabian; Ruppert, David

    2014-01-01

    We introduce the functional generalized additive model (FGAM), a novel regression model for association studies between a scalar response and a functional predictor. We model the link-transformed mean response as the integral with respect to t of F{X(t), t} where F(·,·) is an unknown regression function and X(t) is a functional covariate. Rather than having an additive model in a finite number of principal components as in Müller and Yao (2008), our model incorporates the functional predictor directly and thus our model can be viewed as the natural functional extension of generalized additive models. We estimate F(·,·) using tensor-product B-splines with roughness penalties. A pointwise quantile transformation of the functional predictor is also considered to ensure each tensor-product B-spline has observed data on its support. The methods are evaluated using simulated data and their predictive performance is compared with other competing scalar-on-function regression alternatives. We illustrate the usefulness of our approach through an application to brain tractography, where X(t) is a signal from diffusion tensor imaging at position, t, along a tract in the brain. In one example, the response is disease-status (case or control) and in a second example, it is the score on a cognitive test. R code for performing the simulations and fitting the FGAM can be found in supplemental materials available online.

  15. Western forests and air pollution

    SciTech Connect

    Olson, R.K.; Binkley, D.; Boehm, M.

    1992-01-01

    The book addresses the relationships between air pollution in the western United States and trends in the growth and condition of Western coniferous forests. The major atmospheric pollutants to which forest in the region are exposed are sulfur and nitrogen compounds and ozone. The potential effects of atmospheric pollution on these forests include foliar injury, alteration of growth rates and patterns, soil acidification, shifts in species composition, and modification of the effects of natural stresses.

  16. Comparison of Western immunoblotting and microimmunofluorescence for diagnosis of Mediterranean spotted fever.

    PubMed Central

    Teysseire, N; Raoult, D

    1992-01-01

    One-hundred serum samples from 41 patients suffering from Mediterranean spotted fever (MSF) were tested by microimmunofluorescence (MIF) and Western blot (WB; immunoblot). Immunoglobulin G (IgG), IgM, and IgA antibody-specific responses to the high-molecular-mass species-specific protein antigens (115 kDa and 135 kDa) of Rickettsia conorii, as well as to cross-reactive lipopolysaccharide (LPS) antigens, were observed. The WB assay detected IgM-type antibodies earlier than did the MIF assay. These antibodies were often directed against nonspecific LPS and may have a questionable positive predictive value. In addition, an IgG reaction to a 60-kDa protein was observed in four cases of malignant forms of MSF but was never observed in cases of mild forms. This reaction could be correlated with a marker of the severity of the development of MSF. From a previous MIF survey of blood donors, 9 negative, 11 IgG-positive, and 6 IgM-positive serum samples were selected for comparison by WB. Sera negative by MIF were also negative by WB. MIF IgG-positive sera showed a specific response to R. conorii in the WB assay, but the six serum samples from this seroepidemiological study positive for IgM by MIF were almost all negative by the WB assay. One was positive for IgM against the LPS but was considered a false positive. The WB is shown to provide a new tool for serodiagnosis. Images PMID:1537916

  17. Isolation and RNA gel blot analysis of genes that could serve as potential molecular markers for leaf senescence in Arabidopsis thaliana.

    PubMed

    Yoshida, S; Ito, M; Nishida, I; Watanabe, A

    2001-02-01

    Nine cDNAs, representing genes in which the transcripts accumulated in senescent leaves of Arabidopsis thaliana, were isolated by differential display reverse transcription polymerase chain reaction (DDRT-PCR) and the genes were designated yellow-leaf-specific gene 1 to 9 (YLS1-YLS9). Sequence analysis revealed that none of the YLS genes, except YLS6, had been reported as senescence-up-regulated genes. RNA gel blot analysis revealed that the transcripts of YLS3 accumulated at the highest level at an early senescence stage, whereas the transcripts from the other YLS genes reached their maximum levels in late senescence stages. Transcripts of YLS genes showed various accumulation patterns under natural senescence, and under artificial senescence induced by darkness, ethylene or ABA. These expression characteristics of YLS genes will be useful as potential molecular markers, which will enhance our understanding of natural and artificial senescence processes.

  18. Detection of Ca(2+)-binding proteins by electrophoretic migration in the presence of Ca2+ combined with 45Ca2+ overlay of protein blots

    SciTech Connect

    Garrigos, M.; Deschamps, S.; Viel, A.; Lund, S.; Champeil, P.; Moller, J.V.; le Maire, M. , Gif-sur-Yvette )

    1991-04-01

    When high affinity Ca(2+)-binding proteins like calmodulin, or proteins with a high Ca(2+)-binding capacity like calsequestrin, underwent sodium dodecyl sulfate-gel electrophoresis in Laemmli systems, their electrophoretic migration rates were much higher in gels containing 1 mM Ca2+ than in gels containing ethylene glycol bis(beta-aminoethyl ether) N,N{prime}-tetraacetic acid (EGTA). Replacement of EGTA by Ca2+ in the gel, combined with the blotting of electrophoretically separated proteins on polyvinylidene difluoride membranes and subsequent 45Ca2+ overlay, proved a very effective means of detecting Ca(2+)-binding proteins. This combined approach is important since artifacts occur in both techniques when used separately. We found that the usual procedure of adding Ca2+ to the sample before electrophoresis without including it in the gel itself permitted the detection of only very high affinity Ca(2+)-binding proteins.

  19. Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

    PubMed

    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. PMID:25946013

  20. Localization and Distribution of 'Candidatus Liberibacter asiaticus’ in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody

    PubMed Central

    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. PMID:25946013

  1. Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

    PubMed

    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.

  2. Detection of O6-carboxymethyl-2'-deoxyguanosine in DNA following reaction of nitric oxide with glycine and in human blood DNA using a quantitative immunoslot blot assay.

    PubMed

    Cupid, Belinda C; Zeng, Zuotao; Singh, Rajinder; Shuker, David E G

    2004-03-01

    Previous research has shown that a range of nitrosated glycine derivatives react with DNA to form O6-carboxymethylguanine and O6-methylguanine DNA adducts [Harrison et al. (1999) Chem. Res. Toxicol. 12, 106-111). Nitrosated glycine derivatives may be formed in the gastrointestinal tract from the reaction of dietary glycine with nitrosating agents. The aim of this study was to further investigate the role of dietary glycine in the formation of O6-guanine adducts at physiologically relevant concentrations. In vitro studies were performed by reacting 10 microM to 50 mM glycine with nitric oxide in the presence of oxygen. An HPLC assay was developed to measure the resulting nitrosated glycine derivative, diazoacetate anion. The amount of nitrosating agent present in the reaction mixture was determined by colorimetric measurement of nitrite, the hydrolysis product of N2O3. Diazoacetate anion formation depended linearly on glycine concentration. Solutions of nitrosated glycine reacted with 2'-deoxyguanosine and calf thymus DNA to give O6-carboxymethyl-2'-deoxyguanosine and, at high concentrations of glycine and nitric oxide, O6-methyl-2'-deoxyguanosine. At physiological concentrations of glycine and nitric oxide, diazoacetate anion was not detectable. Studies with synthetic diazoacetate anion showed that concentrations < 14 microM did not give detectable O6-carboxyethylguanine in DNA, even when a sensitive immunoslot blot assay was used. However, O6-carboxymethylguanine was detected in human blood DNA samples obtained from three volunteers consuming a standardized high meat diet, using the immunoslot blot assay. O6-Carboxymethylguanine levels ranged from 35 to 80 (detection limit = 15) O6-carboxymethylguanine per 10(8) bases. These studies provide further evidence that nitrosated amino acids may be risk factors for gastrointestinal tract cancers.

  3. Performance Boosting Additive

    NASA Technical Reports Server (NTRS)

    1999-01-01

    Mainstream Engineering Corporation was awarded Phase I and Phase II contracts from Goddard Space Flight Center's Small Business Innovation Research (SBIR) program in early 1990. With support from the SBIR program, Mainstream Engineering Corporation has developed a unique low cost additive, QwikBoost (TM), that increases the performance of air conditioners, heat pumps, refrigerators, and freezers. Because of the energy and environmental benefits of QwikBoost, Mainstream received the Tibbetts Award at a White House Ceremony on October 16, 1997. QwikBoost was introduced at the 1998 International Air Conditioning, Heating, and Refrigeration Exposition. QwikBoost is packaged in a handy 3-ounce can (pressurized with R-134a) and will be available for automotive air conditioning systems in summer 1998.

  4. Sewage sludge additive

    NASA Technical Reports Server (NTRS)

    Kalvinskas, J. J.; Mueller, W. A.; Ingham, J. D. (Inventor)

    1980-01-01

    The additive is for a raw sewage treatment process of the type where settling tanks are used for the purpose of permitting the suspended matter in the raw sewage to be settled as well as to permit adsorption of the dissolved contaminants in the water of the sewage. The sludge, which settles down to the bottom of the settling tank is extracted, pyrolyzed and activated to form activated carbon and ash which is mixed with the sewage prior to its introduction into the settling tank. The sludge does not provide all of the activated carbon and ash required for adequate treatment of the raw sewage. It is necessary to add carbon to the process and instead of expensive commercial carbon, coal is used to provide the carbon supplement.

  5. Perspectives on Additive Manufacturing

    NASA Astrophysics Data System (ADS)

    Bourell, David L.

    2016-07-01

    Additive manufacturing (AM) has skyrocketed in visibility commercially and in the public sector. This article describes the development of this field from early layered manufacturing approaches of photosculpture, topography, and material deposition. Certain precursors to modern AM processes are also briefly described. The growth of the field over the last 30 years is presented. Included is the standard delineation of AM technologies into seven broad categories. The economics of AM part generation is considered, and the impacts of the economics on application sectors are described. On the basis of current trends, the future outlook will include a convergence of AM fabricators, mass-produced AM fabricators, enabling of topology optimization designs, and specialization in the AM legal arena. Long-term developments with huge impact are organ printing and volume-based printing.

  6. Sarks as additional fermions

    NASA Astrophysics Data System (ADS)

    Agrawal, Jyoti; Frampton, Paul H.; Jack Ng, Y.; Nishino, Hitoshi; Yasuda, Osamu

    1991-03-01

    An extension of the standard model is proposed. The gauge group is SU(2) X ⊗ SU(3) C ⊗ SU(2) S ⊗ U(1) Q, where all gauge symmetries are unbroken. The colour and electric charge are combined with SU(2) S which becomes strongly coupled at approximately 500 GeV and binds preons to form fermionic and vector bound states. The usual quarks and leptons are singlets under SU(2) X but additional fermions, called sarks. transform under it and the electroweak group. The present model explains why no more than three light quark-lepton families can exist. Neutral sark baryons, called narks, are candidates for the cosmological dark matter having the characteristics designed for WIMPS. Further phenomenological implications of sarks are analyzed i including electron-positron annihilation. Z 0 decay, flavor-changing neutral currents. baryon-number non-conservation, sarkonium and the neutron electric dipole moment.

  7. Molecular and Kinetic Properties of Two Acetylcholinesterases from the Western Honey Bee, Apis mellifera

    PubMed Central

    Kim, Young Ho; Cha, Deok Jea; Jung, Je Won; Kwon, Hyung Wook; Lee, Si Hyeock

    2012-01-01

    We investigated the molecular and kinetic properties of two acetylcholinesterases (AmAChE1 and AmAChE2) from the Western honey bee, Apis mellifera. Western blot analysis revealed that AmAChE2 has most of catalytic activity rather than AmAChE1, further suggesting that AmAChE2 is responsible for synaptic transmission in A. mellifera, in contrast to most other insects. AmAChE2 was predominately expressed in the ganglia and head containing the central nervous system (CNS), while AmAChE1 was abundantly observed not only in the CNS but also in the peripheral nervous system/non-neuronal tissues. Both AmAChEs exist as homodimers; the monomers are covalently connected via a disulfide bond under native conditions. However, AmAChE2 was associated with the cell membrane via the glycophosphatidylinositol anchor, while AmAChE1 was present as a soluble form. The two AmAChEs were functionally expressed with a baculovirus system. Kinetic analysis revealed that AmAChE2 has approximately 2,500-fold greater catalytic efficiency toward acetylthiocholine and butyrylthiocholine than AmAChE1, supporting the synaptic function of AmAChE2. In addition, AmAChE2 likely serves as the main target of the organophosphate (OP) and carbamate (CB) insecticides as judged by the lower IC50 values against AmAChE2 than against AmAChE1. When OP and CB insecticides were pre-incubated with a mixture of AmAChE1 and AmAChE2, a significant reduction in the inhibition of AmAChE2 was observed, suggesting a protective role of AmAChE1 against xenobiotics. Taken together, based on their tissue distribution pattern, molecular and kinetic properties, AmAChE2 plays a major role in synaptic transmission, while AmAChE1 has non-neuronal functions, including chemical defense. PMID:23144990

  8. Population models of burrowing mayfly recolonization in Western Lake Erie

    USGS Publications Warehouse

    Madenjian, C.P.; Schloesser, D.W.; Krieger, K.A.

    1998-01-01

    Burrowing mayflies, Hexagenia spp. (H. limbata and H. rigida), began recolonizing western Lake Erie during the 1990s. Survey data for mayfly nymph densities indicated that the population experienced exponential growth between 1991 and 1997. To predict the time to full recovery of the mayfly population, we fitted logistic models, ranging in carrying capacity from 600 to 2000 nymphs/m2, to these survey data. Based on the fitted logistic curves, we forecast that the mayfly population in western Lake Erie would achieve full recovery between years 1998 and 2000, depending on the carrying capacity of the western basin. Additionally, we estimated the mortality rate of nymphs in western Lake Erie during 1994 and then applied an age-based matrix model to the mayfly population. The results of the matrix population modeling corroborated the exponential growth model application in that both methods yielded an estimate of the population growth rate, r, in excess of 0.8 yr-1. This was the first evidence that mayfly populations are capable of recolonizing large aquatic ecosystems at rates comparable with those observed in much smaller lentic ecosystems. Our model predictions should prove valuable to managers of power plant facilities along the western basin in planning for mayfly emergences and to managers of the yellow perch (Perca flavescens) fishery in western Lake Erie.

  9. Additive lattice kirigami

    PubMed Central

    Castle, Toen; Sussman, Daniel M.; Tanis, Michael; Kamien, Randall D.

    2016-01-01

    Kirigami uses bending, folding, cutting, and pasting to create complex three-dimensional (3D) structures from a flat sheet. In the case of lattice kirigami, this cutting and rejoining introduces defects into an underlying 2D lattice in the form of points of nonzero Gaussian curvature. A set of simple rules was previously used to generate a wide variety of stepped structures; we now pare back these rules to their minimum. This allows us to describe a set of techniques that unify a wide variety of cut-and-paste actions under the rubric of lattice kirigami, including adding new material and rejoining material across arbitrary cuts in the sheet. We also explore the use of more complex lattices and the different structures that consequently arise. Regardless of the choice of lattice, creating complex structures may require multiple overlapping kirigami cuts, where subsequent cuts are not performed on a locally flat lattice. Our additive kirigami method describes such cuts, providing a simple methodology and a set of techniques to build a huge variety of complex 3D shapes. PMID:27679822

  10. Additive lattice kirigami

    PubMed Central

    Castle, Toen; Sussman, Daniel M.; Tanis, Michael; Kamien, Randall D.

    2016-01-01

    Kirigami uses bending, folding, cutting, and pasting to create complex three-dimensional (3D) structures from a flat sheet. In the case of lattice kirigami, this cutting and rejoining introduces defects into an underlying 2D lattice in the form of points of nonzero Gaussian curvature. A set of simple rules was previously used to generate a wide variety of stepped structures; we now pare back these rules to their minimum. This allows us to describe a set of techniques that unify a wide variety of cut-and-paste actions under the rubric of lattice kirigami, including adding new material and rejoining material across arbitrary cuts in the sheet. We also explore the use of more complex lattices and the different structures that consequently arise. Regardless of the choice of lattice, creating complex structures may require multiple overlapping kirigami cuts, where subsequent cuts are not performed on a locally flat lattice. Our additive kirigami method describes such cuts, providing a simple methodology and a set of techniques to build a huge variety of complex 3D shapes.

  11. MicroRNA fate upon targeting with anti-miRNA oligonucleotides as revealed by an improved Northern-blot-based method for miRNA detection

    PubMed Central

    Torres, Adrian G.; Fabani, Martin M.; Vigorito, Elena; Gait, Michael J.

    2011-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs) are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such anti-miR ONs include 2′-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs. However, miRNA quantification in the presence of anti-miR ONs has proved to be challenging, due to detection artifacts, which has led to poor understanding of miRNA fate upon anti-miR binding. Here we show that anti-miR ON bound to miR-122 can prevent the miRNA from being properly precipitated into the purified RNA fraction using the standard RNA extraction protocol (TRI-Reagent), yielding an RNA extract that does not reflect the real cellular levels of the miRNA. An increase in the numbers of equivalents of isopropanol during the precipitation step leads to full recovery of the targeted miRNA back into the purified RNA extract. Following our improved protocol, we demonstrate by Northern blotting, in conjunction with a PNA decoy strategy and use of high denaturing PAGE, that high-affinity anti-miRs (K-PNA-K3, LNA/DNA, and LNA/OMe) sequester miR-122 without causing miRNA degradation, while miR-122 targeting with a lower-affinity anti-miR (OMe) seems to promote degradation of the miRNA. The technical issues explored in this work will have relevance for other hybridization-based techniques for miRNA quantification in the presence of anti-miR ONs. PMID:21441346

  12. Delineation of groundwater recharge areas, western Cape Cod, Massachusetts

    USGS Publications Warehouse

    Masterson, John P.; Walter, Donald A.

    2000-01-01

    For additional information on the hydrology and geology of western Cape Cod, the reader is referred to the following reports: LeBlanc and others (1986), Barlow and Hess (1993), Masterson and others (1997a), Masterson and others (1997b), Masterson and others (1998), Ogden Environmental and Energy Services, Inc. (1998) and Jacobs Engineering Group, Inc. (1999).

  13. 77 FR 11071 - Procurement List; Additions and Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-24

    ...: Western Idaho Training Company, Caldwell, ID. Contracting Activity: Dept of the Navy, Navy Region...@AbilityOne.gov . SUPPLEMENTARY INFORMATION: Additions On 12/23/2011 (76 FR 80346); 12/30/2011 (76 FR 82282-82283); and 1/ 6/2012 (77 FR 780), the Committee for Purchase From People Who Are Blind or...

  14. Takahashi Yoshitoki and Western astronomy

    NASA Astrophysics Data System (ADS)

    Yoshida, Tadashi

    2005-05-01

    When we discuss the achievements of Takahashi Yoshitoki (1764-1804), we must pay attention to the two "Western" astronomical treatises, for they contributed a great deal to the advancement of his astronomical knowledge. They are a sequel to the Li-xiang Kao-cheng, a Sino-Western work by a German Jesuit I. Koegler, et al. and the Dutch version of J. J. de Lalande's Astronomie. Both books introduced the latest astronomical knowledge from the West. This paper traces Yoshitoki's life and achievements, focusing on his encounter with these two works.

  15. 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.

    PubMed

    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.

  16. Direct measurement of tubulin and bulk message distributions on polysomes of growing, starved and deciliated Tetrahymena using RNA gel blots of sucrose gradients containing acrylamide.

    PubMed

    Calzone, F J; Callahan, R; Gorovsky, M A

    1988-10-25

    A method was developed using sucrose gradients containing acrylamide which greatly simplifies the measurement of the polysomal distribution of messages. After centrifugation, the acrylamide was polymerized, forming a "polysome gel". RNA gel blots of polysome gels were used to determine the polysomal distributions of alpha-tubulin and total polyadenylated mRNA in growing, starved (nongrowing) and starved-deciliated Tetrahymena and the number of messages loaded onto polysomes was calculated. These measurements indicated that the translational efficiencies of alpha-tubulin mRNA and total polyadenylated mRNA are largely unaffected when the rates of tubulin and total protein synthesis vary dramatically. Thus, differential regulation of alpha-tubulin mRNA translation initiation does not contribute to the greater than 100-fold induction of tubulin synthesis observed during cilia regeneration and in growing cells. The major translation-level process regulating tubulin synthesis in Tetrahymena appears to be a change in message loading mediated by a non-specific message recruitment or unmasking factor.

  17. Application of the Reverse Line Blot Assay for the Molecular Detection of Theileria and Babesia sp. in Sheep and Goat Blood Samples from Pakistan

    PubMed Central

    Iqbal, F; Khattak, RM; Ozubek, S; Khattak, MNK; Rasul, A; Aktas, M

    2013-01-01

    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. PMID:23914243

  18. Effects of histoplasmin M antigen chemical and enzymatic deglycosylation on cross-reactivity in the enzyme-linked immunoelectrotransfer blot method.

    PubMed Central

    Zancopé-Oliveira, R M; Bragg, S L; Reiss, E; Wanke, B; Peralta, J M

    1994-01-01

    The enzyme-linked immunoelectrotransfer blot (EITB) method was evaluated as a suitable method for detecting antibodies against M antigen of Histoplasma capsulatum by use of both glycosylated and deglycosylated M protein of histoplasmin (HMIN). Sera from patients with histoplasmosis, paracoccidioidomycosis, blastomycosis, coccidioidomycosis, and aspergillosis were tested by the EITB with glycosylated M protein of HMIN. This assay demonstrated 100% sensitivity with histoplasmosis serum samples, all of which reacted with the 94-kDa glycoprotein (M antigen). Although the EITB is highly sensitive, it is not specific for histoplasmosis when glycosylated M protein is used as an antigen. A total of 81% of paracoccidioidomycosis, 25% of blastomycosis, 33% of coccidioidomycosis, 73% of aspergillosis, and 16% of tuberculosis serum samples cross-reacted with M protein of HMIN and yielded patterns indistinguishable from those obtained with histoplasmosis serum samples. The EITB reactions with both untreated M antigen and M antigen altered by periodate oxidation or by deglycosylation with endoglycosidases were compared. Cross-reactions with heterologous sera in the EITB could be attributed to periodate-sensitive carbohydrate epitopes, as reflected by the increase in the test specificity from 46.1 to 91.2% after periodate treatment of M protein. The EITB for the detection of antibodies to M antigen is a potential diagnostic test for histoplasmosis, provided that periodate-treated M protein is used as an antigen. Images PMID:8556474

  19. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins.

    PubMed

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities.

  20. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

    PubMed Central

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

  1. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins.

    PubMed

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

  2. Consuming a Western diet for two weeks suppresses fetal genes in mouse hearts

    PubMed Central

    Medford, Heidi M.; Cox, Emily J.; Miller, Lindsey E.

    2014-01-01

    Diets high in sugar and saturated fat (Western diet) contribute to obesity and pathophysiology of metabolic syndrome. A common physiological response to obesity is hypertension, which induces cardiac remodeling and hypertrophy. Hypertrophy is regulated at the level of chromatin by repressor element 1-silencing transcription factor (REST), and pathological hypertrophy is associated with reexpression of a fetal cardiac gene program. Reactivation of fetal genes is commonly observed in hypertension-induced hypertrophy; however, this response is blunted in diabetic hearts, partially due to upregulation of the posttranslational modification O-linked-β-N-acetylglucosamine (O-GlcNAc) to proteins by O-GlcNAc transferase (OGT). OGT and O-GlcNAc are found in chromatin-modifying complexes, but it is unknown whether they play a role in Western diet-induced hypertrophic remodeling. Therefore, we investigated the interactions between O-GlcNAc, OGT, and the fetal gene-regulating transcription factor complex REST/mammalian switch-independent 3A/histone deacetylase (HDAC). Five-week-old male C57BL/6 mice were fed a Western (n = 12) or control diet (n = 12) for 2 wk to examine the early hypertrophic response. Western diet-fed mice exhibited fasting hyperglycemia and increased body weight (P < 0.05). As expected for this short duration of feeding, cardiac hypertrophy was not yet evident. We found that REST is O-GlcNAcylated and physically interacts with OGT in mouse hearts. Western blot analysis showed that HDAC protein levels were not different between groups; however, relative to controls, Western diet hearts showed increased REST and decreased ANP and skeletal α-actin. Transcript levels of HDAC2 and cardiac α-actin were decreased in Western diet hearts. These data suggest that REST coordinates regulation of diet-induced hypertrophy at the level of chromatin. PMID:24523346

  3. U.S. Heritage-Seeking Students Discover Minority Communities in Western Europe

    ERIC Educational Resources Information Center

    Comp, David

    2008-01-01

    This research article examines quantitative data relevant to an increasingly multiethnic Western Europe and investigates European opportunities for U.S. minority heritage-seeking students. In addition to analyzing the demographic data of Western Europe, a review of U.S. higher education enrollment demographics derived from current national…

  4. Thoughts on "Two Exemptions and One Subsidy" (TEOS) in China's Western Region

    ERIC Educational Resources Information Center

    Xiangyang, Tian

    2008-01-01

    The policy of "two exemptions and one subsidy" (TEOS), which is a "popular sentiment" project, has brought life to the western region's rural education and light to impoverished families. In addition to launching the battle for the "two basics" in the western region, the overall popularization of distance education for rural schools in the western…

  5. NEWE: A Western Shoshone History.

    ERIC Educational Resources Information Center

    Inter-Tribal Council of Nevada, Reno.

    One in a series of four histories of native Nevadans, this volume relates the history of the Western Shoshone, or Newe, whose territory included parts of the Great Basin area which extends from southern California to Idaho. Based on the spoken word of tribal elders and research conducted at numerous archives, the history begins with ancient…

  6. Skywatch: The Western Weather Guide.

    ERIC Educational Resources Information Center

    Keen, Richard A.

    The western United States is a region of mountains and valleys with the world's largest ocean next door. Its weather is unique. This book discusses how water, wind, and environmental conditions combine to create the climatic conditions of the region. Included are sections describing: fronts; cyclones; precipitation; storms; tornadoes; hurricanes;…

  7. Fleximode: Within Western Australia TAFE.

    ERIC Educational Resources Information Center

    Toussaint, Dorothy

    After fleximode was introduced into the Western Australian TAFE system, its cost and effectiveness compared with traditional delivery systems were evaluated. Fleximode, as practiced in Australia, was adapted from a mode of study pioneered in England. It offered students the independence of off-campus study in combination with access to college…

  8. Adult Education in Western Germany.

    ERIC Educational Resources Information Center

    Knoll, Joachim H.; And Others

    Here are abstracts of three books on adult education in Western Germany, where the institutions and methods of continuing education have been nearly unknown. The first, ERWACHSENENBILDUNG IN DER BUNDESREPUBLIK (ADULT EDUCATION IN THE FEDERAL REPUBLIC), 167 pages, justifies regarding adult education today as a complete changeover from its forms in…

  9. Multicultural Education in Western Societies.

    ERIC Educational Resources Information Center

    Banks, James A., Ed.; Lynch, James, Ed.

    Western democratic societies share an egalitarian ideology which maintains that a major goal of the state is to protect human rights and promote equality and the structural inclusion of all racial, ethnic, and cultural groups into the fabric of society. Educational initiatives taken to implement reforms that reflect ethnic diversity and promote…

  10. ALIENS IN WESTERN STREAM ECOSYSTEMS

    EPA Science Inventory

    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 ...

  11. Race Relations in Western Nebraska.

    ERIC Educational Resources Information Center

    Jenkins, Melvin L.

    This report is derived from a community forum held in Scottsbluff (Nebraska) on April 28-29, 1993, by the Nebraska Advisory Committee to the United States Commission on Civil Rights. Views on race relations in selected areas of western Nebraska were expressed by concerned citizens; community leaders; government officials (federal, state, and…

  12. Fires Across the Western United States

    NASA Technical Reports Server (NTRS)

    2007-01-01

    Days of record heat made the western United States tinder dry in early July 2007. Numerous wildfires raced across the dry terrain during the weekend of July 7. From Washington to Arizona, firefighters were battling fast-moving wildfires that threatened residences, businesses, gas wells, coal mines, communications equipment, and municipal watersheds. This image of the West was captured by the Moderate Resolution Imaging Spectroradiometer (MODIS) on NASA's Terra satellite on Sunday, July 8. Places where MODIS detected actively burning fires are marked in red. Some of the largest blazes are labeled. Utah's Milford Flat was the largest; according to the July 9 morning report from the National Interagency Fire Center, the blaze was more than 280,000 acres, having grown more than 124,000 acres in the previous 24 hours. The fires have destroyed homes, forced evacuations, shut down trains and highways, and killed several people. Weather conditions were not expected to improve significantly across much of the area for several days, with hot temperatures and dry thunderstorms (lightning and winds, but little rain) likely in many places. Nearly the entire western United States was experiencing some level of drought as of July 3, according to the U.S. Drought Monitor. The drought had reached the 'extreme' category in southern California and western Arizona, and ranged from moderate to severe across most of the rest of the Southwest and Great Basin. The large image provided above has a spatial resolution (level of detail) of 500 meters per pixel. The MODIS Rapid Response Team provides twice-daily images of the region in additional resolutions and formats, including an infrared-enhanced version that makes burned terrain appear brick red. NASA image courtesy the MODIS Rapid Response Team, Goddard Space Flight Center

  13. Genotyping of Canine parvovirus in western Mexico.

    PubMed

    Pedroza-Roldán, César; Páez-Magallan, Varinia; Charles-Niño, Claudia; Elizondo-Quiroga, Darwin; De Cervantes-Mireles, Raúl Leonel; López-Amezcua, Mario Alberto

    2015-01-01

    Canine parvovirus (CPV) is one of the most common infectious agents related to high morbidity rates in dogs. In addition, the virus is associated with severe gastroenteritis, diarrhea, and vomiting, resulting in high death rates, especially in puppies and nonvaccinated dogs. To date, there are 3 variants of the virus (CPV-2a, CPV-2b, and CPV-2c) circulating worldwide. In Mexico, reports describing the viral variants circulating in dog populations are lacking. In response to this deficiency, a total of 41 fecal samples of suspected dogs were collected from October 2013 through April 2014 in the Veterinary Hospital of the University of Guadalajara in western Mexico. From these, 24 samples resulted positive by polymerase chain reaction, and the viral variant was determined by restriction fragment length polymorphism. Five positive diagnosed samples were selected for partial sequencing of the vp2 gene and codon analysis. The results demonstrated that the current dominant viral variant in Mexico is CPV-2c. The current study describes the genotyping of CPV strains, providing valuable evidence of the dominant frequency of this virus in a dog population from western Mexico.

  14. Evaluation of a New Dot Blot Enzyme Immunoassay (Directigen Flu A+B) for Simultaneous and Differential Detection of Influenza A and B Virus Antigens from Respiratory Samples

    PubMed Central

    Reina, Jordi; Padilla, Emma; Alonso, Fermin; Ruiz de Gopegui, Enrique; Munar, Maria; Mari, Margarita

    2002-01-01

    We report a prospective evaluation of a new dot blot enzyme immunoassay (EIA) method for the direct, rapid, qualitative, simultaneous, and differential detection of the influenza A (IA) and B (IB) virus antigen in different respiratory samples. The EIA method was compared with the shell vial culture system (MDCK cell line) used with the same samples. We studied 160 samples from 93 (58.1%) pediatric patients (hospital emergency room) and from 67 (41.9%) adult patients (sentinel network). Seventy-four(46.2%) samples were considered positive; of them, 46 (62.2%) were from pediatric patients and 28 (37.8%) were from an adult group (P < 0.05), with overall positive values of 49.9% and 41.7%, respectively. All 74 (100%) of the positive samples were isolated in cell culture versus the 68.9% that were detected as positive by the new EIA method (P < 0.05). Of the 41 samples positive for the IA virus, the EIA detected 34 (82.9%) positive samples; of the 33 samples positive for the IB virus, the EIA detected 17 (51.5%) positive samples (P < 0.05). No false-positive reaction was detected with the EIA method (specificity and positive predictive value of 100%). The overall results obtained in the comparison between the new EIA and the shell vial culture had a sensibility of 82.9% and predictive negative values of 92.4% for the IA virus and 51.5% and 84.3%, respectively, for the IB virus. This evaluation shows sensitivity and specificity percentages for the new EIA method that is acceptable for routine use in IA virus detection. The results obtained were worse for IB virus detection, but this new EIA method is actually the only one with the capacity to differentiate between the two influenza viruses. PMID:12202608

  15. Evaluation of the Punch-it™ NA-Sample kit for detecting microbial DNA in blood culture bottles using PCR-reverse blot hybridization assay.

    PubMed

    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. PMID:27263831

  16. The prevalence of urogenital micro-organisms detected by a multiplex PCR-reverse line blot assay in women attending three sexual health clinics in Sydney, Australia.

    PubMed

    McKechnie, Michelle L; Hillman, Richard J; Jones, Rachel; Lowe, Penelope C; Couldwell, Deborah L; Davies, Stephen C; King, Fiona; Kong, Fanrong; Gilbert, Gwendolyn L

    2011-07-01

    This study used a previously described multiplex PCR-based reverse line blot (mPCR/RLB) assay to assess the prevalence and distribution of 14 urogenital pathogens or putative pathogens, namely Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Trichomonas vaginalis, Gardnerella vaginalis, Ureaplasma parvum, Ureaplasma urealyticum, Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, herpes simplex virus types 1 and 2, and human adenovirus. First-voided urine specimens and endocervical and self-collected vaginal swabs from each of 216 women attending three sexual health clinics in Sydney, Australia, were tested and the results were compared with those of reference methods for each organism. One hundred and sixty-eight women (77.7 %) had at least one and 105 (48.6 %) had more than one target organism, most commonly G. vaginalis and Ureaplasma spp. The prevalence of each of the four known sexually transmissible pathogens was <5 %. Of the 216 women, 111 (51.4 %) reported at least one symptom consistent with genital or urethral infection, including discharge, pain or discomfort. Only G. vaginalis was detected more frequently in women with symptoms (P = 0.05). The specificity of the mPCR/RLB assay compared with that of the reference methods for each organism and for all specimen types was 100 %. The mean sensitivities of the mPCR/RLB assay compared with those of the reference methods for self-collected vaginal swabs, cervical swabs and first-voided urine specimens for all organisms were 99.3, 98.1 and 84.6 %, respectively; however, these differences were not significant. There were no differences in sensitivities between specimen types for C. trachomatis, N. gonorrhoeae, T. vaginalis and H. influenzae, although all were found infrequently. Overall, the mPCR/RLB platform was found to be an accurate testing platform in a sexual health clinic setting.

  17. 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.

    PubMed

    Wang, He; Xiao, Meng; Kong, Fanrong; Chen, Sharon; Dou, Hong-Tao; Sorrell, Tania; Li, Ruo-Yu; Xu, Ying-Chun

    2011-05-01

    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

  18. Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

    SciTech Connect

    Okita, Naoyuki; Ashizawa, Daisuke; Ohta, Ryo; Abe, Hideaki; Tanuma, Sei-ichi

    2010-02-19

    Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V{sub max}) and the michaelis constant (k{sub m}) of PARG reaction were 4.46 {mu}M and 128.33 {mu}mol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 {mu}M. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.

  19. Genome of turbot rhabdovirus exhibits unusual non-coding regions and an additional ORF that could be expressed in fish cell.

    PubMed

    Zhu, Ruo-Lin; Lei, Xiao-Ying; Ke, Fei; Yuan, Xiu-Ping; Zhang, Qi-Ya

    2011-02-01

    Genomic sequence of Scophthalmus maximus rhabdovirus (SMRV) isolated from diseased turbot has been characterized. The complete genome of SMRV comprises 11,492 nucleotides and encodes five typical rhabdovirus genes N, P, M, G and L. In addition, two open reading frames (ORF) are predicted overlapping with P gene, one upstream of P and smaller than P (temporarily called Ps), and another in P gene which may encodes a protein similar to the vesicular stomatitis virus C protein. The C ORF is contained within the P ORF. The five typical proteins share the highest sequence identities (48.9%) with the corresponding proteins of rhabdoviruses in genus Vesiculovirus. Phylogenetic analysis of partial L protein sequence indicates that SMRV is close to genus Vesiculovirus. The first 13 nucleotides at the ends of the SMRV genome are absolutely inverse complementarity. The gene junctions between the five genes show conserved polyadenylation signal (CATGA(7)) and intergenic dinucleotide (CT) followed by putative transcription initiation sequence A(A/G)(C/G)A(A/G/T), which are different from known rhabdoviruses. The entire Ps ORF was cloned and expressed, and used to generate polyclonal antibody in mice. One obvious band could be detected in SMRV-infected carp leucocyte cells (CLCs) by anti-Ps/C serum via Western blot, and the subcellular localization of Ps-GFP fusion protein exhibited cytoplasm distribution as multiple punctuate or doughnut shaped foci of uneven size.

  20. Transcriptome analysis provides new insights into liver changes induced in the rat upon dietary administration of the food additives butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole.

    PubMed

    Stierum, Rob; Conesa, Ana; Heijne, Wilbert; Ommen, Ben van; Junker, Karin; Scott, Mary P; Price, Roger J; Meredith, Clive; Lake, Brian G; Groten, John

    2008-08-01

    Transcriptomics was performed to gain insight into mechanisms of food additives butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG), and thiabendazole (TB), additives for which interactions in the liver can not be excluded. Additives were administered in diets for 28 days to Sprague-Dawley rats and cDNA microarray experiments were performed on hepatic RNA. BHT induced changes in the expression of 10 genes, including phase I (CYP2B1/2; CYP3A9; CYP2C6) and phase II metabolism (GST mu2). The CYP2B1/2 and GST expression findings were confirmed by real time RT-PCR, western blotting, and increased GST activity towards DCNB. CC altered the expression of 12 genes. Three out of these were related to peroxisomes (phytanoyl-CoA dioxygenase, enoyl-CoA hydratase; CYP4A3). Increased cyanide insensitive palmitoyl-CoA oxidation was observed, suggesting that CC is a weak peroxisome proliferator. TB changed the expression of 12 genes, including CYP1A2. In line, CYP1A2 protein expression was increased. The expression level of five genes, associated with p53 was found to change upon TB treatment, including p53 itself, GADD45alpha, DN-7, protein kinase C beta and serum albumin. These array experiments led to the novel finding that TB is capable of inducing p53 at the protein level, at least at the highest dose levels employed above the current NOAEL. The expression of eight genes changed upon PG administration. This study shows the value of gene expression profiling in food toxicology in terms of generating novel hypotheses on the mechanisms of action of food additives in relation to pathology.

  1. Use of the Falcon assay screening test--enzyme-linked immunosorbent assay (FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) to determine the prevalence of human fascioliasis in the Bolivian Altiplano.

    PubMed

    Hillyer, G V; Soler de Galanes, M; Rodriguez-Perez, J; Bjorland, J; Silva de Lagrava, M; Ramirez Guzman, S; Bryan, R T

    1992-05-01

    A collaborative study between the University of Puerto Rico School of Medicine, the Centers for Disease Control, the Bolivian Ministry of Health, and private voluntary organizations (Foster Parents Plan International and Danchurchaid) working in Bolivia has identified a region in the northwestern Altiplano of Bolivia near Lake Titicaca as harboring the highest prevalence of human fascioliasis in the world reported to date. Two serologic techniques (the Falcon assay screening test-enzyme-linked immunosorbent assay [FAST-ELISA] and the enzyme-linked immunoelectrotransfer blot [EITB]) were used in the determination of its prevalence. One hundred serum samples and 73 stool samples were obtained from Aymara Indians from Corapata, Bolivia. Antibody absorbance levels to Fasciola hepatica excretion-secretion antigens were compared with EITB banding patterns using the same antigen preparation. A positive FAST-ELISA result was defined as an absorbance value greater than the mean plus three standard deviations of two sets of normal negative controls (Puerto Rican and Bolivian). Using this criterion, 53 of 100 sera tested were found positive by this technique. Within this group, 19 (95%) of 20 individuals who were parasite positive were also positive by FAST-ELISA. An additional 24 individuals who were negative for F. hepatica eggs and 10 individuals for whom no specimens were received were also positive by FAST-ELISA. Among the 53 individuals negative for F. hepatica eggs, 29 were also negative by FAST-ELISA. The EITB analysis of the sera from confirmed infected individuals revealed at least three F. hepatica (Fh) bands with molecular weights of 12, 17, and 63 kD, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. 76 FR 76704 - Western Technical College; Notice of Application Tendered for Filing With the Commission and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-08

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Federal Energy Regulatory Commission Western Technical College; Notice of Application Tendered for Filing With the Commission and Soliciting Additional Study Requests Take notice that the following...

  3. Shallow magma targets in the western US

    SciTech Connect

    Hardee, H.C.

    1984-10-01

    Within the next few years a hole will be drilled into a shallow magma body in the western US for the purpose of evaluating the engineering feasibility of magma energy. This paper examines potential drilling sites for these engineering feasibility experiments. Target sites high on the list are ones that currently exhibit good geophysical and geological data for shallow magma and also have reasonable operational requirements. Top ranked sites for the first magma energy well are Long Valley, CA, and Coso/Indian Wells, CA. Kilauea, HI, also in the top group, is an attractive site for some limited field experiments. A number of additional sites offer promise as eventual magma energy sites, but sparsity of geophysical data presently prevents these sites from being considered for the first magma energy well.

  4. WESTERN AIRBORNE CONTAMINANTS ASSESSMENT PROJECT RESEARCH PLAN

    EPA Science Inventory

    The goal of the Western Airborne Contaminants Assessment Project (WACAP) is to assess the deposition of airborne contaminants in Western National Parks, providing regional and local information on exposure, accumulation, impacts, and probable sources. This project is being desig...

  5. [Environmental efficiency evaluation under carbon emission constraint in Western China].

    PubMed

    Rong, Jian-bo; Yan, Li-jiao; Huang, Shao-rong; Zhang, Ge

    2015-06-01

    This research used the SBM model based on undesirable outputs to measure the static environmental efficiency of Western China under carbon emission constraint from 2000 to 2012. The researchers also utilized the Malmquist index to further analyze the change tendency of environmental efficiency. Additionally, Tobit regression analysis was used to study the factors relevant to environmental efficiency. Practical solutions to improve environmental quality in Western China were put forward. The study showed that in Western China, environmental efficiency with carbon emission constraint was significantly lower than that without carbon emission constraint, and the difference could be described as an inverse U-shaped curve which increased at first and then decreased. Guang-xi and Inner Mongolia, the two provinces met the effective environmental efficiency levels all the time under carbon emission constraint. However, the five provinces of Guizhou, Gansu, Qinghai, Ningxia and Xinjiang did not. Furthermore, Ningxia had the lowest level of environmental efficiency, with a score between 0.281-0.386. Although the environmental efficiency of most provinces was currently at an ineffective level, the environmental efficiency quality was gradually improving at an average speed of 6.6%. Excessive CO2 emission and a large amount of energy consumption were the primary factors causing environmental inefficiency in Western China, and energy intensity had the most negative impact on the environmental efficiency. The increase of import and export trade reduced the environmental efficiency significantly in Western China, while the increase of foreign direct investment had a positive effect on its environmental efficiency.

  6. Hammersley Range, northern Western Australia

    NASA Technical Reports Server (NTRS)

    1990-01-01

    The oval shaped basin of the sedimentary rocks of the Hammersley Range, northern Western Australia (23.0S, 119.0E) dominates the center of this near nadir view. The Fortescue River is the remarkably straight, fault controlled feature bordering the Hammersley on the north. Sand dunes are the main surface features in the northeast and southwest. Many dry lakebeds can be seen to the east as light grey colored patches along the watercourses.

  7. The western Veil nebula (Image)

    NASA Astrophysics Data System (ADS)

    Glenny, M.

    2009-12-01

    The western Veil nebula in Cygnus. 15-part mosaic by Mike Glenny, Gloucestershire, taken over several months mostly in the autumn of 2008. 200mm LX90/f10 autoguided, Meade UHC filter, 0.3xFR/FF, Canon 20Da DSLR. Exposures each typically 10x360 secs at ISO1600, processed in Registax4, PixInsight (for flat field correction) & Photoshop CS.

  8. Urban landscapes and the western drought

    NASA Astrophysics Data System (ADS)

    Pataki, D. E.

    2015-12-01

    Cities in the western U.S. are heavily irrigated and have increasingly been the focus of water conservation measures. Even cities that previously relied only on voluntary reductions in outdoor water use have been employing stricter mandates to limit irrigation. These cities are in a period of transition and the outcomes are far from certain. There are many tradeoffs in the environmental and social consequences of different urban water management strategies. Here we review recent work studying these tradeoffs in cities of southern California and Utah. We have measured the water use of different types of landscapes ranging from turfgrass to urban trees to xeriscapes. Unshaded turfgrass shows evapotranspiration (ET) rates close to potential ET; however, shaded turfgrass uses substantially less water. On the other hand, plants used in xeriscapes may have surprisingly high transpiration rates if they are heavily watered. In addition, unshaded xeriscapes may substantially alter surface energy balance and have unintended consequences for urban climate. Through whole tree sap flux measurements and scaling of ET estimates, we have found that urban trees generally use less water than turfgrass, and provide additional cooling benefits through interception of radiation. Current measures to reduce outdoor water use through irrigation restrictions and turfgrass removal programs do not include safeguards to ensure that urban trees receive adequate irrigation, and the future of urban tree canopies in western cities is highly uncertain. Although trees and other deep-rooted vegetation may require less irrigation than turfgrass and better withstand periods of drought, this vegetation must still be appropriate managed with water inputs informed by an understanding of plant water relations and urban subsurface hydrology. On the current trajectory, cities may see a substantial loss of vegetative cover and leaf area unless an understanding of ecohydrology is better integrated into

  9. Enhancement of recombinant erythropoietin production in CHO cells in an incubator without CO(2) addition.

    PubMed

    Yoon, S K; Ahn, Y H; Han, K

    2001-10-01

    The effect of low levels of carbon dioxide (CO(2)) in the gas phase on the production of recombinant human erythropoietin (EPO)in CHO cells was explored. A T-flask culture in an incubator without CO(2) addition showed a slow cell growth initially followed by the cessation of growth, while other cultures incubated under 0.5-5% CO(2) concentrations grew normally at the same rate during the entire period of cultivation. Interestingly, the production of EPO in the culture incubated under no CO(2) supply was highest among the tested cultures. The cell specific secretion rate of EPO (q(EPO)) of the culture under no CO(2) supply was about 3 times higher than that of the culture under 5% CO(2) supply. Western blot analysis and in vivo bioassay of EPO showed no apparent changes in EPO quality between the two cases of different CO(2) environments (air vs. 5% CO(2)), suggesting robust glycosylation of EPO by CHO cells even under very reduced CO(2) environment. Various combinations of the two extreme cases, with 5% CO(2) supply (suitable for cell growth) and no CO(2) addition (better for EPO production), were made in order to maximize the volumetric productivity of EPO secretion (P(V)) in CHO cells. The P(V) of the cultures programmed with initial incubation under 5% CO(2) followed by no CO(2) supply was about 2 times superior to that of the culture incubated only under no CO(2) supply. The P(V) of the culture under no CO(2) supply was slightly lower than that of culture grown under 5% CO(2). However, the q(EPO) of the no CO(2) supply case was more than 5 times higher than that of the culture under 5% CO(2) supply. In conclusion, we have demonstrated that a simple programming of CO(2) supply to an incubator can enhance the production of EPO in CHO cells remarkably, without any apparent change of the EPO quality. PMID:19002908

  10. Buddha philosophy and western psychology

    PubMed Central

    Aich, Tapas Kumar

    2013-01-01

    Four noble truths as preached by Buddha are that the life is full of suffering (Duhkha), that there is a cause of this suffering (Duhkha-samudaya), it is possible to stop suffering (Duhkha-nirodha), and there is a way to extinguish suffering (Duhkha-nirodha-marga). Eight fold Path (astangika-marga) as advocated by Buddha as a way to extinguish the sufferings are right views, right resolve/aspiration, right speech, right action/conduct, right livelihood, right effort right mindfulness and right concentration. Mid-twentieth century saw the collaborations between many psychoanalysts and Buddhist scholars as a meeting between “two of the most powerful forces” operating in the Western mind. Buddhism and Western Psychology overlap in theory and in practice. Over the last century, experts have written on many commonalities between Buddhism and various branches of modern western psychology like phenomenological psychology, psychoanalytical psychotherapy, humanistic psychology, cognitive psychology and existential psychology. Orientalist Alan Watts wrote ‘if we look deeply into such ways of life as Buddhism, we do not find either philosophy or religion as these are understood in the West. We find something more nearly resembling psychotherapy’. Buddha was a unique psychotherapist. His therapeutic methods helped millions of people throughout the centuries. This essay is just an expression of what little the current author has understood on Buddha philosophy and an opportunity to offer his deep tribute to one of the greatest psychotherapists the world has ever produced! PMID:23858249

  11. The seismicity of western Scandinavia

    SciTech Connect

    Abrraseys, N.N.

    1985-05-01

    The purpose of this paper is to present the results of an evaluation of the seismicity of western Scandinavia. Intensities, with reference to the MSK scale, have been assessed for the larger earthquakes, in most cases from primary sources, and isoseismal maps have been constructed for the most important events, while for smaller shocks one or more isoseismal radii were estimated. In all, over 3,200 earthquakes have been retrieved for Northwest Europe, including about 300 artificial events such as chemical explosions, rock-bursts and mine explosions. Of these, 500 events occurred in western Scandinavia. Surface-wave magnitudes were reassessed for 205 events, using surface-wave amplitude-period data, and all earthquakes recorded by more than 6 stations were relocated for the period prior to 1955. A calibration formula, obtained from the combination of macroseismic and instrumental data of the 20th century, has been used to assign magnitudes to all events for which there is macroseismic information available, thus deriving a homogeneous body of data covering a period of 180 years. It is shown that the largest earthquake in the region since 1800 occurred on land and that it had magnitude in excess of 6.0. This is contrary to the current belief, based on short-term data, that the larger events in western Fennoscandia occur offshore in the continental shelf areas.

  12. Buddha philosophy and western psychology.

    PubMed

    Aich, Tapas Kumar

    2013-01-01

    Four noble truths as preached by Buddha are that the life is full of suffering (Duhkha), that there is a cause of this suffering (Duhkha-samudaya), it is possible to stop suffering (Duhkha-nirodha), and there is a way to extinguish suffering (Duhkha-nirodha-marga). Eight fold Path (astangika-marga) as advocated by Buddha as a way to extinguish the sufferings are right views, right resolve/aspiration, right speech, right action/conduct, right livelihood, right effort right mindfulness and right concentration. Mid-twentieth century saw the collaborations between many psychoanalysts and Buddhist scholars as a meeting between "two of the most powerful forces" operating in the Western mind. Buddhism and Western Psychology overlap in theory and in practice. Over the last century, experts have written on many commonalities between Buddhism and various branches of modern western psychology like phenomenological psychology, psychoanalytical psychotherapy, humanistic psychology, cognitive psychology and existential psychology. Orientalist Alan Watts wrote 'if we look deeply into such ways of life as Buddhism, we do not find either philosophy or religion as these are understood in the West. We find something more nearly resembling psychotherapy'. Buddha was a unique psychotherapist. His therapeutic methods helped millions of people throughout the centuries. This essay is just an expression of what little the current author has understood on Buddha philosophy and an opportunity to offer his deep tribute to one of the greatest psychotherapists the world has ever produced!

  13. 33 CFR 125.06 - Western rivers.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 2 2014-07-01 2014-07-01 false Western rivers. 125.06 Section... VESSELS § 125.06 Western rivers. The term western rivers as used in the regulations in this subchapter shall include only the Red River of the North, the Mississippi River and its tributaries above the...

  14. 33 CFR 125.06 - Western rivers.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false Western rivers. 125.06 Section... VESSELS § 125.06 Western rivers. The term western rivers as used in the regulations in this subchapter shall include only the Red River of the North, the Mississippi River and its tributaries above the...

  15. 33 CFR 125.06 - Western rivers.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 2 2012-07-01 2012-07-01 false Western rivers. 125.06 Section... VESSELS § 125.06 Western rivers. The term western rivers as used in the regulations in this subchapter shall include only the Red River of the North, the Mississippi River and its tributaries above the...

  16. 33 CFR 125.06 - Western rivers.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Western rivers. 125.06 Section... VESSELS § 125.06 Western rivers. The term western rivers as used in the regulations in this subchapter shall include only the Red River of the North, the Mississippi River and its tributaries above the...

  17. Effect of bovine lactoferrin addition to milk in yogurt manufacturing.

    PubMed

    Franco, I; Castillo, E; Pérez, M D; Calvo, M; Sánchez, L

    2010-10-01

    The aim of this work was to study the effect of milk supplementation with lactoferrin of different iron saturation on the manufacturing and characteristics of yogurt. Bovine lactoferrin was added at concentrations of 0.5, 1, and 2 mg/mL in the holo (iron saturated) and apo (without iron) forms. Some physicochemical properties, such as pH, concentration of lactic acid, and texture of supplemented yogurts, were determined throughout the shelf-life period storage (28 d) at 4°C. We also evaluated the stability of lactoferrin in supplemented yogurt throughout the storage time. The supplementation of milk with bovine lactoferrin did not greatly affect the physical properties of the yogurt, though apo-lactoferrin slightly delayed the decrease of pH. This could be attributed to the partial inhibition observed on the growth of Streptococcus thermophilus. The integrity and immunoreactive concentration of lactoferrin, determined by Western blotting and noncompetitive ELISA, respectively, remained constant throughout the shelf life of yogurt.

  18. Tectonic evolution of the western Kunlun orogenic belt, western China

    NASA Astrophysics Data System (ADS)

    Zhihong, Wang

    2004-11-01

    The western Kunlun orogenic belt in western China evolved through the development of a large subduction-accretionary complex, including flysch sediments and granitic plutons, and by collision of three terranes, namely the North and South Kunlun and Karakorum-Qiangtang blocks from the early Paleozoic to the early Mesozoic. North-dipping subduction of the Paleo-Tethys ocean beneath the Kunlun terranes, which may have commenced in the Cambrian, produced an early Paleozoic Andean-type magmatic arc on the South Kunlun, and a marginal back arc basin, represented by the early Paleozoic Oytag-Kudi ophiolite belt, between the North and South Kunlun. A northward subduction zone consumed the basin and the young, hot upper plate lithosphere was obducted southward onto the South Kunlun following closure of the basin. This resulted in collision of the North and South Kunlun in the early Devonian. Continuous northward subduction of Paleo-Tethys resulted in the development of a Carboniferous-Triassic magmatic arc, and a back arc rifting sequence composed of the Carboniferous to Permian carbonates and clastic sediments on the North and South Kunlun terranes. The Paleo-Tethys ocean finally closed in the late Triassic-early Jurassic, when the Kunlun and Karakorum-Qiangtang blocks were accreted, with the Kara-Kunlun accretionary prism marking their suture zone.

  19. Western Slope of Andes, Peru

    NASA Technical Reports Server (NTRS)

    2008-01-01

    Along the western flank of the Andes, 400 km SE of Lima Peru, erosion has carved the mountain slopes into long, narrow serpentine ridges. The gently-sloping sediments have been turned into a plate of worms wiggling their way downhill to the ocean.

    The image was acquired September 28, 2004, covers an area of 38 x 31.6 km, and is located near 14.7 degrees south latitude, 74.5 degrees west longitude.

    The U.S. science team is located at NASA's Jet Propulsion Laboratory, Pasadena, Calif. The Terra mission is part of NASA's Science Mission Directorate.

  20. The Overthrust Belt of Western North America

    SciTech Connect

    Verrall, P.

    1993-02-01

    The Overthrust Belt extends for 5000 mi (8000 km) from the Brooks Range in Alaska to the Sierra Madre Oriental in Mexico. It consists of northeastward vergent thrust and fold structures involving late Precambrian to early Tertiary sedimentary section. These sediments represent deposition off the western rift margin, formed in late Precambrian time, of the North American Precambrian craton. The northeastward thrusting continued throughout the Mesozoic as a response to the convergence of the East Pacific Plate with the North American Plate. This convergence resulted in subduction beneath the North American Plate except at the northwest end (the Brooks Range) where the result was obduction. Convergence ceased when the west edge of the East Pacific Plate reached the subduction zone. The sedimentary section involved in the Thrust Belt contains good Devonian to Cretaceous hydrocarbon source rocks, and Ordovician to traps related to the thrusting (simple thrust sheets, imbricate thrust sheets, folded thrust sheets, step anticlines, footwall cutoffs, footwall anticlines, etc.). Field methods involved in exploration for hydrocarbons include field geological mapping, remote sensing (aerial photography and Landsat imagery), various seismic refraction and seismic reflection techniques (including modern detailed three dimension surveys) and potential field methods such as gravity and magnetic surveying. Studies of the field data include paleontology, source rock and hydrocarbon migration studies, structural and stratigraphic analyses, and the processing of geophysical data. This work has succeeded in two major areas: the Western Canadian Rocky Mountain Foothills, a major gas province producing mainly from Paleozoic reservoirs; and the Wyoming-Idaho-Utah portion of the thrust belt, also a major gas producer from Paleozoic reservoirs and, in addition, a major oil producer from the Jurassic Nugget Sandstone.

  1. 78 FR 21916 - Procurement List; Addition And Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-12

    ... INFORMATION: Addition On 2/22/2013 (78 FR 12296-12297), the Committee for Purchase From People Who Are Blind... Activity: DEPT OF THE ARMY, W40M WESTERN RGNL CNTRG OFC, TACOMA, WA Deletions On 3/23/2012 (77 FR 17035); 3/30/2012 (77 FR 19263); 4/6/2012 (77 FR 20795); 4/27/2012 (77 FR 25146-25147); 5/11/2012 (77 FR...

  2. Incorporation of additives into polymers

    DOEpatents

    McCleskey, T. Mark; Yates, Matthew Z.

    2003-07-29

    There has been invented a method for incorporating additives into polymers comprising: (a) forming an aqueous or alcohol-based colloidal system of the polymer; (b) emulsifying the colloidal system with a compressed fluid; and (c) contacting the colloidal polymer with the additive in the presence of the compressed fluid. The colloidal polymer can be contacted with the additive by having the additive in the compressed fluid used for emulsification or by adding the additive to the colloidal system before or after emulsification with the compressed fluid. The invention process can be carried out either as a batch process or as a continuous on-line process.

  3. [Patch-testing methods: additional specialised or additional series].

    PubMed

    Cleenewerck, M-B

    2009-01-01

    The tests in the European standard battery must occasionally be supplemented by specialised or additional batteries, particularly where the contact allergy is thought to be of occupational origin. These additional batteries cover all allergens associated with various professional activities (hairdressing, baking, dentistry, printing, etc.) and with different classes of materials and chemical products (glue, plastic, rubber...). These additional tests may also include personal items used by patients on a daily basis such as cosmetics, shoes, plants, textiles and so on.

  4. Additive manufacturing of optical components

    NASA Astrophysics Data System (ADS)

    Heinrich, Andreas; Rank, Manuel; Maillard, Philippe; Suckow, Anne; Bauckhage, Yannick; Rößler, Patrick; Lang, Johannes; Shariff, Fatin; Pekrul, Sven

    2016-08-01

    The development of additive manufacturing methods has enlarged rapidly in recent years. Thereby, the work mainly focuses on the realization of mechanical components, but the additive manufacturing technology offers a high potential in the field of optics as well. Owing to new design possibilities, completely new solutions are possible. This article briefly reviews and compares the most important additive manufacturing methods for polymer optics. Additionally, it points out the characteristics of additive manufactured polymer optics. Thereby, surface quality is of crucial importance. In order to improve it, appropriate post-processing steps are necessary (e.g. robot polishing or coating), which will be discussed. An essential part of this paper deals with various additive manufactured optical components and their use, especially in optical systems for shape metrology (e.g. borehole sensor, tilt sensor, freeform surface sensor, fisheye lens). The examples should demonstrate the potentials and limitations of optical components produced by additive manufacturing.

  5. Additive inhibition of porcine reproductive and respiratory syndrome virus infection with the soluble sialoadhesin and CD163 receptors.

    PubMed

    Chen, Yang; Guo, Rui; He, Shan; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

    2014-01-22

    Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease to the swine industry worldwide. Current PRRS vaccines are only partially effective and new vaccine development faces great challenges. Sialoadhesin (Sn) and CD163 are the two essential receptors for PRRSV infection of porcine alveolar macrophage (PAM). To investigate the feasibility of the soluble viral receptors for PRRS control, in the present study we generated recombinant adenovirus (rAd) expressing the four N-terminal Ig-like domains of porcine Sn (Sn4D), the fifth SRCR domain (SRCR5) or domains 5-9 (SRCR59) of porcine CD163 as porcine Fc (pFc) fusion proteins. Efficient expression of the soluble viral receptors in the rAd-transduced cells was confirmed by RT-PCR and Western blotting. To detect their antiviral activities, the soluble viral receptors were purified from the media of rAd-transduced cells and identified by Western blotting. The viral binding assay showed that the soluble receptors Sn4D-Fc and SRCR59-Fc, but not SRCR5-Fc and the control pFc, were able to bind to PRRSV particles. The viral infection blocking assays showed that co-treatment of PRRSV with different concentrations of Sn4D-Fc and SRCR59-Fc proteins resulted in a much higher (72.1%-77.6%) reduction in PRRSV-positive cell number than the single protein treatment (45.1%-60.0% or 44.0%-56.2%). To investigate the feasibility of delivering the soluble viral receptors to PAM, two pig cell lines were transduced with rAd-Sn4D-Fc and/or rAd-SRCR59-Fc using a transwell culture system. PAM cells were infected with PRRSV and then co-cultured with the rAd-transduced cells. Viral titration assay showed that co-cultivation of the infected PAM with rAd-Sn4D-Fc- and rAd-SRCR59-Fc-transduced cells resulted in much higher (by ∼3.5 log) reduction in the viral titers (TCID50) than that of co-cultivation with the single vector-transduced cells (by ∼1.0 log). Further studies showed that the rAd co

  6. Schooling in western culture promotes context-free processing.

    PubMed

    Ventura, Paulo; Pattamadilok, Chotiga; Fernandes, Tânia; Klein, Olivier; Morais, José; Kolinsky, Régine

    2008-06-01

    Culture has been shown to influence the way people apprehend their physical environment. Cognitive orientation is more holistic in East Asian cultures, which emphasize relationships and connectedness among objects in the field, than in Western cultures, which are more prone to focus exclusively on the object and its attributes. We investigated whether, beyond, or in conjunction with culture, literacy and/or schooling may also have an influence on this cognitive orientation. Using the Framed-Line Test both in Portugal and in Thailand, we compared literate schooled adults with two groups of unschooled adults: one of illiterates and one of ex-illiterates. As in former studies on Western people, Portuguese-schooled literates were more accurate in the absolute task than in the relative task. In contrast, Portuguese illiterates and ex-illiterates were more accurate in the relative task than in the absolute task. Such an effect of schooling was not observed in the Thai groups, all of whom performed better on the relative task. Thus, the capacity to abstract from contextual information does not stem only from passive exposure to the culture or the physical environment of Western countries. Western schooling, as part of or in addition to culture, is a crucial factor. PMID:18343399

  7. The distribution of aromatic hydrocarbons in western Lake Erie

    SciTech Connect

    Metcalfe, C.D.; Metcalfe, T.L.; Koenig, B.G.; Haffner, G.D.

    1995-12-31

    In order to determine whether biota in western Lake Erie are exposed to elevated concentrations of aromatic hydrocarbons, the authors collected biota along a corridor extending from Peche Island in the Detroit River to Pelee Island in western Lake Erie and determined concentrations of polynuclear aromatic hydrocarbons (PAHs) in zebra mussels, and levels of fluorescent aromatic compounds (FACs) in the bile of freshwater drum and gizzard shad. In addition, they deployed semi-permeable membrane devices (SPMDs) at various locations along this corridor to monitor for PAHs in water. PAHs were elevated in SPMDs and zebra mussels from the Detroit River, and PAH concentrations declined from west to east in Lake Erie. There were elevated levels of bile FACs in drum and gizzard shad from highly contaminated regions of the Detroit River. These data are consistent with the Detroit River being a significant source of PAH contamination in western Lake Erie. Since the ratios of concentrations of PAHs and PCBs in zebra mussels did not vary throughout the study area, it appears that both classes of aromatic contaminants are distributed by similar mechanisms throughout western Lake Erie.

  8. Comparison of Two Widely Used Human Papillomavirus Detection and Genotyping Methods, GP5+/6+-Based PCR Followed by Reverse Line Blot Hybridization and Multiplex Type-Specific E7-Based PCR.

    PubMed

    Clifford, Gary M; Vaccarella, Salvatore; Franceschi, Silvia; Tenet, Vanessa; Umulisa, M Chantal; Tshomo, Ugyen; Dondog, Bolormaa; Vorsters, Alex; Tommasino, Massimo; Heideman, Daniëlle A M; Snijders, Peter J F; Gheit, Tarik

    2016-08-01

    GP5+/6+-based PCR followed by reverse line blot hybridization (GP5+/6+RLB) and multiplex type-specific PCR (E7-MPG) are two human papillomavirus (HPV) genotyping methodologies widely applied in epidemiological research. We investigated their relative analytical performance in 4,662 samples derived from five studies in Bhutan, Rwanda, and Mongolia coordinated by the International Agency for Research on Cancer (IARC). A total of 630 samples were positive by E7-MPG only (13.5%), 24 were positive by GP5+/6+RLB only (0.5%), and 1,014 were positive (21.8%) by both methods. Ratios of HPV type-specific positivity of the two tests (E7-MPG:GP5+/6+RLB ratio) were calculated among 1,668 samples that were HPV positive by one or both tests. E7-MPG:GP5+/6+RLB ratios were >1 for all types and highly reproducible across populations and sample types. E7-MPG:GP5+/6+RLB ratios were highest for HPV53 (7.5) and HPV68 (7.1). HPV16 (1.6) and HPV18 (1.7) had lower than average E7-MPG:GP5+/6+RLB ratios. Among E7-MPG positive infections, median mean fluorescence intensity (MFI; a semiquantitative measure of viral load) tended to be higher among samples positive for the same virus type by GP5+/6+RLB than for those negative for the same type by GP5+/6+RLB. Exceptions, however, included HPV53, -59, and -82, for which the chances of being undetected by GP5+/6+RLB appeared to be MFI independent. Furthermore, the probability of detecting an additional type by E7-MPG was higher when another type was already detected by GP5+/6+RLB, suggesting the existence of masking effects due to competition for GP5+/6+ PCR primers. In conclusion, this analysis is not an evaluation of clinical performance but may inform choices for HPV genotyping methods in epidemiological studies, when the relative merits and dangers of sensitivity versus specificity for individual types should be considered, as well as the potential to unmask nonvaccine types following HPV vaccination. PMID:27225411

  9. Biomass Distribution in Western Hemlock, Douglas-Fir and Western Redcedar.

    SciTech Connect

    Pong, W.Y.; Waddell, Dale R.; Biomass and Energy Project

    1986-05-30

    This report presents results from a study characterizing the weight and volume of trees from overstocked (doghair) mixed conifer stands of Douglas-fir (Pseudotsuga menziesii), western hemlock (Tsuga heterophylla) and western redcedar (Thuja plicata) located on the Quilcene Ranger District, Olympic National Forest. There are approximately 20,000 acres classed as doghair on the Quilcene District. In order to evaluate their harvesting and utilization potential accurate estimates of volume and weight of entire trees and stands are needed. The objective of this study was to characterize the biomass of the three major tree species located in overstocked stands by providing estimators to predict the green and dry weight of the total tree, as well as separate predictions for the components of stem and crown. Additional objectives included estimates for the weight of the 'standing dead', the weight of the dead material on the forest floor, and estimators to predict the cubic volume of the wood and bark in the stems. 11 refs., 9 figs., 15 tabs.

  10. Habitat Suitability Index Models: Western grebe

    USGS Publications Warehouse

    Short, Henry L.

    1984-01-01

    The western grebe (Aechmophorus occi denta 1is) "breeds from southeastern Alaska, south-central British Columbia, central Alberta, central Saskatchewan and southwestern Manitoba south to southern Californi a, north-central Utah, southwestern Colorado, southwest~rn and northeastern New Mexico, western Nebraska, northwestern Iowa, and western Minnesota; and locally in Mexico from Chihuahua and Durango south to northern Guerrero, Puebla and San Luis Potosi" (American Ornithologists' Union 1983:10).

  11. Enantioselective Michael Addition of Water

    PubMed Central

    Chen, Bi-Shuang; Resch, Verena; Otten, Linda G; Hanefeld, Ulf

    2015-01-01

    The enantioselective Michael addition using water as both nucleophile and solvent has to date proved beyond the ability of synthetic chemists. Herein, the direct, enantioselective Michael addition of water in water to prepare important β-hydroxy carbonyl compounds using whole cells of Rhodococcus strains is described. Good yields and excellent enantioselectivities were achieved with this method. Deuterium labeling studies demonstrate that a Michael hydratase catalyzes the water addition exclusively with anti-stereochemistry. PMID:25529526

  12. Enantioselective Michael addition of water.

    PubMed

    Chen, Bi-Shuang; Resch, Verena; Otten, Linda G; Hanefeld, Ulf

    2015-02-01

    The enantioselective Michael addition using water as both nucleophile and solvent has to date proved beyond the ability of synthetic chemists. Herein, the direct, enantioselective Michael addition of water in water to prepare important β-hydroxy carbonyl compounds using whole cells of Rhodococcus strains is described. Good yields and excellent enantioselectivities were achieved with this method. Deuterium labeling studies demonstrate that a Michael hydratase catalyzes the water addition exclusively with anti-stereochemistry.

  13. Nodding syndrome, western Uganda, 1994.

    PubMed

    Kaiser, Christoph; Rubaale, Tom; Tukesiga, Ephraim; Kipp, Walter; Asaba, George

    2015-07-01

    Nodding syndrome (NS) is a poorly understood condition, which was delineated in 2008 as a new epilepsy syndrome. So far, confirmed cases of NS have been observed in three circumscribed African areas: southern Tanzania, southern Sudan, and northern Uganda. Case-control studies have provided evidence of an association between NS and infection with Onchocerca volvulus, but the causation of NS is still not fully clarified. We report a case of a 15-year old boy with head nodding seizures and other characteristic features of NS from an onchocerciasis endemic area in western Uganda, with no contiguity to the hitherto known areas. We suggest that the existence of NS should be systematically investigated in other areas. PMID:25918208

  14. Lunar western limb pyroclastic deposits

    NASA Technical Reports Server (NTRS)

    Coombs, Cassandra R.; Hawke, B. Ray

    1991-01-01

    It has become increasingly evident that the lunar pyroclastic volcanism played an important role in the formation and resurfacing of many areas of the Moon. On-going analysis of lunar Orbiter and Apollo photographs continues to locate and identify pyroclastic deposits and suggests that they just may be more ubiquitous than once thought. Located near mare/highland boundaries, many of these deposits formed contemporaneously with effusive mare volcanism. The mantling deposits formed as products of fire-fountaining. Probable source vents for these deposits include irregular depressions at the head of associated sinuous rilles and/or along irregular fractures in the floors of ancient craters. Here, researchers provide a brief synopsis of the nature of the dark mantling deposits and briefly discuss several newly identified deposits on the western limb.

  15. Gasoline additives, emissions, and performance

    SciTech Connect

    1995-12-31

    The papers included in this publication deal with the influence of fuel, additive, and hardware changes on a variety of vehicle performance characteristics. Advanced techniques for measuring these performance parameters are also described. Contents include: Fleet test evaluation of gasoline additives for intake valve and combustion chamber deposit clean up; A technique for evaluating octane requirement additives in modern engines on dynamometer test stands; A fleet test of two additive technologies comparing their effects on tailpipe emissions; Investigation into the vehicle exhaust emissions of high percentage ethanol blends; Variability in hydrocarbon speciation measurements at low emission (ULEV) levels; and more.

  16. Single particle quantum dot imaging achieves ultrasensitive detection capabilities for Western immunoblot analysis.

    PubMed

    Scholl, Benjamin; Liu, Hong Yan; Long, Brian R; McCarty, Owen J T; O'Hare, Thomas; Druker, Brian J; Vu, Tania Q

    2009-06-23

    Substantially improved detection methods are needed to detect fractionated protein samples present at trace concentrations in complex, heterogeneous tissue and biofluid samples. Here we describe a modification of traditional Western immunoblotting using a technique to count quantum-dot-tagged proteins on optically transparent PVDF membranes. Counts of quantum-dot-tagged proteins on immunoblots achieved optimal detection sensitivity of 0.2 pg and a sample size of 100 cells. This translates to a 10(3)-fold improvement in detection sensitivity and a 10(2)-fold reduction in required cell sample, compared to traditional Westerns processed using the same membrane immunoblots. Quantum dot fluorescent blinking analysis showed that detection of single QD-tagged proteins is possible and that detected points of fluorescence consist of one or a few (<9) QDs. The application of single nanoparticle detection capabilities to Western blotting technologies may provide a new solution to a broad range of applications currently limited by insufficient detection sensitivity and/or sample availability.

  17. Color Addition and Subtraction Apps

    ERIC Educational Resources Information Center

    Ruiz, Frances; Ruiz, Michael J.

    2015-01-01

    Color addition and subtraction apps in HTML5 have been developed for students as an online hands-on experience so that they can more easily master principles introduced through traditional classroom demonstrations. The evolution of the additive RGB color model is traced through the early IBM color adapters so that students can proceed step by step…

  18. Prevalence of Eating Disorders: A Comparison of Western and Non-Western Countries

    PubMed Central

    Makino, Maria; Tsuboi, Koji; Dennerstein, Lorraine

    2004-01-01

    Objective To compare the prevalence of eating disorders between Western and non-Western countries. Method Potential references were identified through an English-language literature search using Medline and Medscape articles. Results Prevalence rates in Western countries for anorexia nervosa ranged from 0.1% to 5.7% in female subjects. Prevalence rates for bulimia nervosa ranged from 0% to 2.1% in males and from 0.3% to 7.3% in female subjects in Western countries. Prevalence rates in non-Western countries for bulimia nervosa ranged from 0.46% to 3.2% in female subjects. Studies of eating attitudes indicate abnormal eating attitudes in non-Western countries have been gradually increasing. Conclusion The prevalence of eating disorders in non-Western countries is lower than that of the Western countries but appears to be increasing. PMID:15520673

  19. Comparing the nature of the western and eastern Azores mantle

    NASA Astrophysics Data System (ADS)

    Genske, Felix S.; Beier, Christoph; Stracke, Andreas; Turner, Simon P.; Pearson, Norman J.; Hauff, Folkmar; Schaefer, Bruce F.; Haase, Karsten M.

    2016-01-01

    The Azores islands in the central North-Atlantic originate from a regional melting anomaly, probably created by melting hot, unusually hydrous and geochemically enriched mantle. Here, we present Hf, Pb and Os isotopic data in geochemically well-characterised primitive lavas from the islands Flores and Corvo that are located west of the Mid-Atlantic Ridge (MAR), as well as submarine samples from a subsided island west of Flores and from Deep Sea Drilling Project (DSDP) holes drilled in the western part of the Azores platform and beyond. These are compared to existing data from the Azores islands east of the MAR. The geodynamic origin of the two islands west of the ridge axis and furthest from the inferred plume centre in the central part of the plateau is enigmatic. The new data constrain the source compositions of the Flores and Corvo lavas and show that the western and eastern Azores mantle is isotopically similar, with the exception of an enriched component found exclusively on eastern São Miguel. Trace element ratios involving high field strength elements (HFSE) are distinctly different in the western islands (e.g. twofold higher Nb/Zr) compared to any of the islands east of the MAR. A similar signature is observed in MAR basalts to the south of the Azores platform and inferred to originate from (auto-) metasomatic enrichment of the sub-ridge mantle (Gale et al., 2011, 2013). In a similar fashion, low degree melts from an enriched source component may metasomatise the ambient plume mantle underneath the western Azores islands. Melting such a modified plume mantle can explain the chemical differences between lavas from the western and eastern Azores islands without the need for additional plume components. Recent re-enrichment and intra melting column modification of the upwelling mantle can cause local to regional scale geochemical differences in mantle-derived melts.

  20. Analysis of Cycling Costs in Western Wind and Solar Integration Study

    SciTech Connect

    Jordan, G.; Venkataraman, S.

    2012-06-01

    The Western Wind and Solar Integration Study (WWSIS) examined the impact of up to 30% penetration of variable renewable generation on the Western Electricity Coordinating Council system. Although start-up costs and higher operating costs because of part-load operation of thermal generators were included in the analysis, further investigation of additional costs associated with thermal unit cycling was deemed worthwhile. These additional cycling costs can be attributed to increases in capital as well as operations and maintenance costs because of wear and tear associated with increased unit cycling. This analysis examines the additional cycling costs of the thermal fleet by leveraging the results of WWSIS Phase 1 study.

  1. Color Addition and Subtraction Apps

    NASA Astrophysics Data System (ADS)

    Ruiz, Frances; Ruiz, Michael J.

    2015-10-01

    Color addition and subtraction apps in HTML5 have been developed for students as an online hands-on experience so that they can more easily master principles introduced through traditional classroom demonstrations. The evolution of the additive RGB color model is traced through the early IBM color adapters so that students can proceed step by step in understanding mathematical representations of RGB color. Finally, color addition and subtraction are presented for the X11 colors from web design to illustrate yet another real-life application of color mixing.

  2. Endemic goiter in western Colombia.

    PubMed

    Gaitan, E

    1983-01-01

    Goiter continues to occur in some areas of western Colombia despite iodine supplementation for 30 years. In 1973-1977, an average goiter prevalence of 15% (range 1-42%) still persisted among schoolchildren of 41 localities. Significant relationships were found between goiter prevalence and both the geological composition of watersheds and bacterial contamination of water supplies. Together, these associations account for 80% of the observed variation in goiter prevalence. The presence of sedimentary rocks rich in organic matter (coals, shales, etc.) was the best indicator of disease. The second best indicator, presence of K. pneumoniae in the water source, was associated with lower goiter prevalence but, as in other investigations, contamination of the pipeline system (households and schools) with gram-negative bacteria was associated with higher disease rates. Thus, epidemiological evidence indicates a cause-effect relationship between sources of drinking water and the persistence and development of goiter in this part of the world. Furthermore, identification of resorcinol, phthalate esters, and sulfur-bearing organic compounds, possibly aliphatic disulfides, in the water supplying the endemic goiter district of Candelaria town in western Colombia adds experimental support to this hypothesis. Resorcinol is derived from coal and humic substances, high molecular weight complex polymeric organic compounds present in sedimentary rocks, soils and water. Resorcinol is goitrogenic in man and experimental animals. Phthalate esters, also related to humic materials, undergo biodegradation by gram-negative bacteria with production of intermediate metabolites possessing antithyroid activity. Like phthalates and resorcinol, organic disulfides have also been identified as water contaminants in other parts of the world, and are known to be potent antithyroid compounds. The goitrogenic effect of organic and bacterial pollutants in water supplies is more pronounced in segments of

  3. Ancient Israel in Western Civ Textbooks

    ERIC Educational Resources Information Center

    Cargill, Jack

    2001-01-01

    The author frequently teaches introductory courses in what was once generally called "Western Civilization" and has often been called upon to referee all or parts of the manuscripts of new editions of "Western Civ" textbooks. Through his own reading, he has become aware that much current scholarship on ancient Israel and Judah is inclined to…

  4. Medusahead management guide for the western states

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Medusahead is an exotic annual grass that is invading rangelands in the western United States. Its invasion decreases livestock forage production, degrades wildlife habitat, and reduces biodiversity. This is a comprehensive guide for management of medusahead in the western United States. This gui...

  5. Western Undergraduate Exchange: Enrollment Report, Fall 2009

    ERIC Educational Resources Information Center

    Western Interstate Commission for Higher Education, 2009

    2009-01-01

    The Western Undergraduate Exchange (WUE) is a regional tuition-reciprocity agreement that enables students from WICHE (Western Interstate Commission for Higher Education) states to enroll in participating two- and four-year public institutions at 150 percent of the enrolling institution's resident tuition. WUE has been operating for more than 20…

  6. Liberty in African and Western Thought.

    ERIC Educational Resources Information Center

    Mudimbe, V. Y.

    The theoretical African mode of thinking about liberty seems to stand in contrast to the view of liberty that has dominated Western philosophy. Western philosophy accepts as its starting point the notion of an unconstrained and uncontextualized "I" that is defined in relation to the self and its inner being. Greek, Cartesian, and classical…

  7. Basic Western Lviv Region Conversational Ukrainian

    ERIC Educational Resources Information Center

    Petryshyn, Ivan

    2015-01-01

    Purpose: To present the first complete Guide for studying the Western-Ukrainian Dialect and its scientific description of Phonology. Methodology: descriptive, contrastive and analytical methods of defining the peculiarities of the Dialect. Results: the regularities and the laws have been defined as to the specifics of the Western-Ukrainian Dialect…

  8. Mid-Western Educational Researcher, 1999.

    ERIC Educational Resources Information Center

    Bainer, Deborah L., Ed.; Kramer, Gene A., Ed.; Smith, Richard M., Ed.

    1999-01-01

    Volume 12 of the Mid-Western Educational Researcher contains four issues. The first issue includes kick-off, keynote, luncheon, and presidential addresses from the annual meeting of the Mid-Western Educational Research Association (MWERA), held in Chicago, Illinois, in October 1998. Issue 3 is the program for MWERA's annual meeting in Chicago,…

  9. Teebi hypertelorism syndrome: additional cases.

    PubMed

    Machado-Paula, Ligiane Alves; Guion-Almeida, Maria Leine

    2003-03-01

    We report on two unrelated Brazilian boys who have craniofacial and digital anomalies resembling those reported with Teebi hypertelorism syndrome. Additional features such as cleft lip and palate, large uvula, atypical chin and abnormal scapulae were observed.

  10. Major Air Pollutants Over the Western Plain in Taiwan

    NASA Astrophysics Data System (ADS)

    Liu, S. C.; Chang, J. S.

    2004-12-01

    The Western Plain of Taiwan where more than two thirds of Taiwan¡¦s 23 million population reside is practically a megacity. Oxidants and particulate matter at levels hazardous to human health are found throughout the Western Plain. In addition, deposition of large amounts of sulfate, nitrate, and ammonium ions has severely stressed the ecosystem. While emission controls have effectively brought down the concentrations of particulate matter in the last decade, ozone concentrations have increased significantly. The cause(s) of the ozone increase is not fully understood. Data taken by Taiwan EPA air quality stations suggest that smaller titration by nitrogen oxide in recent years is a contributor to the ozone increase in urban areas. There is some indication that an increase in synoptic scale background ozone concentration is also a contributor, implying a significant role of long-range transport.

  11. Late Paleozoic extension in the Great Basin, western United States

    SciTech Connect

    Miller, E.L.; Smith, D.L. )

    1990-08-01

    Geologic mapping in the Toiyabe Range in central Nevada has revealed the existence of normal faults of probable mid-Mississippian to Early Permian age that strike roughly east-west and dip northward. Additional evidence of uplift and erosion followed by mafic volcanism and subsidence suggests that much of the central and southern Toiyabe Range was affected by late Paleozoic extension. Similar patterns of late Paleozoic uplift and subsidence, together with local basaltic volcanism, are widespread in the western United States, suggesting that the continental margin was dominated by extension or transtension in Mississippian to Permian time. This extension was coeval with convergence between North America and South America across the Ouachita and Marathon belts, and the dynamic interaction of these two margins may, by analogy with the Cenozoic tectonics of Asia, has given rise to complex late Paleozoic deformation in the Ancestral Rocky Mountains and adjacent areas of the interior western United States.

  12. Abrupt climate shift in the Western Mediterranean Sea

    PubMed Central

    Schroeder, K.; Chiggiato, J.; Bryden, H. L.; Borghini, M.; Ben Ismail, S.

    2016-01-01

    One century of oceanographic measurements has evidenced gradual increases in temperature and salinity of western Mediterranean water masses, even though the vertical stratification has basically remained unchanged. Starting in 2005, the basic structure of the intermediate and deep layers abruptly changed. We report here evidence of reinforced thermohaline variability in the deep western basin with significant dense water formation events producing large amounts of warmer, saltier and denser water masses than ever before. We provide a detailed chronological order to these changes, giving an overview of the new water masses and following their route from the central basin interior to the east (toward the Tyrrhenian) and toward the Atlantic Ocean. As a consequence of this climate shift, new deep waters outflowing through Gibraltar will impact the North Atlantic in terms of salt and heat input. In addition, modifications in the Mediterranean abyssal ecosystems and biogeochemical cycles are to be expected. PMID:26965790

  13. Abrupt climate shift in the Western Mediterranean Sea.

    PubMed

    Schroeder, K; Chiggiato, J; Bryden, H L; Borghini, M; Ben Ismail, S

    2016-01-01

    One century of oceanographic measurements has evidenced gradual increases in temperature and salinity of western Mediterranean water masses, even though the vertical stratification has basically remained unchanged. Starting in 2005, the basic structure of the intermediate and deep layers abruptly changed. We report here evidence of reinforced thermohaline variability in the deep western basin with significant dense water formation events producing large amounts of warmer, saltier and denser water masses than ever before. We provide a detailed chronological order to these changes, giving an overview of the new water masses and following their route from the central basin interior to the east (toward the Tyrrhenian) and toward the Atlantic Ocean. As a consequence of this climate shift, new deep waters outflowing through Gibraltar will impact the North Atlantic in terms of salt and heat input. In addition, modifications in the Mediterranean abyssal ecosystems and biogeochemical cycles are to be expected. PMID:26965790

  14. Abrupt climate shift in the Western Mediterranean Sea

    NASA Astrophysics Data System (ADS)

    Schroeder, K.; Chiggiato, J.; Bryden, H. L.; Borghini, M.; Ben Ismail, S.

    2016-03-01

    One century of oceanographic measurements has evidenced gradual increases in temperature and salinity of western Mediterranean water masses, even though the vertical stratification has basically remained unchanged. Starting in 2005, the basic structure of the intermediate and deep layers abruptly changed. We report here evidence of reinforced thermohaline variability in the deep western basin with significant dense water formation events producing large amounts of warmer, saltier and denser water masses than ever before. We provide a detailed chronological order to these changes, giving an overview of the new water masses and following their route from the central basin interior to the east (toward the Tyrrhenian) and toward the Atlantic Ocean. As a consequence of this climate shift, new deep waters outflowing through Gibraltar will impact the North Atlantic in terms of salt and heat input. In addition, modifications in the Mediterranean abyssal ecosystems and biogeochemical cycles are to be expected.

  15. Polyolefins as additives in plastics

    SciTech Connect

    Deanin, R.D.

    1993-12-31

    Polyolefins are not only major commodity plastics - they are also very useful as additives, both in other polyolefins and also in other types of plastics. This review covers ethylene, propylene, butylene and isobutylene polymers, in blends with each other, and as additives to natural rubber, styrene/butadiene rubber, polystyrene, polyvinyl chloride, polymethyl methacrylate, polyphenylene oxide, polycarbonate, thermoplastic polyesters, polyurethanes, polyamides, and mixed automotive plastics recycling.

  16. Bats of the Western Indian Ocean Islands.

    PubMed

    O'Brien, John

    2011-08-16

    The natural colonisation of many remote oceanic islands by bats, including those of the western Indian Ocean, has been facilitated by their unique capability among mammals for powered flight. In the western Indian Ocean region, only the Malagasy islands of Madagascar and the Comoros archipelago have been naturally colonised by non-volant mammals. Despite their greater potential for inter-island dispersal, and thus gene transfer, endemicity of Chiroptera in the western Indian Ocean islands is high. Given their vulnerability to stochastic and anthropogenic disturbances, greater focus needs to be placed on investigating the demographic and ecological history of bats on Western Indian Ocean islands to safeguard not only their future, but also the ecosystem functioning on these islands, for which they are undoubtedly such an integral part. Here, I summarise the taxonomic and life history information available on bats from Western Indian Ocean islands and highlight knowledge gaps and conservation issues that threaten the continued persistence of some species.

  17. Screening of Glycyrrhiza uralensis Fisch. ex DC. containing high concentrations of glycyrrhizin by Eastern blotting and enzyme-linked immunosorbent assay using anti-glycyrrhizin monoclonal antibody for selective breeding of licorice.

    PubMed

    Fujii, Shunsuke; Tuvshintogtokh, Indree; Mandakh, Bayart; Munkhjargal, Battseren; Uto, Takuhiro; Morinaga, Osamu; Shoyama, Yukihiro

    2014-10-01

    A rapid selection system was used to screen Glycyrrhiza uralensis plants containing high concentrations of glycyrrhizin (GC) by Eastern blotting using anti-GC monoclonal antibody (MAb). Chromatographic fingerprinting by Eastern blotting correlated with the GC concentration analyzed by enzyme-linked immunosorbent assay (ELISA). The roots of wild G. uralensis growing in Mongolia were analyzed by Eastern blotting to identify plants containing high concentrations of GC, and the GC concentration was confirmed by ELISA. G. uralensis plants cultivated in the greenhouse were also analyzed in the same manner. GC concentrations in wild G. uralensis roots and cultivated plants varied widely: between 0.06 and 9.36 percent dry weight (dw%). To confirm the homogeneity of GC concentrations in the cultivated plants, we monitored GC concentrations in the plants over 2 years. Although GC concentrations changed in two plants, they remained comparatively constant in the other five plants, suggesting that GC concentrations are genetically determined. To identify high GC-producing plants, 1025 plants were analyzed, and the highest concentration of GC was 5.36 dw%.

  18. Food additives and preschool children.

    PubMed

    Martyn, Danika M; McNulty, Breige A; Nugent, Anne P; Gibney, Michael J

    2013-02-01

    Food additives have been used throughout history to perform specific functions in foods. A comprehensive framework of legislation is in place within Europe to control the use of additives in the food supply and ensure they pose no risk to human health. Further to this, exposure assessments are regularly carried out to monitor population intakes and verify that intakes are not above acceptable levels (acceptable daily intakes). Young children may have a higher dietary exposure to chemicals than adults due to a combination of rapid growth rates and distinct food intake patterns. For this reason, exposure assessments are particularly important in this age group. The paper will review the use of additives and exposure assessment methods and examine factors that affect dietary exposure by young children. One of the most widely investigated unfavourable health effects associated with food additive intake in preschool-aged children are suggested adverse behavioural effects. Research that has examined this relationship has reported a variety of responses, with many noting an increase in hyperactivity as reported by parents but not when assessed using objective examiners. This review has examined the experimental approaches used in such studies and suggests that efforts are needed to standardise objective methods of measuring behaviour in preschool children. Further to this, a more holistic approach to examining food additive intakes by preschool children is advisable, where overall exposure is considered rather than focusing solely on behavioural effects and possibly examining intakes of food additives other than food colours.

  19. Western rangelands: overgrazed and undermanaged

    SciTech Connect

    Sheridan, D.

    1981-05-01

    Overgrazing and poor management of the western arid lands causes desertification as the levels of water tables and surface waters drop, top soil and surface waters become more saline, soil erodes, and native vegetation disappears. This process had led to severe desertification in an estimated 1.1 million square miles and very severe desertification on 10,500 square miles in the US. The three areas in the very severe category occur in the Navajo Indian reservation in Arizona and New Mexico and on either side of El Paso, Texas. All were subjected to overgrazing. Government policies have only recently tried to bring public land grazing in line with the land's carrying capacity by focusing on long-term productivity. The Public Rangelands Improvement Act of 1978 authorizes better management and multiple use of public lands, but the Bureau of Land Management has not established an effective monitoring system to ensure its implementation or to overcome political constraints against reducing livestock. Ranchers disagree with the assessments made by scientists and support vegetation modification instead of grazing allotments. 58 references, 7 figures. (DCK)

  20. Petroleum systems of western Venezuela

    SciTech Connect

    Passalacqua, H.; Gallango, O.; Capello, M.A.; deToni, B.; Tocco, R.; Truskowski, I.; Loureiro, D.

    1996-08-01

    Integrating multidisciplinary information from western Venezuela Intevep, S.A. developed a comprehensive geological synthesis of the Maracaibo, Barinas and Apure basins which allow the description of 5 petroliferous systems with pods located within the Venezuelan borders: (1) Northeast of Maracaibo Basin, (2) Southwest of Maracaibo Basin, (3) Eastern Zulia, (4) Lara`s Nappes and (5) Barinas Subbasin. These systems comprise different structural styles: (1) basement tectonics, (2) thin-skin tectonics, (3) inversions, (4) triangular zones, (5) {open_quotes}flower and pop-up{close_quotes} structures. The main reservoirs in the region are the fractured Cretaceous limestones (K0 to K5), the Lower to Middle Eocene sandstones (T1 Misoa C and Misoa BC) and the Miocene sandstones of the Lagunillas Formation. Three source rocks were identified: the principal one of Cretaceous age, marine origin and totally located in the Maracaibo Basin (K3-K4-K5, La Luna and Capacho formations) and two located south and southwest of the Maracaibo Basin, with organic material of continental origin, of Paleocene (K7-K8, Orocue and Marcelina formations) and Oligocene age (T4, Carbonera Formation). Three expulsion periods were encountered: (a) Lower to Middle Eocene (52-40 Ma) and (b) Upper Eocene to Lower Miocene (40-21 Ma), with pods located in the northeast region of the Maracaibo Lake and (c) Lower Miocene to Holocene (2 1-0 Ma) with a pod located in the Maracaibo Lake.

  1. Protein western array analysis in human pituitary tumours: insights and limitations.

    PubMed

    Ribeiro-Oliveira, Antônio; Franchi, Giulia; Kola, Blerina; Dalino, Paolo; Pinheiro, Sérgio Veloso Brant; Salahuddin, Nabila; Musat, Madalina; Góth, Miklós I; Czirják, Sándor; Hanzély, Zoltán; da Silva, Deivid Augusto; Paulino, Eduardo; Grossman, Ashley B; Korbonits, Márta

    2008-12-01

    The molecular analysis of pituitary tumours has received a great deal of attention, although the majority of studies have concentrated on the genome and the transcriptome. We aimed to study the proteome of human pituitary adenomas. A protein array using 1005 monoclonal antibodies was used to study GH-, corticotrophin- and prolactin-secreting as well as non-functioning pituitary adenomas (NFPAs). Individual protein expression levels in the tumours were compared with the expression profile of normal pituitary tissue. Out of 316 proteins that were detected in the pituitary tissue samples, 116 proteins had not previously been described in human pituitary tissue. Four prominent differentially expressed proteins with potential importance to tumorigenesis were chosen for validation by immunohistochemistry and western blotting. In the protein array analysis heat shock protein 110 (HSP110), a chaperone associated with protein folding, and B2 bradykinin receptor, a potential regulator of prolactin secretion, were significantly overexpressed in all adenoma subtypes, while C-terminal Src kinase (CSK), an inhibitor of proto-oncogenic enzymes, and annexin II, a calcium-dependent binding protein, were significantly underexpressed in all adenoma subtypes. The immunohistochemical analysis confirmed the overexpression of HSP110 and B2 bradykinin receptor and underexpression of CSK and annexin II in pituitary adenoma cells when compared with their corresponding normal pituitary cells. Western blotting only partially confirmed the proteomics data: HSP110 was significantly overexpressed in prolactinomas and NFPAs, the B2 bradykinin receptor was significantly overexpressed in prolactinomas, annexin II was significantly underexpressed in somatotrophinomas, while CSK did not show significant underexpression in any tumour. Protein expression analysis of pituitary samples disclosed both novel proteins and putative protein candidates for pituitary tumorigenesis, though validation using

  2. Vegetation response to western juniper slash treatments.

    PubMed

    O'Connor, Casey; Miller, Rick; Bates, Jonathan D

    2013-09-01

    The expansion of piñon-juniper woodlands the past 100 years in the western United States has resulted in large scale efforts to kill trees and recover sagebrush steppe rangelands. It is important to evaluate vegetation recovery following woodland control to develop best management practices. In this study, we compared two fuel reduction treatments and a cut-and-leave (CUT) treatment used to control western juniper (Juniperus occidentalis spp. occidentalis Hook.) of the northwestern United States. Treatments were; CUT, cut-and-broadcast burn (BURN), and cut-pile-and-burn the pile (PILE). A randomized complete block design was used with five replicates of each treatment located in a curl leaf mahogany (Cercocarpus ledifolius Nutt. ex Torr. & A. Gray)/mountain big sagebrush (Artemisia tridentata Nutt. spp. vaseyana (Rydb.) Beetle)/Idaho fescue (Festuca idahoensis Elmer) association. In 2010, 4 years after tree control the cover of perennial grasses (PG) [Sandberg's bluegrass (Poa secunda J. Pres) and large bunchgrasses] were about 4 and 5 % less, respectively, in the BURN (7.1 ± 0.6 %) than the PILE (11.4 ± 2.3 %) and CUT (12.4 ± 1.7 %) treatments (P < 0.0015). In 2010, cover of invasive cheatgrass (Bromus tectorum L.) was greater in the BURN (6.3 ± 1.0 %) and was 50 and 100 % greater than PILE and CUT treatments, respectively. However, the increase in perennial bunchgrass density and cover, despite cheatgrass in the BURN treatment, mean it unlikely that cheatgrass will persist as a major understory component. In the CUT treatment mahogany cover increased 12.5 % and density increased in from 172 ± 25 to 404 ± 123 trees/ha. Burning, killed most or all of the adult mahogany, and mahogany recovery consisted of 100 and 67 % seedlings in the PILE and BURN treatments, respectively. After treatment, juniper presence from untreated small trees (<1 m tall; PILE and CUT treatments) and seedling emergence (all treatments) represented 25-33 % of

  3. Extension of the standard addition method by blank addition.

    PubMed

    Steliopoulos, Panagiotis

    2015-01-01

    Standard addition involves adding varying amounts of the analyte to sample portions of fixed mass or fixed volume and submitting those portions to the sample preparation procedure. After measuring the final extract solutions, the observed signals are linearly regressed on the spiked amounts. The original unknown amount is estimated by the opposite of the abscissa intercept of the fitted straight line [1]. A limitation of this method is that only data points with abscissa values equal to and greater than zero are available so that there is no information on whether linearity holds below the spiking level zero. An approach to overcome this limitation is introduced.•Standard addition is combined with blank addition.•Blank addition means that defined mixtures of blank matrix and sample material are subjected to sample preparation to give final extract solutions.•Equations are presented to estimate the original unknown amount and to calculate the 1-2α confidence interval about this estimate using the combined data set.

  4. ADDITIVITY ASSESSMENT OF TRIHALOMETHANE MIXTURES BY PROPORTIONAL RESPONSE ADDITION

    EPA Science Inventory

    If additivity is known or assumed, the toxicity of a chemical mixture may be predicted from the dose response curves of the individual chemicals comprising the mixture. As single chemical data are abundant and mixture data sparse, mixture risk methods that utilize single chemical...

  5. Extension of the standard addition method by blank addition

    PubMed Central

    Steliopoulos, Panagiotis

    2015-01-01

    Standard addition involves adding varying amounts of the analyte to sample portions of fixed mass or fixed volume and submitting those portions to the sample preparation procedure. After measuring the final extract solutions, the observed signals are linearly regressed on the spiked amounts. The original unknown amount is estimated by the opposite of the abscissa intercept of the fitted straight line [1]. A limitation of this method is that only data points with abscissa values equal to and greater than zero are available so that there is no information on whether linearity holds below the spiking level zero. An approach to overcome this limitation is introduced.•Standard addition is combined with blank addition.•Blank addition means that defined mixtures of blank matrix and sample material are subjected to sample preparation to give final extract solutions.•Equations are presented to estimate the original unknown amount and to calculate the 1-2α confidence interval about this estimate using the combined data set. PMID:26844210

  6. [INVITED] Lasers in additive manufacturing

    NASA Astrophysics Data System (ADS)

    Pinkerton, Andrew J.

    2016-04-01

    Additive manufacturing is a topic of considerable ongoing interest, with forecasts predicting it to have major impact on industry in the future. This paper focusses on the current status and potential future development of the technology, with particular reference to the role of lasers within it. It begins by making clear the types and roles of lasers in the different categories of additive manufacturing. This is followed by concise reviews of the economic benefits and disadvantages of the technology, current state of the market and use of additive manufacturing in different industries. Details of these fields are referenced rather than expanded in detail. The paper continues, focusing on current indicators to the future of additive manufacturing. Barriers to its development, trends and opportunities in major industrial sectors, and wider opportunities for its development are covered. Evidence indicates that additive manufacturing may not become the dominant manufacturing technology in all industries, but represents an excellent opportunity for lasers to increase their influence in manufacturing as a whole.

  7. Evaluation of certain food additives.

    PubMed

    2015-01-01

    This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various food additives, including flavouring agents, and to prepare specifications for identity and purity. The first part of the report contains a general discussion of the principles governing the toxicological evaluation of and assessment of dietary exposure to food additives, including flavouring agents. A summary follows of the Committee's evaluations of technical, toxicological and dietary exposure data for eight food additives (Benzoe tonkinensis; carrageenan; citric and fatty acid esters of glycerol; gardenia yellow; lutein esters from Tagetes erecta; octenyl succinic acid-modified gum arabic; octenyl succinic acid-modified starch; paprika extract; and pectin) and eight groups of flavouring agents (aliphatic and alicyclic hydrocarbons; aliphatic and aromatic ethers; ionones and structurally related substances; miscellaneous nitrogen-containing substances; monocyclic and bicyclic secondary alcohols, ketones and related esters; phenol and phenol derivatives; phenyl-substituted aliphatic alcohols and related aldehydes and esters; and sulfur-containing heterocyclic compounds). Specifications for the following food additives were revised: citric acid; gellan gum; polyoxyethylene (20) sorbitan monostearate; potassium aluminium silicate; and Quillaia extract (Type 2). Annexed to the report are tables summarizing the Committee's recommendations for dietary exposures to and toxicological evaluations of all of the food additives and flavouring agents considered at this meeting.

  8. Clinical effects of sulphite additives.

    PubMed

    Vally, H; Misso, N L A; Madan, V

    2009-11-01

    Sulphites are widely used as preservative and antioxidant additives in the food and pharmaceutical industries. Topical, oral or parenteral exposure to sulphites has been reported to induce a range of adverse clinical effects in sensitive individuals, ranging from dermatitis, urticaria, flushing, hypotension, abdominal pain and diarrhoea to life-threatening anaphylactic and asthmatic reactions. Exposure to the sulphites arises mainly from the consumption of foods and drinks that contain these additives; however, exposure may also occur through the use of pharmaceutical products, as well as in occupational settings. While contact sensitivity to sulphite additives in topical medications is increasingly being recognized, skin reactions also occur after ingestion of or parenteral exposure to sulphites. Most studies report a 3-10% prevalence of sulphite sensitivity among asthmatic subjects following ingestion of these additives. However, the severity of these reactions varies, and steroid-dependent asthmatics, those with marked airway hyperresponsiveness, and children with chronic asthma, appear to be at greater risk. In addition to episodic and acute symptoms, sulphites may also contribute to chronic skin and respiratory symptoms. To date, the mechanisms underlying sulphite sensitivity remain unclear, although a number of potential mechanisms have been proposed. Physicians should be aware of the range of clinical manifestations of sulphite sensitivity, as well as the potential sources of exposure. Minor modifications to diet or behaviour lead to excellent clinical outcomes for sulphite-sensitive individuals.

  9. Additive Manufacturing of Hybrid Circuits

    NASA Astrophysics Data System (ADS)

    Sarobol, Pylin; Cook, Adam; Clem, Paul G.; Keicher, David; Hirschfeld, Deidre; Hall, Aaron C.; Bell, Nelson S.

    2016-07-01

    There is a rising interest in developing functional electronics using additively manufactured components. Considerations in materials selection and pathways to forming hybrid circuits and devices must demonstrate useful electronic function; must enable integration; and must complement the complex shape, low cost, high volume, and high functionality of structural but generally electronically passive additively manufactured components. This article reviews several emerging technologies being used in industry and research/development to provide integration advantages of fabricating multilayer hybrid circuits or devices. First, we review a maskless, noncontact, direct write (DW) technology that excels in the deposition of metallic colloid inks for electrical interconnects. Second, we review a complementary technology, aerosol deposition (AD), which excels in the deposition of metallic and ceramic powder as consolidated, thick conformal coatings and is additionally patternable through masking. Finally, we show examples of hybrid circuits/devices integrated beyond 2-D planes, using combinations of DW or AD processes and conventional, established processes.

  10. Postmarketing surveillance of food additives.

    PubMed

    Butchko, H H; Tschanz, C; Kotsonis, F N

    1994-08-01

    Postmarketing surveillance of consumption and of anecdotal reports of adverse health effects has been recognized by a number of regulatory authorities as a potentially useful method to provide further assurance of the safety of new food additives. Surveillance of consumption is used to estimate more reliably actual consumption levels relative to the acceptable daily intake of a food additive. Surveillance of anecdotal reports of adverse health effects is used to determine the presence of infrequent idiosyncratic responses that may not be predictable from premarket evaluations. The high-intensity sweetner, aspartame, is a food additive that has been the subject of extensive evaluation during the postmarketing period and is thus used as an example to discuss postmarketing surveillance.

  11. RNA interference tools for the western flower thrips, Frankliniella occidentalis.

    PubMed

    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

  12. Tougher Addition Polyimides Containing Siloxane

    NASA Technical Reports Server (NTRS)

    St. Clair, T. L.; Maudgal, S.

    1986-01-01

    Laminates show increased impact resistances and other desirable mechanical properties. Bismaleamic acid extended by reaction of diaminosiloxane with maleic anhydride in 1:1 molar ratio, followed by reaction with half this molar ratio of aromatic dianhydride. Bismaleamic acid also extended by reaction of diaminosiloxane with maleic anhydride in 1:2 molar ratio, followed by reaction with half this molar ratio of aromatic diamine (Michael-addition reaction). Impact resistances improved over those of unmodified bismaleimide, showing significant increase in toughness. Aromatic addition polyimides developed as both matrix and adhesive resins for applications on future aircraft and spacecraft.

  13. Preparation for upgrading western subbituminous coal

    SciTech Connect

    Grimes, R.W.; Cha, C.Y.; Sheesley, D.C.

    1990-11-01

    The objective of this project was to establish the physical and chemical characteristics of western coal and determine the best preparation technologies for upgrading this resource. Western coal was characterized as an abundant, easily mineable, clean, low-sulfur coal with low heating value, high moisture, susceptibility to spontaneous ignition, and considerable transit distances from major markets. Project support was provided by the Morgantown Energy Technology Center (METC) of the US Department of Energy (DOE). The research was conducted by the Western Research Institute, (WRI) in Laramie, Wyoming. The project scope of work required the completion of four tasks: (1) project planning, (2) literature searches and verbal contacts with consumers and producers of western coal, (3) selection of the best technologies to upgrade western coal, and (4) identification of research needed to develop the best technologies for upgrading western coals. The results of this research suggest that thermal drying is the best technology for upgrading western coals. There is a significant need for further research in areas involving physical and chemical stabilization of the dried coal product. Excessive particle-size degradation and resulting dustiness, moisture reabsorption, and high susceptibility to spontaneous combustion are key areas requiring further research. Improved testing methods for the determination of equilibrium moisture and susceptibility to spontaneous ignition under various ambient conditions are recommended.

  14. Lubricating additive for drilling muds

    SciTech Connect

    Gutierrez, A.; Brois, S. J.; Brownawell, D. W.; Walker, T. O.

    1985-01-01

    Aqueous drilling fluids containing a minor amount of an additive composition featuring oxazolines of C/sub 1/-C/sub 30/ alkylthioglycolic acid. Such fluids are especially useful where reduced torque drilling fluids are needed. Another embodiment of this invention relates to a method of drilling utilizing the above-described fluids.

  15. Tetrasulfide extreme pressure lubricant additives

    SciTech Connect

    Gast, L.E.; Kenney, H.E.; Schwab, A.W.

    1980-08-19

    A novel class of compounds has been prepared comprising the tetrasulfides of /sup 18/C hydrocarbons, /sup 18/C fatty acids, and /sup 18/C fatty and alkyl and triglyceride esters. These tetrasulfides are useful as extreme pressure lubricant additives and show potential as replacements for sulfurized sperm whale oil.

  16. Promoting Additive Acculturation in Schools.

    ERIC Educational Resources Information Center

    Gibson, Margaret A.

    1995-01-01

    A study focusing on 113 ninth graders of Mexican descent indicates that most students and their parents adhere to a strategy of additive acculturation (incorporating skills of the new culture and language), but that the school curriculum and general school climate devalue Mexican culture. (SLD)

  17. Individualized Additional Instruction for Calculus

    ERIC Educational Resources Information Center

    Takata, Ken

    2010-01-01

    College students enrolling in the calculus sequence have a wide variance in their preparation and abilities, yet they are usually taught from the same lecture. We describe another pedagogical model of Individualized Additional Instruction (IAI) that assesses each student frequently and prescribes further instruction and homework based on the…

  18. Out of bounds additive manufacturing

    DOE PAGES

    Holshouser, Chris; Newell, Clint; Palas, Sid; Love, Lonnie J.; Kunc, Vlastimil; Lind, Randall F.; Lloyd, Peter D.; Rowe, John C.; Blue, Craig A.; Duty, Chad E.; et al

    2013-03-01

    Lockheed Martin and Oak Ridge National Laboratory are working on an additive manufacturing system capable of manufacturing components measured not in terms of inches or feet, but multiple yards in all dimensions with the potential to manufacture parts that are completely unbounded in size.

  19. Tinkertoy Color-Addition Device.

    ERIC Educational Resources Information Center

    Ferguson, Joe L.

    1995-01-01

    Describes construction and use of a simple home-built device, using an overhead projector, for use in demonstrations of the addition of various combinations of red, green, and blue light. Useful in connection with discussions of color, color vision, or color television. (JRH)

  20. Additional Financial Resources for Education.

    ERIC Educational Resources Information Center

    Hubbard, Ben C.

    This paper discusses the continuing need for additional educational funds and suggests that the only way to gain these funds is through concerted and persistent political efforts by supporters of education at both the federal and state levels. The author first points out that for many reasons declining enrollment may not decrease operating costs…

  1. New analysis reexamines the value of cancer care in the United States compared to Western Europe.

    PubMed

    Soneji, Samir; Yang, JaeWon

    2015-03-01

    Despite sharp increases in spending on cancer treatment since 1970 in the United States compared to Western Europe, US cancer mortality rates have decreased only modestly. This has raised questions about the additional value of US cancer care derived from this additional spending. We calculated the number of US cancer deaths averted, compared to the situation in Western Europe, between 1982 and 2010 for twelve cancer types. We also assessed the value of US cancer care, compared to that in Western Europe, by estimating the ratio of additional spending on cancer to the number of quality-adjusted life-years saved. Compared to Western Europe, for three of the four costliest US cancers-breast, colorectal, and prostate-there were approximately 67,000, 265,000, and 60,000 averted US deaths, respectively, and for lung cancer there were roughly 1,120,000 excess deaths in the study period. The ratio of incremental cost to quality-adjusted life-years saved equaled $402,000 for breast cancer, $110,000 for colorectal cancer, and $1,979,000 for prostate cancer-amounts that exceed most accepted thresholds for cost-effective medical care. The United States lost quality-adjusted life-years despite additional spending for lung cancer: -$19,000 per quality-adjusted life-year saved. Our results suggest that cancer care in the United States may provide less value than corresponding cancer care in Western Europe for many leading cancers.

  2. Evaluation of certain food additives.

    PubMed

    2012-01-01

    This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various food additives, including flavouring agents, with a view to concluding as to safety concerns and to preparing specifications for identity and purity. The first part of the report contains a general discussion of the principles governing the toxicological evaluation of and assessment of dietary exposure to food additives, including flavouring agents. A summary follows of the Committee's evaluations of technical, toxicological and dietary exposure data for five food additives (magnesium dihydrogen diphosphate; mineral oil (medium and low viscosity) classes II and III; 3-phytase from Aspergillus niger expressed in Aspergillus niger; serine protease (chymotrypsin) from Nocardiopsis prasina expressed in Bacillus licheniformis; and serine protease (trypsin) from Fusarium oxysporum expressed in Fusarium venenatum) and 16 groups of flavouring agents (aliphatic and aromatic amines and amides; aliphatic and aromatic ethers; aliphatic hydrocarbons, alcohols, aldehydes, ketones, carboxylic acids and related esters, sulfides, disulfides and ethers containing furan substitution; aliphatic linear alpha,beta-unsaturated aldehydes, acids and related alcohols, acetals and esters; amino acids and related substances; epoxides; furfuryl alcohol and related substances; linear and branched-chain aliphatic, unsaturated, unconjugated alcohols, aldehydes, acids and related esters; miscellaneous nitrogen-containing substances; phenol and phenol derivatives; pyrazine derivatives; pyridine, pyrrole and quinoline derivatives; saturated aliphatic acyclic branched-chain primary alcohols, aldehydes and acids; simple aliphatic and aromatic sulfides and thiols; sulfur-containing heterocyclic compounds; and sulfur-substituted furan derivatives). Specifications for the following food additives were revised: ethyl cellulose, mineral oil (medium viscosity), modified starches and titanium

  3. Evaluation of certain food additives.

    PubMed

    2012-01-01

    This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various food additives, including flavouring agents, with a view to concluding as to safety concerns and to preparing specifications for identity and purity. The first part of the report contains a general discussion of the principles governing the toxicological evaluation of and assessment of dietary exposure to food additives, including flavouring agents. A summary follows of the Committee's evaluations of technical, toxicological and dietary exposure data for five food additives (magnesium dihydrogen diphosphate; mineral oil (medium and low viscosity) classes II and III; 3-phytase from Aspergillus niger expressed in Aspergillus niger; serine protease (chymotrypsin) from Nocardiopsis prasina expressed in Bacillus licheniformis; and serine protease (trypsin) from Fusarium oxysporum expressed in Fusarium venenatum) and 16 groups of flavouring agents (aliphatic and aromatic amines and amides; aliphatic and aromatic ethers; aliphatic hydrocarbons, alcohols, aldehydes, ketones, carboxylic acids and related esters, sulfides, disulfides and ethers containing furan substitution; aliphatic linear alpha,beta-unsaturated aldehydes, acids and related alcohols, acetals and esters; amino acids and related substances; epoxides; furfuryl alcohol and related substances; linear and branched-chain aliphatic, unsaturated, unconjugated alcohols, aldehydes, acids and related esters; miscellaneous nitrogen-containing substances; phenol and phenol derivatives; pyrazine derivatives; pyridine, pyrrole and quinoline derivatives; saturated aliphatic acyclic branched-chain primary alcohols, aldehydes and acids; simple aliphatic and aromatic sulfides and thiols; sulfur-containing heterocyclic compounds; and sulfur-substituted furan derivatives). Specifications for the following food additives were revised: ethyl cellulose, mineral oil (medium viscosity), modified starches and titanium

  4. 75 FR 19942 - Western Pacific Fishery Management Council; Public Meetings

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-16

    ... National Oceanic and Atmospheric Administration RIN 0648-XV86 Western Pacific Fishery Management Council... Administration (NOAA), Commerce. ACTION: Notice of public meetings. SUMMARY: The Western Pacific Fishery... and Training Program and Western Pacific Community Demonstration Project Program for Council...

  5. Western teachers of science or teachers of Western science: On the influence of Western modern science in a post-colonial context

    NASA Astrophysics Data System (ADS)

    Burke, Lydia E. Carol-Ann

    An expanding body of research explores the social, political, cultural and personal challenges presented by the Western emphasis of curricula around the world. The aim of my study is to advance this field of inquiry by gaining insight into perceptions of Western modern science presented by students, teachers and administrators in a given Caribbean setting. Through this study I asked how my research participants described the nature of scientific knowledge, how they related scientific knowledge to other culturally-valued knowledges and the meanings they attached to the geographic origins of science teachers. Situating this work firmly within the practice of Foucauldian critical discourse analysis, I have utilised a conceptual framework defined by the power/knowledge and complicity/resistance themes of post-colonial theory to support my interpretation of participant commentary in an overall quest that is concerned about the ways in which Western modern science might be exerting a colonising influence. Fourteen students, nine teachers (both expatriate and local) and three administrators participated in the study. I combined a semi-structured question and answer interview format with a card sort activity. I used a procedure based on my own adaptation of Stephenson's Q methodology, where the respondents placed 24 statements hierarchically along a continuum of increasing strength of agreement, presenting their rationalisations, personal stories and illustrations as they sorted. I used an inverse factor analysis, in combination with the interview transcripts, to assist me in the identification of three discourse positions described by my research participants: The truth value of scientific knowledge, The pragmatic use of science to promote progress, and The priority of cultural preservation. The interview transcripts were also analysed for emergent themes, providing an additional layer of data interpretation. The research findings raise concerns regarding the hegemonic

  6. Publications of the Western Earth Surface Processes Team, 1999

    USGS Publications Warehouse

    Stone, Paul; Powell, Charles L.

    2000-01-01

    The Western Earth Surfaces Processes Team (WESPT) of the U.S. Geological Survey, Geologic Division (USGS, GD), conducts geologic mapping and related topical earth- science studies in the western United States. This work is focused on areas where modern geologic maps and associated earth-science data are needed to address key societal and environmental issues such as ground-water quality, potential geologic hazards, and land-use decisions. Areas of primary emphasis currently include southern California, the San Francisco Bay region, and the Pacific Northwest. The team has its headquarters in Menlo Park, California, and maintains field offices at several other locations in the western United States. The results of research conducted by the WESPT are released to the public as a variety of databases, maps, text reports, and abstracts, both through the internal publication system of the USGS and in diverse external publications such as scientific journals and books. This report lists publications of the WESPT released in 1999 as well as additional 1997 and 1998 publications that were not included in the previous list (USGS Open-file Report 99-302). Most of the publications listed were authored or coauthored by WESPT staff. The list also includes some publications authored by non-USGS cooperators with the WESPT, as well as some authored by USGS staff outside the WESPT in cooperation with WESPT projects.

  7. Spatial distribution of tropospheric ozone in western Washington, USA

    USGS Publications Warehouse

    Cooper, S.M.; Peterson, D.L.

    2000-01-01

    We quantified the distribution of tropospheric ozone in topographically complex western Washington state, USA (total area a??6000 km2), using passive ozone samplers along nine river drainages to measure ozone exposure from near sea level to high-elevation mountain sites. Weekly average ozone concentrations were higher with increasing distance from the urban core and at higher elevations, increasing a mean of 1.3 ppbv per 100 m elevation gain for all mountain transects. Weekly average ozone concentrations were generally highest in Cascade Mountains drainages east and southeast of Seattle (maximum=55a??67 pbv) and in the Columbia River Gorge east of Portland (maximum=59 ppbv), and lowest in the western Olympic Peninsula (maximum=34 ppbv). Higher ozone concentrations in the Cascade Mountains and Columbia River locations downwind of large cities indicate that significant quantities of ozone and ozone precursors are being transported eastward toward rural wildland areas by prevailing westerly winds. In addition, temporal (week to week) variation in ozone distribution is synchronous within and between all drainages sampled, which indicates that there is regional coherence in air pollution detectable with weekly averages. These data provide insight on large-scale spatial variation of ozone distribution in western Washington, and will help regulatory agencies optimize future monitoring networks and identify locations where human health and natural resources could be at risk.

  8. Spatial distribution of tropospheric ozone in western Washington, USA.

    PubMed

    Cooper, S M; Peterson, D L

    2000-03-01

    We quantified the distribution of tropospheric ozone in topographically complex western Washington state, USA (total area approximately 6000 km(2)), using passive ozone samplers along nine river drainages to measure ozone exposure from near sea level to high-elevation mountain sites. Weekly average ozone concentrations were higher with increasing distance from the urban core and at higher elevations, increasing a mean of 1.3 ppbv per 100 m elevation gain for all mountain transects. Weekly average ozone concentrations were generally highest in Cascade Mountains drainages east and southeast of Seattle (maximum=55-67 pbv) and in the Columbia River Gorge east of Portland (maximum=59 ppbv), and lowest in the western Olympic Peninsula (maximum=34 ppbv). Higher ozone concentrations in the Cascade Mountains and Columbia River locations downwind of large cities indicate that significant quantities of ozone and ozone precursors are being transported eastward toward rural wildland areas by prevailing westerly winds. In addition, temporal (week to week) variation in ozone distribution is synchronous within and between all drainages sampled, which indicates that there is regional coherence in air pollution detectable with weekly averages. These data provide insight on large-scale spatial variation of ozone distribution in western Washington, and will help regulatory agencies optimize future monitoring networks and identify locations where human health and natural resources could be at risk. PMID:15092980

  9. Per- and polyfluoroalkyl substances in the Western Mediterranean Sea waters.

    PubMed

    Brumovský, Miroslav; Karásková, Pavlína; Borghini, Mireno; Nizzetto, Luca

    2016-09-01

    The spatial and temporal distribution of per- and polyfluoroalkyl substances (PFASs) in the open Western Mediterranean Sea waters was investigated in this study for the first time. In addition to surface water samples, a deep water sample (1390 m depth) collected in the center of the western basin was analyzed. Perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorohexanesulfonate (PFHxS) and perfluorooctanesulfonate (PFOS) were detected in all samples and were the dominant PFASs found. The sum of PFAS concentrations (ΣPFASs) ranged 246-515 pg/L for surface water samples. PFASs in surface water had a relatively homogeneous distribution with levels similar to those previously measured in the Atlantic near the Strait of Gibraltar, in water masses feeding the inflow to the Mediterranean Sea. Higher concentrations of PFHxA, PFHpA and PFHxS were, however, found in the present study. Inflowing Atlantic water and river/coastal discharges are likely the major sources of PFASs to the Western Mediterranean basin. Slightly lower (factor of 2) ΣPFASs was found in the deep water sample (141 pg/L). Such a relatively high contamination of deep water is likely to be linked to recurring deep water renewal fed by downwelling events in the Gulf of Lion and/or Ligurian Sea. PMID:27314632

  10. Crustal imprints of Precambrian orogenesis in western Laurentia

    NASA Astrophysics Data System (ADS)

    Chen, Yunfeng; Gu, Yu Jeffrey; Dokht, Ramin M. H.; Sacchi, Mauricio D.

    2015-10-01

    Crustal low-velocity zones (LVZs) have been reported in active orogens such as the Himalayas and the Andes but rarely in stable cratonic regions. In this study, we provide compelling evidence for a significant midcrustal LVZ beneath eastern-central Alberta, an integral part of the Precambrian Canadian Shield covered by thick Phanerozoic sedimentary deposits. This 200 km wide, over 10 km thick midcrustal LVZ is well resolved by shear velocity inversions using P-to-S receiver functions from more than 4600 earthquakes. It is generally overlain by a high-velocity upper crust in the depth range of 8-15 km, especially in western-central Alberta, which coincides with the previously documented Winagami reflection sequence. We interpret the LVZ to be of granitic composition, potentially in connection with the crystallization of partially molten crust during the Paleoproterozoic eon. In addition to the Precambrian tectonic history of western Laurentia, which featured plate convergence conducive to crustal melting, our crustal model is further supported by (1) a moderate spatial correlation between the LVZ and heat flow, and (2) shear velocities consistent with that of granite. The well preserved Winagami reflection sequence and the LVZ are potential evidence of distinct episodes of magmatism and crust modification in the Precambrian basement of the Western Canada Sedimentary Basin. The existence of a broad crustal LVZ suggests extensive subduction, orogenesis, and crustal melting during the Precambrian assembly of the North American craton.

  11. Western water and climate change.

    PubMed

    Dettinger, Michael; Udall, Bradley; Georgakakos, Aris

    2015-12-01

    The western United States is a region long defined by water challenges. Climate change adds to those historical challenges, but does not, for the most part, introduce entirely new challenges; rather climate change is likely to stress water supplies and resources already in many cases stretched to, or beyond, natural limits. Projections are for continued and, likely, increased warming trends across the region, with a near certainty of continuing changes in seasonality of snowmelt and streamflows, and a strong potential for attendant increases in evaporative demands. Projections of future precipitation are less conclusive, although likely the northern-most West will see precipitation increases while the southernmost West sees declines. However, most of the region lies in a broad area where some climate models project precipitation increases while others project declines, so that only increases in precipitation uncertainties can be projected with any confidence. Changes in annual and seasonal hydrographs are likely to challenge water managers, users, and attempts to protect or restore environmental flows, even where annual volumes change little. Other impacts from climate change (e.g., floods and water-quality changes) are poorly understood and will likely be location dependent. In this context, four iconic river basins offer glimpses into specific challenges that climate change may bring to the West. The Colorado River is a system in which overuse and growing demands are projected to be even more challenging than climate-change-induced flow reductions. The Rio Grande offers the best example of how climate-change-induced flow declines might sink a major system into permanent drought. The Klamath is currently projected to face the more benign precipitation future, but fisheries and irrigation management may face dire straits due to warming air temperatures, rising irrigation demands, and warming waters in a basin already hobbled by tensions between endangered fisheries

  12. Western water and climate change.

    PubMed

    Dettinger, Michael; Udall, Bradley; Georgakakos, Aris

    2015-12-01

    The western United States is a region long defined by water challenges. Climate change adds to those historical challenges, but does not, for the most part, introduce entirely new challenges; rather climate change is likely to stress water supplies and resources already in many cases stretched to, or beyond, natural limits. Projections are for continued and, likely, increased warming trends across the region, with a near certainty of continuing changes in seasonality of snowmelt and streamflows, and a strong potential for attendant increases in evaporative demands. Projections of future precipitation are less conclusive, although likely the northern-most West will see precipitation increases while the southernmost West sees declines. However, most of the region lies in a broad area where some climate models project precipitation increases while others project declines, so that only increases in precipitation uncertainties can be projected with any confidence. Changes in annual and seasonal hydrographs are likely to challenge water managers, users, and attempts to protect or restore environmental flows, even where annual volumes change little. Other impacts from climate change (e.g., floods and water-quality changes) are poorly understood and will likely be location dependent. In this context, four iconic river basins offer glimpses into specific challenges that climate change may bring to the West. The Colorado River is a system in which overuse and growing demands are projected to be even more challenging than climate-change-induced flow reductions. The Rio Grande offers the best example of how climate-change-induced flow declines might sink a major system into permanent drought. The Klamath is currently projected to face the more benign precipitation future, but fisheries and irrigation management may face dire straits due to warming air temperatures, rising irrigation demands, and warming waters in a basin already hobbled by tensions between endangered fisheries

  13. Decontamination formulation with sorbent additive

    DOEpatents

    Tucker; Mark D. , Comstock; Robert H.

    2007-10-16

    A decontamination formulation and method of making that neutralizes the adverse health effects of both chemical and biological compounds, especially chemical warfare (CW) and biological warfare (BW) agents, and toxic industrial chemicals. The formulation provides solubilizing compounds that serve to effectively render the chemical and biological compounds, particularly CW and BW compounds, susceptible to attack, and at least one reactive compound that serves to attack (and detoxify or kill) the compound. The formulation includes at least one solubilizing agent, a reactive compound, a bleaching activator, a sorbent additive, and water. The highly adsorbent, water-soluble sorbent additive (e.g., sorbitol or mannitol) is used to "dry out" one or more liquid ingredients, such as the liquid bleaching activator (e.g., propylene glycol diacetate or glycerol diacetate) and convert the activator into a dry, free-flowing powder that has an extended shelf life, and is more convenient to handle and mix in the field.

  14. Additive-free digital microfluidics.

    PubMed

    Freire, Sergio L S; Tanner, Brendan

    2013-07-16

    Digital microfluidics, a technique for manipulation of droplets, is becoming increasingly important for the development of miniaturized platforms for laboratory processes. Despite the enthusiasm, droplet motion is frequently hindered by the desorption of proteins or other analytes to surfaces. Current approaches to minimize this unwanted surface fouling involve the addition of extra species to the droplet or its surroundings, which might be problematic depending on the droplet content. Here, a new strategy is introduced to move droplets containing cells and other analytes on solid substrates, without extra moieties; in particular, droplets with bovine serum albumin could be moved at a concentration 2000 times higher than previously reported (without additives). This capability is achieved by using a soot-based superamphiphobic surface combined with a new device geometry, which favors droplet rolling. Contrasting with electrowetting, wetting forces are not required for droplet motion.

  15. Deer Lodge Valley investigations, western Montana

    SciTech Connect

    Wideman, C.J.; Sonderegger, J.; Crase, E.; Peterson, J.; Ruscetta, C.A.

    1982-07-01

    A review of the geothermal investigations conducted in the Deer Lodge Valley of Western Montana is briefly presented. Maps of the generalized geology and Bouguer gravity and graphs of selected geothermal gradients and resistivity sounding profiles are presented. (MJF)

  16. Day Pass through Western United States

    NASA Video Gallery

    This video over the Western United States was taken by the crew of Expedition 29 aboard the International Space Station. This sequence of shots was taken on Sept. 24, 2011, from 17:45:14 to 17:54:1...

  17. WESTERN ECOLOGY DIVISION - GENERAL INFORMATION SHEET

    EPA Science Inventory

    abstract for flyer - general information The Western Ecology Division (WED), part of EPAs National Health and Environmental Effects Research Laboratory, provides information to EPA offices and regions nationwide to improve understanding of how human activities affect estuarine,...

  18. Additive concentrates for distillate fuels

    SciTech Connect

    Rossi, A.; Lewtas, K.

    1985-08-27

    An additive concentrate for incorporation into wax containing petroleum fuel oil compositions to improve low temperature flow properties comprising an oil solution containing: 3% to 90 wt. % of a C30-C300 oil-soluble nitrogen compound wax crystal growth inhibitor having at least one straight C8-C40 alkyl chain and partial esters, and at least one mole per mole of an organic acid capable of hydrogen bonding to improve the solubility in the oil.

  19. Additive manufacturing of hybrid circuits

    DOE PAGES

    Bell, Nelson S.; Sarobol, Pylin; Cook, Adam; Clem, Paul G.; Keicher, David M.; Hirschfeld, Deidre; Hall, Aaron Christopher

    2016-03-26

    There is a rising interest in developing functional electronics using additively manufactured components. Considerations in materials selection and pathways to forming hybrid circuits and devices must demonstrate useful electronic function; must enable integration; and must complement the complex shape, low cost, high volume, and high functionality of structural but generally electronically passive additively manufactured components. This article reviews several emerging technologies being used in industry and research/development to provide integration advantages of fabricating multilayer hybrid circuits or devices. First, we review a maskless, noncontact, direct write (DW) technology that excels in the deposition of metallic colloid inks for electrical interconnects.more » Second, we review a complementary technology, aerosol deposition (AD), which excels in the deposition of metallic and ceramic powder as consolidated, thick conformal coatings and is additionally patternable through masking. As a result, we show examples of hybrid circuits/devices integrated beyond 2-D planes, using combinations of DW or AD processes and conventional, established processes.« less

  20. Beet western yellows virus infects the carnivorous plant Nepenthes mirabilis.

    PubMed

    Miguel, Sissi; Biteau, Flore; Mignard, Benoit; Marais, Armelle; Candresse, Thierry; Theil, Sébastien; Bourgaud, Frédéric; Hehn, Alain

    2016-08-01

    Although poleroviruses are known to infect a broad range of higher plants, carnivorous plants have not yet been reported as hosts. Here, we describe the first polerovirus naturally infecting the pitcher plant Nepenthes mirabilis. The virus was identified through bioinformatic analysis of NGS transcriptome data. The complete viral genome sequence was assembled from overlapping PCR fragments and shown to share 91.1 % nucleotide sequence identity with the US isolate of beet western yellows virus (BWYV). Further analysis of other N. mirabilis plants revealed the presence of additional BWYV isolates differing by several insertion/deletion mutations in ORF5.

  1. Valve, compressor contracts awarded for Western Hemisphere projects

    SciTech Connect

    1998-01-19

    Major valve and compressor contracts have been let for projects in the Western Hemisphere. Petrobras has awarded Nuovo Pignone, Florence, a $10.5 million contract to supply 400 valves for the 1,975-mile natural-gas pipeline being constructed from Bolivia into Brazil. Additionally, Brazilian company Maritima Petroleo and TransCanada PipeLines Ltd., Calgary, have awarded Nuovo Pignone separate contracts to supply turbocompressor packages. The Brazilian contract is for offshore Campos Basin; the Canadian, for a major expansion of TCPL`s system delivering natural gas out of Alberta. The paper discusses the Bolivia-Brazil pipeline, compressor orders, and the companies.

  2. Western Interconnection Energy Imbalance Market Status and Prospects (Presentation)

    SciTech Connect

    Milligan, M.; Kirby, B.; King, J.; Beuning, S.

    2011-10-01

    This presentation describes how a new wholesale electricity market for energy imbalance ancillary services could be implemented and operated. Some conclusions of this presentation are: (1) Method for calculating additional reserve requirements due to wind and solar production; (2) EIM results in substantial reduction in reserves requirements and ramping demand; (3) Reduced participation reduces benefits for all but reduces the benefits to non-participants the most; (4) Full participation leads to maximum benefit across the Western Interconnection, up to 42% of total reserve requirement; and (5) Regional EIM implementations have smaller but substantial benefits.

  3. Beet western yellows virus infects the carnivorous plant Nepenthes mirabilis.

    PubMed

    Miguel, Sissi; Biteau, Flore; Mignard, Benoit; Marais, Armelle; Candresse, Thierry; Theil, Sébastien; Bourgaud, Frédéric; Hehn, Alain

    2016-08-01

    Although poleroviruses are known to infect a broad range of higher plants, carnivorous plants have not yet been reported as hosts. Here, we describe the first polerovirus naturally infecting the pitcher plant Nepenthes mirabilis. The virus was identified through bioinformatic analysis of NGS transcriptome data. The complete viral genome sequence was assembled from overlapping PCR fragments and shown to share 91.1 % nucleotide sequence identity with the US isolate of beet western yellows virus (BWYV). Further analysis of other N. mirabilis plants revealed the presence of additional BWYV isolates differing by several insertion/deletion mutations in ORF5. PMID:27180098

  4. Obstetrical events that shaped Western European history.

    PubMed

    Ober, W B

    1992-01-01

    Taking into account that marriage, the family as a social unit, and concepts of legitimacy developed to ensure the devolution of property and that, when these concepts apply in a society based on hierarchically organized monarchies, they also involve the devolution of power, this essay furnishes examples of dislocations in such devolutions, in terms of familiar incidents in western European history. That Jane Seymour died in childbirth but her son Edward VI survived long enough to ensure the stability of the Church of England is the first example. The infertility of Mary Tudor, when married to Philip II of Spain, prevented the formation of an Anglo-Spanish dynasty that would have been Roman Catholic is the second example of such a dislocation. Likewise, the infertility of Charles II's wife, Catherine of Braganza, led to the succession of James II, a practicing Roman Catholic, whose attempts to undermine the Church of England led to the Glorious Revolution of 1788 and the preservation of English Protestantism. Another example is the death in 1817 of Princess Charlotte, in childbirth, which led to the scramble of George III's aging sons to marry and beget an heir to the throne. The only success led to the birth of the future Queen Victoria, whose dynastic competence remains unquestionable, but who herself had some passing involvement with obstetrical developments. Finally, the delivery of Kaiser Wilhelm II, who sustained a brachial plexus injury that produced Erb's palsy of the left arm, is considered, and the question of intrapartum fetal hypoxia is raised as a hypothesis, in addition to the mechanical trauma and its effect on his personality.

  5. Groundwater sapping processes, Western Desert, Egypt.

    SciTech Connect

    Luo, W.; Arvidson, R.E.; Sultan, M.; Becker, R.; Crombie, M.K.; Sturchio, N.; El Alfy, Z.; Environmental Research; Washington Univ.; Egyptian Geological Survey and Mining Authority

    1997-01-01

    Depressions of the Western Desert of Egypt (specifically, Kharga, Farafra, and Kurkur regions) are mainly occupied by shales that are impermeable, but easily erodible by rainfall and runoff, whereas the surrounding plateaus are composed of limestones that are permeable and more resistant to fluvial erosion under semiarid to arid conditions. Scallop-shaped escarpment edges and stubby-looking channels that cut into the plateau units are suggestive of slumping of limestones by ground-water sapping at the limestone-shale interfaces, removal of slump blocks by weathering and fluvial erosion, and consequent scarp retreat. Spring-derived tufa deposits found near the limestone escarpments provide additional evidence for possible ground-water sapping during previous wet periods. A computer simulation model was developed to quantify the ground-water sapping processes, using a cellular automata algorithm with coupled surface runoff and ground-water flow for a permeable, resistant layer over an impermeable, friable unit. Erosion, deposition, slumping, and generation of spring-derived tufas were parametrically modeled. Simulations using geologically reasonable parameters demonstrate that relatively rapid erosion of the shales by surface runoff, ground-water sapping, and slumping of the limestones, and detailed control by hydraulic conductivity inhomogeneities associated with structures explain the depressions, escarpments, and associated landforms and deposits. Using episodic wet pulses, keyed by {delta}{sup 18}O deep-sea core record, the model produced tufa ages that are statistically consistent with the observed U/Th tufa ages. This result supports the hypothesis that northeastern African wet periods occurred during interglacial maxima. The {delta}{sup 18}O-forced model also replicates the decrease in fluvial and sapping activity over the past million years, as northeastern Africa became hyperarid. The model thus provides a promising predictive tool for studying long

  6. Quantification of Knickpoint Migration in Western Iowa

    NASA Astrophysics Data System (ADS)

    Wilson, C. G.; Thomas, J. T.; Papanicolaou, T.; Elhakeem, M.

    2009-12-01

    Knickpoints threaten the stability of bridge structures in Western Iowa, thus the overarching goal of this research was to provide a predictive tool for knickpoint propagation. The study involved detailed field investigations over two years in order to monitor the upstream migration of a knickpoint on Mud Creek in Mills County, IA and identify the key mechanisms triggering knickpoint propagation. A state-of-the-art laser level system mounted on a movable truss provided continuous measurements of the knickpoint front for different flow conditions. A pressure transducer in proximity of the truss provided simultaneous flow depth measurements. The laser and pressure transducer measurements led to the identification of the conditions, at which the knickpoint migration commenced. It is suggested that negative pressures developed by the reverse roller flow near the toe of the knickpoint face triggered undercutting of the knickpoint at this location. The pressure differential between the negative pressure and the atmospheric pressure also pulled the impinging jet closer to the knickpoint face producing scour. In addition, the pressure differential may have induced suction of sediment from the face. Other contributing factors included slump failure, seepage effects, and local fluvial erosion due to the exerted fluid shear. The prevailing flow conditions and soil information along with the channel cross-sectional geometry and gradient were used as inputs to a transcritical, one dimensional, hydraulic/geomorphic numerical model, which was used to map the flow characteristics and shear stress conditions near the knickpoint. Such detailed flow calculations do not exist in the published literature. The coupling of field and modeling work resulted in the development of a blueprint methodology, which can be adopted in different parts of the country for evaluating knickpoint evolution. This information will assist local government agencies in better understanding the principal

  7. Egypt western desert activity hits high gear

    SciTech Connect

    Petzet, G.A.

    1996-11-04

    Exploration in Egypt`s western desert is turning up large oil and gas reserves in several sub-basins. Though not a threat to the Gulf of Suez`s oil production dominance, the western desert`s output is climbing. And gas production is also set to rise as more pipelines are built. The paper describes the geology, Qarun field production, further exploration at Qarun and the Khalda/Obaiyed gas.

  8. USGS Western Coastal and Marine Geology Team

    USGS Publications Warehouse

    Johnson, Sam; Gibbons, Helen

    2007-01-01

    The Western Coastal and Marine Geology Team of the U.S. Geological Survey (USGS) studies the coasts of the western United States, including Alaska and Hawai‘i. Team scientists conduct research, monitor processes, and develop information about coastal and marine geologic hazards, environmental conditions, habitats, and energy and mineral resources. This information helps managers at all levels of government and in the private sector make informed decisions about the use and protection of national coastal and marine resources.

  9. Evaluation of certain food additives.

    PubMed

    2009-01-01

    This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various food additives, including flavouring agents, with a view to recommending acceptable daily intakes (ADIs) and to preparing specifications for identity and purity. The first part of the report contains a general discussion of the principles governing the toxicological evaluation and assessment of intake of food additives (in particular, flavouring agents). A summary follows of the Committee's evaluations of technical, toxicological and intake data for certain food additives (asparaginase from Aspergillus niger expressed in A. niger, calcium lignosulfonate (40-65), ethyl lauroyl arginate, paprika extract, phospholipase C expressed in Pichia pastoris, phytosterols, phytostanols and their esters, polydimethylsiloxane, steviol glycosides and sulfites [assessment of dietary exposure]) and 10 groups of related flavouring agents (aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids and related esters; aliphatic linear alpha,beta-unsaturated aldehydes, acids and related alcohols, acetals and esters; aliphatic secondary alcohols, ketones and related esters; alkoxy-substituted allylbenzenes present in foods and essential oils and used as flavouring agents; esters of aliphatic acyclic primary alcohols with aliphatic linear saturated carboxylic acids; furan-substituted aliphatic hydrocarbons, alcohols, aldehydes, ketones, carboxylic acids and related esters, sulfides, disulfides and ethers; miscellaneous nitrogen-containing substances; monocyclic and bicyclic secondary alcohols, ketones and related esters; hydroxy- and alkoxy-substituted benzyl derivatives; and substances structurally related to menthol). Specifications for the following food additives were revised: canthaxanthin; carob bean gum and carob bean gum (clarified); chlorophyllin copper complexes, sodium and potassium salts; Fast Green FCF; guar gum and guar gum (clarified

  10. Fire-Retardant Polymeric Additives

    NASA Technical Reports Server (NTRS)

    Williams, Martha K.; Smith, Trent M.

    2011-01-01

    Polyhydroxyamide (PHA) and polymethoxyamide (PMeOA) are fire-retardant (FR) thermoplastic polymers and have been found to be useful as an additive for imparting fire retardant properties to other compatible, thermoplastic polymers (including some elastomers). Examples of compatible flammable polymers include nylons, polyesters, and acrylics. Unlike most prior additives, PHA and PMeOA do not appreciably degrade the mechanical properties of the matrix polymer; indeed, in some cases, mechanical properties are enhanced. Also, unlike some prior additives, PHA and PMeOA do not decompose into large amounts of corrosive or toxic compounds during combustion and can be processed at elevated temperatures. PMeOA derivative formulations were synthesized and used as an FR additive in the fabrication of polyamide (PA) and polystyrene (PS) composites with notable reduction (>30 percent for PS) in peak heat release rates compared to the neat polymer as measured by a Cone Calorimeter (ASTM E1354). Synergistic effects were noted with nanosilica composites. These nanosilica composites had more than 50-percent reduction in peak heat release rates. In a typical application, a flammable thermoplastic, thermoplastic blend, or elastomer that one seeks to render flame-retardant is first dry-mixed with PHA or PMeOA or derivative thereof. The proportion of PHA or PMeOA or derivative in the mixture is typically chosen to lie between 1 and 20 weight percent. The dry blend can then be melt-extruded. The extruded polymer blend can further be extruded and/or molded into fibers, pipes, or any other of a variety of objects that may be required to be fire-retardant. The physical and chemical mechanisms which impart flame retardancy of the additive include inhibiting free-radical oxidation in the vapor phase, preventing vaporization of fuel (the polymer), and cooling through the formation of chemical bonds in either the vapor or the condensed phase. Under thermal stress, the cyclic hydroxyl/ methoxy

  11. Factors affecting spruce establishment and recruitment near western treeline, Alaska

    NASA Astrophysics Data System (ADS)

    Miller, A. E.; Sherriff, R.; Wilson, T. L.

    2015-12-01

    Regional warming and increases in tree growth are contributing to increased productivity near the western forest margin in Alaska. The effects of warming on seedling recruitment has received little attention, in spite of forecasted forest expansion near western treeline. Here, we used stand structure and environmental data from white spruce (Picea glauca) stands (n = 95) sampled across a longitudinal gradient to explore factors influencing white spruce growth, establishment and recruitment in southwest Alaska. Using tree-ring chronologies developed from a subset of the plots (n = 30), we estimated establishment dates and basal area increment (BAI) for trees of all age classes across a range of site conditions. We used GLMs (generalized linear models) to explore the relationship between tree growth and temperature in undisturbed, low elevation sites along the gradient, using BAI averaged over the years 1975-2000. In addition, we examined the relationship between growing degree days (GDD) and seedling establishment over the previous three decades. We used total counts of live seedlings, saplings and live and dead trees, representing four cohorts, to evaluate whether geospatial, climate, and measured plot covariates predicted abundance of the different size classes. We hypothesized that the relationship between abundance and longitude would vary by size class, and that this relationship would be mediated by growing season temperature. We found that mean BAI for trees in undisturbed, low elevation sites increased with July maximum temperature, and that the slope of the relationship with temperature changed with longitude (interaction significant with 90% confidence). White spruce establishment was positively associated with longer summers and/or greater heat accumulation, as inferred from GDD. Seedling, sapling and tree abundance were also positively correlated with temperature across the study area. The response to longitude was mixed, with smaller size classes

  12. Communication and relationship satisfaction in Chinese, Western, and intercultural Chinese-Western couples.

    PubMed

    Hiew, Danika N; Halford, W Kim; van de Vijver, Fons J R; Liu, Shuang

    2016-03-01

    The current study compared Chinese, Western, and intercultural Chinese-Western couples' communication and examined how culture moderates the association of communication with relationship satisfaction. We coded the communication of 33 Western couples, 36 Chinese couples, and 54 intercultural Chinese-Western couples when discussing a relationship problem and when reminiscing about positive relationship events. Couples with Chinese female partners showed fewer positive behaviors and more negative behaviors (as classified in existing Western coding systems) than couples with Western female partners. The male partner's culture had few associations with couples' rates of communication behavior. Relationship satisfaction was associated with low rates of negative behaviors and high rates of most of the positive behaviors across cultural groups, and these associations were more evident in problem discussions than positive reminiscences.

  13. Communication and relationship satisfaction in Chinese, Western, and intercultural Chinese-Western couples.

    PubMed

    Hiew, Danika N; Halford, W Kim; van de Vijver, Fons J R; Liu, Shuang

    2016-03-01

    The current study compared Chinese, Western, and intercultural Chinese-Western couples' communication and examined how culture moderates the association of communication with relationship satisfaction. We coded the communication of 33 Western couples, 36 Chinese couples, and 54 intercultural Chinese-Western couples when discussing a relationship problem and when reminiscing about positive relationship events. Couples with Chinese female partners showed fewer positive behaviors and more negative behaviors (as classified in existing Western coding systems) than couples with Western female partners. The male partner's culture had few associations with couples' rates of communication behavior. Relationship satisfaction was associated with low rates of negative behaviors and high rates of most of the positive behaviors across cultural groups, and these associations were more evident in problem discussions than positive reminiscences. PMID:26371449

  14. Bats of the Western Indian Ocean Islands

    PubMed Central

    O'Brien, John

    2011-01-01

    Simple Summary The purpose of this paper is to review the literature pertaining to the bat faunas of the western Indian Ocean islands, particularly in light of the identification of many new species on Madagascar and the taxonomic reassignment of others, and to summarise details of their general biology, feeding ecology, reproduction and conservation. Abstract The natural colonisation of many remote oceanic islands by bats, including those of the western Indian Ocean, has been facilitated by their unique capability among mammals for powered flight. In the western Indian Ocean region, only the Malagasy islands of Madagascar and the Comoros archipelago have been naturally colonised by non-volant mammals. Despite their greater potential for inter-island dispersal, and thus gene transfer, endemicity of Chiroptera in the western Indian Ocean islands is high. Given their vulnerability to stochastic and anthropogenic disturbances, greater focus needs to be placed on investigating the demographic and ecological history of bats on Western Indian Ocean islands to safeguard not only their future, but also the ecosystem functioning on these islands, for which they are undoubtedly such an integral part. Here, I summarise the taxonomic and life history information available on bats from Western Indian Ocean islands and highlight knowledge gaps and conservation issues that threaten the continued persistence of some species. PMID:26486500

  15. A systematic review of integrated traditional Chinese and Western medicine for managing irritable bowel syndrome.

    PubMed

    Li, Chun-Yan; Ain Mohd Tahir, Nurul; Li, Shu-Chuen

    2015-01-01

    Traditional Chinese medicine (TCM) has been commonly used by Chinese practitioners to treat irritable bowel syndrome (IBS). However, the effectiveness of combining TCM with Western medicine in managing IBS has not been evaluated systematically. In this study, we evaluated the clinical effectiveness of combining TCM and Western medicine in the treatment of IBS via meta-analyses. We reviewed 72 eligible randomized controlled trials from January 2009 to December 2013 investigating the effectiveness of integrated TCM and Western medicine in the management of IBS. In the meta-analyses, the relative risks (RRs) and 95% confidence interval (95%CI) were calculated using raw data from each study, and low heterogeneity was detected. When compared to the Western medicine treatment alone, our result showed TCM combined with Western interventions significantly improved IBS global symptoms (RR, 1.21; 95%CI: 1.18-1.24). Additionally, there was no significant difference in therapeutic effects of the integrated approach in the meta-analyses involving the various IBS subtypes. Likewise, both Chinese proprietary herbal medicine plus conventional treatment and compound herbal preparations plus conventional treatment showed similar and statistically significant effects on global improvement compared with western treatment alone, with RRs of 1.22 (95%CI: 1.14-1.30) and 1.22 (95%CI: 1.18-1.27), respectively. These results demonstrated that treating IBS with integrated traditional Chinese and Western medicines showed better effectiveness than conventional Western medicine alone. Although due to the quality of the included studies, our results might possess a high risk of bias. TCM, particularly Chinese proprietary medicine, with the benefits of low-cost, easy to use, and good palatability, would be an attractive option to be used in conjunction with conventional Western medicine to manage IBS patients.

  16. Metal Additive Manufacturing: A Review

    NASA Astrophysics Data System (ADS)

    Frazier, William E.

    2014-06-01

    This paper reviews the state-of-the-art of an important, rapidly emerging, manufacturing technology that is alternatively called additive manufacturing (AM), direct digital manufacturing, free form fabrication, or 3D printing, etc. A broad contextual overview of metallic AM is provided. AM has the potential to revolutionize the global parts manufacturing and logistics landscape. It enables distributed manufacturing and the productions of parts-on-demand while offering the potential to reduce cost, energy consumption, and carbon footprint. This paper explores the material science, processes, and business consideration associated with achieving these performance gains. It is concluded that a paradigm shift is required in order to fully exploit AM potential.

  17. Individualized additional instruction for calculus

    NASA Astrophysics Data System (ADS)

    Takata, Ken

    2010-10-01

    College students enrolling in the calculus sequence have a wide variance in their preparation and abilities, yet they are usually taught from the same lecture. We describe another pedagogical model of Individualized Additional Instruction (IAI) that assesses each student frequently and prescribes further instruction and homework based on the student's performance. Our study compares two calculus classes, one taught with mandatory remedial IAI and the other without. The class with mandatory remedial IAI did significantly better on comprehensive multiple-choice exams, participated more frequently in classroom discussion and showed greater interest in theorem-proving and other advanced topics.

  18. The Mozart Effect: Additional Data.

    PubMed

    Hughes, John R.

    2002-04-01

    After the review of the Mozart effect was published in this journal (Hughes JR. Epilepsy Behav 2001;2:369-417), additional data from the music of Haydn and Liszt have been analyzed that may account for the decrease in seizure activity originally reported during Mozart music. Even with these added data Mozart music continued to score significantly higher than the selections from the other six composers in one of the important characteristics of this music, namely, the repetition of the melody. However Haydn's values were second highest among Mozart, J. S. Bach, Wagner, Beethoven, Chopin, and Liszt.

  19. Identification and characterization of plasmids from the western aster yellows mycoplasmalike organism.

    PubMed Central

    Kuske, C R; Kirkpatrick, B C

    1990-01-01

    Supercoiled double-stranded DNA molecules (plasmids) were isolated from plants infected with three laboratory strains of western aster yellows mycoplasma-like organism (AY-MLO) by using cesium chloride-ethidium bromide density gradients. Southern blot analysis, using plasmids from the severe strain of AY-MLO (SAY-MLO) as the probe, identified at least four plasmids in celery, aster, and periwinkle plants and in Macrosteles severini leafhopper vectors infected with either the dwarf AY-MLO, Tulelake AY-MLO, or SAY-MLO strain. Plasmids were also detected in two California field isolates of AY-MLO but not in plants infected with the beet leafhopper-transmitted virescence agent, western X, or elm yellows MLOs. SAY-MLO plasmids were 5.2, 4.9, 3.4, and 1.7 kilobase pairs in size. Plasmids isolated from dwarf AY- and Tulelake AY-MLOs were 7.4, 5.1, 3.5, and 1.7 kilobase pairs in size. No evidence was obtained for integration of SAY-MLO plasmids into the MLO chromosome. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 PMID:2307660

  20. Additive effects of ulinastatin and docetaxel on growth of breast cancer xenograft in nude mice and expression of PGE2, IL-10, and IL-2 in primary breast cancer cells.

    PubMed

    Zhong, Biao; Shen, Hongyan; Sun, Xin; Wang, Hong; Zhang, Yonghua; Sun, Zhijun

    2012-05-01

    Ulinastatin is a broad-spectrum enzyme inhibitor extracted from urine. Previous data from our group suggested that ulinastatin could significantly inhibit proliferation of human breast MDA-MB-231 cells, growth of tumor xenograft in nude mice, and expression of interleukin (IL)-6 and IL-8. In the present study, we investigated whether there is an additive effect of ulinastatin and docetaxel on growth of breast cancer xenografts in nude mice and its possible mechanisms. Nude mice and primary human breast cancer cells were treated with phosphate buffered saline (PBS), ulinastatin, docetaxel, or ulinastatin plus docetaxel, respectively. Their effects on xenograft growth; expressions of cyclooxygenase-2 (COX2), prostaglandin E2 receptor 2 (EP2), IL-10, and IL-2; and secretion of prostaglandin E2 (PGE2) were examined using variety of methods, including semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent (ELISA) assay, and immunohistochemistry SP method. The treatment with ulinastatin, docetaxel, or ulinastatin plus docetaxel could significantly (1) inhibit COX2 and IL-10 expression in primary tumor cells at both mRNA and protein levels, (2) reduce PGE2 secretion in culture supernatant (p<0.05), (3) inhibit COX2, EP2, and IL-10 protein levels in primary xenograft of nude mice, and (4) increase IL-2 expression (p<0.05) in primary xenografts of nude mice. In addition, ulinastatin and docetaxel had additive effects. We suggest that ulinastatin had similar effects of docetaxel and can enhance docetaxel's anticancer effects possibly by inhibiting COX2 expression, reducing PGE2 and EP2 expression and their binding, upregulating IL-2, and downregulating IL-10.

  1. Additive manufacturing of RF absorbers

    NASA Astrophysics Data System (ADS)

    Mills, Matthew S.

    The ability of additive manufacturing techniques to fabricate integrated electromagnetic absorbers tuned for specific radio frequency bands within structural composites allows for unique combinations of mechanical and electromagnetic properties. These composites and films can be used for RF shielding of sensitive electromagnetic components through in-plane and out-of-plane RF absorption. Structural composites are a common building block of many commercial platforms. These platforms may be placed in situations in which there is a need for embedded RF absorbing properties along with structural properties. Instead of adding radar absorbing treatments to the external surface of existing structures, which adds increased size, weight and cost; it could prove to be advantageous to integrate the microwave absorbing properties directly into the composite during the fabrication process. In this thesis, a method based on additive manufacturing techniques of composites structures with prescribed electromagnetic loss, within the frequency range 1 to 26GHz, is presented. This method utilizes screen printing and nScrypt micro dispensing to pattern a carbon based ink onto low loss substrates. The materials chosen for this study will be presented, and the fabrication technique that these materials went through to create RF absorbing structures will be described. The calibration methods used, the modeling of the RF structures, and the applications in which this technology can be utilized will also be presented.

  2. Fuel Additives: Canada bans MMT

    SciTech Connect

    Sissell, K.

    1997-04-16

    The Canadian Senate voted late last week to ban use of the manganese-based fuel additive MMT, produced only in the US by Ethyl. MMT, which has been sold in Canada for the past 20 years and accounts for about half of Ethyl`s Canadian sales, has been criticized by environmentalists, who have raised public health concerns, and automakers, who say it harms emission control systems. {open_quotes}Canada`s vote is a great victory for public health and the environment,{close_quotes} says Environmental Defense Fund executive director Fred Krupp. {open_quotes}The US should move swiftly to follow suit and suspend sales of MMT until adequate toxicity testing on the additive is completed.{close_quotes} EPA had refused to approve MMT for sale because of health concerns but was compelled to do so by a December 1995 court ruling. Ethyl asserts the ban violates Canada`s obligations under Nafta and says it will file a damage claim with the Nafta arbitration panel.

  3. Neutron Characterization for Additive Manufacturing

    NASA Technical Reports Server (NTRS)

    Watkins, Thomas; Bilheux, Hassina; An, Ke; Payzant, Andrew; DeHoff, Ryan; Duty, Chad; Peter, William; Blue, Craig; Brice, Craig A.

    2013-01-01

    Oak Ridge National Laboratory (ORNL) is leveraging decades of experience in neutron characterization of advanced materials together with resources such as the Spallation Neutron Source (SNS) and the High Flux Isotope Reactor (HFIR) shown in Fig. 1 to solve challenging problems in additive manufacturing (AM). Additive manufacturing, or three-dimensional (3-D) printing, is a rapidly maturing technology wherein components are built by selectively adding feedstock material at locations specified by a computer model. The majority of these technologies use thermally driven phase change mechanisms to convert the feedstock into functioning material. As the molten material cools and solidifies, the component is subjected to significant thermal gradients, generating significant internal stresses throughout the part (Fig. 2). As layers are added, inherent residual stresses cause warping and distortions that lead to geometrical differences between the final part and the original computer generated design. This effect also limits geometries that can be fabricated using AM, such as thin-walled, high-aspect- ratio, and overhanging structures. Distortion may be minimized by intelligent toolpath planning or strategic placement of support structures, but these approaches are not well understood and often "Edisonian" in nature. Residual stresses can also impact component performance during operation. For example, in a thermally cycled environment such as a high-pressure turbine engine, residual stresses can cause components to distort unpredictably. Different thermal treatments on as-fabricated AM components have been used to minimize residual stress, but components still retain a nonhomogeneous stress state and/or demonstrate a relaxation-derived geometric distortion. Industry, federal laboratory, and university collaboration is needed to address these challenges and enable the U.S. to compete in the global market. Work is currently being conducted on AM technologies at the ORNL

  4. Additives in fibers and fabrics.

    PubMed

    Barker, R H

    1975-06-01

    The additives and contaminants which occur in textile fibers vary widely, depending on the type of fiber and the pretreatment which it has received. Synthetic fibers such as nylon and polyester contain trace amounts of contaminants such as catalysts and catalyst deactivators which remain after the synthesis of the basic polymers. In addition, there are frequently a number of materials which are added to perform specific functions in almost all man-made fibers. Examples of these would include traces of metals or metal salts used as tracers for identification of specific lots of fiber, TiO2 or similar materials added as delustrants, and a host of organic species added for such special purposes as antistatic agents or flame retardants. There may also be considerable quantities of residual monomer or small oligomers dissolved in the polymer matrix. The situation becomes even more complex after the fibers are converted into fabric form. Numerous materials are applied at various stages of fabric preparation to act as lubricants, sizing agents, antistats, bleaches, and wetting agents to facilitate the processing, but these are normally removed before the fabric reaches the cutters of the ultimate consumers and therefore usually do not constitute potential hazards. However, there are many other chemical agents which are frequently added during the later stages of fabric preparation and which are not designed to be removed. Aside from dyes and printing pigments, the most common additive for apparel fabrics is a durable press treatment. This generally involves the use of materials capable of crosslinking cellulosics by reacting through such functions as N-methylolated amides or related compounds such as ureas and carbamates. These materials pose some potential hazards due to both the nitrogenous bases and the formaldehyde which they usually release. There is usually also some residual catalyst in fabrics which have received such treatments. Other types of chemical treatments

  5. Additives in fibers and fabrics.

    PubMed Central

    Barker, R H

    1975-01-01

    The additives and contaminants which occur in textile fibers vary widely, depending on the type of fiber and the pretreatment which it has received. Synthetic fibers such as nylon and polyester contain trace amounts of contaminants such as catalysts and catalyst deactivators which remain after the synthesis of the basic polymers. In addition, there are frequently a number of materials which are added to perform specific functions in almost all man-made fibers. Examples of these would include traces of metals or metal salts used as tracers for identification of specific lots of fiber, TiO2 or similar materials added as delustrants, and a host of organic species added for such special purposes as antistatic agents or flame retardants. There may also be considerable quantities of residual monomer or small oligomers dissolved in the polymer matrix. The situation becomes even more complex after the fibers are converted into fabric form. Numerous materials are applied at various stages of fabric preparation to act as lubricants, sizing agents, antistats, bleaches, and wetting agents to facilitate the processing, but these are normally removed before the fabric reaches the cutters of the ultimate consumers and therefore usually do not constitute potential hazards. However, there are many other chemical agents which are frequently added during the later stages of fabric preparation and which are not designed to be removed. Aside from dyes and printing pigments, the most common additive for apparel fabrics is a durable press treatment. This generally involves the use of materials capable of crosslinking cellulosics by reacting through such functions as N-methylolated amides or related compounds such as ureas and carbamates. These materials pose some potential hazards due to both the nitrogenous bases and the formaldehyde which they usually release. There is usually also some residual catalyst in fabrics which have received such treatments. Other types of chemical treatments

  6. Expansion and methylation status at FRAXE can be detected on EcoRI blots used for FRAXA diagnosis: Analysis of four FRAXE families with mild mental retardation in males

    SciTech Connect

    Biancalana, V.; Bouix, J.C.; Mandel, J.L.

    1996-10-01

    The original test for the analysis of the CCG expansion at the FRAXE locus involves Southern blot analysis of HindIII digests. We show that, by using a different probe, the FRAXE mutation can be detected easily on the same EcoRI or EagI+EcoRI blots as are used for detection of FRAXA. Unexpectedly, we found that both the expansion and methylation status can be determined on a single EcoRI digest, because of the presence of a methylation-sensitive EcoRI site very close to the CCG repeat. We thus detected in a series of mentally retarded individuals previously tested for FRAXA expansion a FRAXE proband who led to the identification of a large sibship (7 of 10 children carrying a mutation). We also show that two fragile X families without FRAXA mutation that previously have been described by Oberle et al. have the FRAXE expansion. In another family also ascertained initially by cytogenetic finding of a fragile X site, we performed the combined cytogenetic and molecular prenatal diagnosis of a mutated male fetus. All nine males (>3 years old) in whom we found a methylated mutation had mild mental retardation. Our results suggest that the threshold of repeat length for abnormal methylation and fragile-site expression may be smaller at FRAXE than at FRAXA. 20 refs., 5 figs., 1 tab.

  7. 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.

    PubMed

    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.

  8. An Additive Manufacturing Test Artifact.

    PubMed

    Moylan, Shawn; Slotwinski, John; Cooke, April; Jurrens, Kevin; Donmez, M Alkan

    2014-01-01

    A test artifact, intended for standardization, is proposed for the purpose of evaluating the performance of additive manufacturing (AM) systems. A thorough analysis of previously proposed AM test artifacts as well as experience with machining test artifacts have inspired the design of the proposed test artifact. This new artifact is designed to provide a characterization of the capabilities and limitations of an AM system, as well as to allow system improvement by linking specific errors measured in the test artifact to specific sources in the AM system. The proposed test artifact has been built in multiple materials using multiple AM technologies. The results of several of the builds are discussed, demonstrating how the measurement results can be used to characterize and improve a specific AM system. PMID:26601039

  9. An Additive Manufacturing Test Artifact

    PubMed Central

    Moylan, Shawn; Slotwinski, John; Cooke, April; Jurrens, Kevin; Donmez, M Alkan

    2014-01-01

    A test artifact, intended for standardization, is proposed for the purpose of evaluating the performance of additive manufacturing (AM) systems. A thorough analysis of previously proposed AM test artifacts as well as experience with machining test artifacts have inspired the design of the proposed test artifact. This new artifact is designed to provide a characterization of the capabilities and limitations of an AM system, as well as to allow system improvement by linking specific errors measured in the test artifact to specific sources in the AM system. The proposed test artifact has been built in multiple materials using multiple AM technologies. The results of several of the builds are discussed, demonstrating how the measurement results can be used to characterize and improve a specific AM system. PMID:26601039

  10. SIPSEY WILDERNESS AND ADDITIONS, ALABAMA.

    USGS Publications Warehouse

    Schweinfurth, Stanley P.; Mory, Peter C.

    1984-01-01

    On the basis of geologic, geochemical, and mineral surveys the Sipsey Wilderness and additions are deemed to have little promise for the occurrence of metallic mineral resources. Although limestone, shale, and sandstone resources that occur in the area are physically suitable for a variety of uses, similar materials are available outside the area closer to transportation routes and potential markets. A small amount of coal has been identified in the area, occurring as nonpersistent beds less than 28 in. thick. Oil and (or) natural gas resources may be present if suitable structural traps exist in the subsurface. Therefore, the area has a probable oil and gas potential. Small amounts of asphaltic sandstone and limestone, commonly referred to as tar sands, may also occur in the subsurface. 5 refs.

  11. An Additive Manufacturing Test Artifact.

    PubMed

    Moylan, Shawn; Slotwinski, John; Cooke, April; Jurrens, Kevin; Donmez, M Alkan

    2014-01-01

    A test artifact, intended for standardization, is proposed for the purpose of evaluating the performance of additive manufacturing (AM) systems. A thorough analysis of previously proposed AM test artifacts as well as experience with machining test artifacts have inspired the design of the proposed test artifact. This new artifact is designed to provide a characterization of the capabilities and limitations of an AM system, as well as to allow system improvement by linking specific errors measured in the test artifact to specific sources in the AM system. The proposed test artifact has been built in multiple materials using multiple AM technologies. The results of several of the builds are discussed, demonstrating how the measurement results can be used to characterize and improve a specific AM system.

  12. A novel addition polyimide adhesive

    NASA Technical Reports Server (NTRS)

    St.clair, T. L.; Progar, D. J.

    1981-01-01

    An addition polyimide adhesive, LARC 13, was developed which shows promise for bonding both titanium and composites for applications which require service temperatures in excess of 533 K. The LARC 13 is based on an oligomeric bis nadimide containing a meta linked aromatic diamine. The adhesive melts prior to polymerization due to its oligomeric nature, thereby allowing it to be processed at 344 kPa or less. Therefore, LARC 13 is ideal for the bonding of honeycomb sandwich structures. After melting, the resin thermosets during the cure of the nadic endcaps to a highly crosslinked system. Few volatiles are evolved, thus allowing large enclosed structures to be bonded. Preparation of the adhesive as well as bonding, aging, and testing of lap shear and honeycomb samples are discussed.

  13. Adverse reactions to food additives.

    PubMed

    Simon, R A

    1986-01-01

    There are thousands of agents that are intentionally added to the food that we consume. These include preservatives, stabilizers, conditioners, thickeners, colorings, flavorings, sweeteners, antioxidants, etc. etc. Yet only a surprisingly small number have been associated with hypersensitivity reactions. Amongst all the additives, FD&C dyes have been most frequently associated with adverse reactions. Tartrazine is the most notorious of them all; however, critical review of the medical literature and current Scripps Clinic studies would indicate that tartrazine has been confirmed to be at best only occasionally associated with flares of urticaria or asthma. There is no convincing evidence in the literature of reactivity to the other azo or nonazo dyes. This can also be said of BHA/BHT, nitrites/nitrates and sorbates. Parabens have been shown to elicit IgE mediated hypersensitivity reactions when used as pharmaceutical preservatives; however, as with the other additives noted above, ingested parabens have only occasionally been associated with adverse reactions. MSG, the cause of the 'Chinese restaurant syndrome' has only been linked to asthma in one report. Sulfiting agents used primarily as food fresheners and to control microbial growth in fermented beverages have been established as the cause of any where from mild to severe and even fatal reactions in at least 5% of the asthmatic population. Other reactions reported to follow sulfite ingestion include anaphylaxis, gastro intestinal complaints and dermatological eruptions. The prevalence of these non asthmatic reactions is unknown. The mechanism of sulfite sensitive asthma is also unknown but most likely involves hyperreactivity to inhale SO2 in the great majority of cases; however, there are reports of IgE mediated reactions and other sulfite sensitive asthmatics have been found with low levels of sulfite oxidase; necessary to oxidize endogenous sulfite to sulfate.

  14. 78 FR 55062 - Western Pacific Fishery Management Council; Public Meetings

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-09

    .... Western Pacific resource agencies c. Western Pacific Regional Fishery Management Council 3. About the Non... Marine Recreational Fisheries Survey b. Western Pacific creel surveys c. Marine Recreational Information... National Oceanic and Atmospheric Administration RIN 0648-XC858 Western Pacific Fishery Management...

  15. 15 CFR 754.4 - Unprocessed western red cedar.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 15 Commerce and Foreign Trade 2 2012-01-01 2012-01-01 false Unprocessed western red cedar. 754.4... CONTROLS § 754.4 Unprocessed western red cedar. (a) License requirement. As indicated by the letters “SS... the export of unprocessed western red cedar covered by ECCN 1C988 (Western red cedar (thuja...

  16. 15 CFR 754.4 - Unprocessed western red cedar.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 15 Commerce and Foreign Trade 2 2013-01-01 2013-01-01 false Unprocessed western red cedar. 754.4... CONTROLS § 754.4 Unprocessed western red cedar. (a) License requirement. As indicated by the letters “SS... the export of unprocessed western red cedar covered by ECCN 1C988 (Western red cedar (thuja...

  17. 15 CFR 754.4 - Unprocessed western red cedar.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 15 Commerce and Foreign Trade 2 2014-01-01 2014-01-01 false Unprocessed western red cedar. 754.4... CONTROLS § 754.4 Unprocessed western red cedar. (a) License requirement. As indicated by the letters “SS... the export of unprocessed western red cedar covered by ECCN 1C988 (Western red cedar (thuja...

  18. Communication Patterns in Adult-Infant Interactions in Western and Non-Western Cultures.

    ERIC Educational Resources Information Center

    Keller, Heidi; And Others

    1988-01-01

    Analyzes the early communication structure in adult-child interactions with two- to six-month old babies in Western (West Germany, Greece) and non-Western (Yanomami, Trobriand) societies. Discusses universal international verbal and non-verbal structures reflecting intuitive parenting programs. (FMW)

  19. Relationship Between Atmospheric circulation and Snowpack in theWestern United States

    SciTech Connect

    Jin, Jiming; Miller, Norman L.; Sorooshian, Soroosh; Gao, Xiaogang

    2004-06-02

    Snow anomalies in the western United States (U.S.) have beenwidely investigated by many researchers due to its impact on wateravailability. This study focuses on how anomalous atmospheric circulationaffects snowpack accumulation in the western U.S. using observations andoutput from the National Center for Atmospheric Research (NCAR) CommunityClimate Model version 3 (CCM3). Our results indicate that themid-latitude atmospheric circulation anomalies induced by the ElNino-Southern Oscillation (ENSO) tend to drive winter precipitationshifts, leading to an anomalous snowpack distribution in the western U.S.The warm phase of ENSO produces increased snowpack in the Southwest,while the cold phase of ENSO generates increased snowpack in theNorthwest. Temperature has a secondary impact on the anomalous snowpackdistribution during ENSO episodes. Additionally, the non-linearatmospheric dynamics-related Pacific-North American (PNA) pattern isfound to strongly affect snow anomalies in the western U.S. independentfrom ENSO. The positive phase of the PNA pattern produces coldertemperature and stronger precipitation due to the lower pressure in theregion, leading to an above normal snowpack. Conversely, the negativephase of the PNA pattern generates warmer temperature and weakerprecipitation resulting from the higher pressure, producing a below thannormal snowpack in the western U.S. In general, the NCAR-CCM3 reproducesthe observed processes. However, model biases are identified andreported. The information provided in this study strengthens ourunderstanding of climate and water supply variability in the westernU.S.

  20. Additive attacks on speaker recognition

    NASA Astrophysics Data System (ADS)

    Farrokh Baroughi, Alireza; Craver, Scott

    2014-02-01

    Speaker recognition is used to identify a speaker's voice from among a group of known speakers. A common method of speaker recognition is a classification based on cepstral coefficients of the speaker's voice, using a Gaussian mixture model (GMM) to model each speaker. In this paper we try to fool a speaker recognition system using additive noise such that an intruder is recognized as a target user. Our attack uses a mixture selected from a target user's GMM model, inverting the cepstral transformation to produce noise samples. In our 5 speaker data base, we achieve an attack success rate of 50% with a noise signal at 10dB SNR, and 95% by increasing noise power to 0dB SNR. The importance of this attack is its simplicity and flexibility: it can be employed in real time with no processing of an attacker's voice, and little computation is needed at the moment of detection, allowing the attack to be performed by a small portable device. For any target user, knowing that user's model or voice sample is sufficient to compute the attack signal, and it is enough that the intruder plays it while he/she is uttering to be classiffed as the victim.