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  1. A survey of tools for the analysis of quantitative PCR (qPCR) data.

    Science.gov (United States)

    Pabinger, Stephan; Rödiger, Stefan; Kriegner, Albert; Vierlinger, Klemens; Weinhäusel, Andreas

    2014-09-01

    Real-time quantitative polymerase-chain-reaction (qPCR) is a standard technique in most laboratories used for various applications in basic research. Analysis of qPCR data is a crucial part of the entire experiment, which has led to the development of a plethora of methods. The released tools either cover specific parts of the workflow or provide complete analysis solutions. Here, we surveyed 27 open-access software packages and tools for the analysis of qPCR data. The survey includes 8 Microsoft Windows, 5 web-based, 9 R-based and 5 tools from other platforms. Reviewed packages and tools support the analysis of different qPCR applications, such as RNA quantification, DNA methylation, genotyping, identification of copy number variations, and digital PCR. We report an overview of the functionality, features and specific requirements of the individual software tools, such as data exchange formats, availability of a graphical user interface, included procedures for graphical data presentation, and offered statistical methods. In addition, we provide an overview about quantification strategies, and report various applications of qPCR. Our comprehensive survey showed that most tools use their own file format and only a fraction of the currently existing tools support the standardized data exchange format RDML. To allow a more streamlined and comparable analysis of qPCR data, more vendors and tools need to adapt the standardized format to encourage the exchange of data between instrument software, analysis tools, and researchers.

  2. A survey of tools for the analysis of quantitative PCR (qPCR data

    Directory of Open Access Journals (Sweden)

    Stephan Pabinger

    2014-09-01

    Our comprehensive survey showed that most tools use their own file format and only a fraction of the currently existing tools support the standardized data exchange format RDML. To allow a more streamlined and comparable analysis of qPCR data, more vendors and tools need to adapt the standardized format to encourage the exchange of data between instrument software, analysis tools, and researchers.

  3. A common base method for analysis of qPCR data and the application of simple blocking in qPCR experiments.

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    Ganger, Michael T; Dietz, Geoffrey D; Ewing, Sarah J

    2017-12-01

    qPCR has established itself as the technique of choice for the quantification of gene expression. Procedures for conducting qPCR have received significant attention; however, more rigorous approaches to the statistical analysis of qPCR data are needed. Here we develop a mathematical model, termed the Common Base Method, for analysis of qPCR data based on threshold cycle values (C q ) and efficiencies of reactions (E). The Common Base Method keeps all calculations in the logscale as long as possible by working with log 10 (E) ∙ C q , which we call the efficiency-weighted C q value; subsequent statistical analyses are then applied in the logscale. We show how efficiency-weighted C q values may be analyzed using a simple paired or unpaired experimental design and develop blocking methods to help reduce unexplained variation. The Common Base Method has several advantages. It allows for the incorporation of well-specific efficiencies and multiple reference genes. The method does not necessitate the pairing of samples that must be performed using traditional analysis methods in order to calculate relative expression ratios. Our method is also simple enough to be implemented in any spreadsheet or statistical software without additional scripts or proprietary components.

  4. QPCR: Application for real-time PCR data management and analysis

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    Eichhorn Heiko

    2009-08-01

    Full Text Available Abstract Background Since its introduction quantitative real-time polymerase chain reaction (qPCR has become the standard method for quantification of gene expression. Its high sensitivity, large dynamic range, and accuracy led to the development of numerous applications with an increasing number of samples to be analyzed. Data analysis consists of a number of steps, which have to be carried out in several different applications. Currently, no single tool is available which incorporates storage, management, and multiple methods covering the complete analysis pipeline. Results QPCR is a versatile web-based Java application that allows to store, manage, and analyze data from relative quantification qPCR experiments. It comprises a parser to import generated data from qPCR instruments and includes a variety of analysis methods to calculate cycle-threshold and amplification efficiency values. The analysis pipeline includes technical and biological replicate handling, incorporation of sample or gene specific efficiency, normalization using single or multiple reference genes, inter-run calibration, and fold change calculation. Moreover, the application supports assessment of error propagation throughout all analysis steps and allows conducting statistical tests on biological replicates. Results can be visualized in customizable charts and exported for further investigation. Conclusion We have developed a web-based system designed to enhance and facilitate the analysis of qPCR experiments. It covers the complete analysis workflow combining parsing, analysis, and generation of charts into one single application. The system is freely available at http://genome.tugraz.at/QPCR

  5. Identification of Optimal Reference Genes for Normalization of qPCR Analysis during Pepper Fruit Development

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    Yuan Cheng

    2017-06-01

    Full Text Available Due to its high sensitivity and reproducibility, quantitative real-time PCR (qPCR is practiced as a useful research tool for targeted gene expression analysis. For qPCR operations, the normalization with suitable reference genes (RGs is a crucial step that eventually determines the reliability of the obtained results. Although pepper is considered an ideal model plant for the study of non-climacteric fruit development, at present no specific RG have been developed or validated for the qPCR analyses of pepper fruit. Therefore, this study aimed to identify stably expressed genes for their potential use as RGs in pepper fruit studies. Initially, a total of 35 putative RGs were selected by mining the pepper transcriptome data sets derived from the PGP (Pepper Genome Platform and PGD (Pepper Genome Database. Their expression stabilities were further measured in a set of pepper (Capsicum annuum L. var. 007e fruit samples, which represented four different fruit developmental stages (IM: Immature; MG: Mature green; B: Break; MR: Mature red using the qPCR analysis. Then, based on the qPCR results, three different statistical algorithms, namely geNorm, Normfinder, and boxplot, were chosen to evaluate the expression stabilities of these putative RGs. It should be noted that nine genes were proven to be qualified as RGs during pepper fruit development, namely CaREV05 (CA00g79660; CaREV08 (CA06g02180; CaREV09 (CA06g05650; CaREV16 (Capana12g002666; CaREV21 (Capana10g001439; CaREV23 (Capana05g000680; CaREV26 (Capana01g002973; CaREV27 (Capana11g000123; CaREV31 (Capana04g002411; and CaREV33 (Capana08g001826. Further analysis based on geNorm suggested that the application of the two most stably expressed genes (CaREV05 and CaREV08 would provide optimal transcript normalization in the qPCR experiments. Therefore, a new and comprehensive strategy for the identification of optimal RGs was developed. This strategy allowed for the effective normalization of the qPCR

  6. MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay

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    Nagy, Alexander; Černíková, Lenka; Vitásková, Eliška; Křivda, Vlastimil; Dán, Ádám; Dirbáková, Zuzana; Jiřincová, Helena; Procházka, Bohumír; Sedlák, Kamil; Havlíčková, Martina

    2016-01-01

    In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence. PMID:27031831

  7. Miltenberger blood group typing by real-time polymerase chain reaction (qPCR) melting curve analysis in Thai population.

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    Vongsakulyanon, A; Kitpoka, P; Kunakorn, M; Srikhirin, T

    2015-12-01

    To develop reliable and convenient methods for Miltenberger (Mi(a) ) blood group typing. To apply real-time polymerase chain reaction (qPCR) melting curve analysis to Mi(a) blood group typing. The Mi(a) blood group is the collective set of glycophorin hybrids in the MNS blood group system. Mi(a+) blood is common among East Asians and is also found in the Thai population. Incompatible Mi(a) blood transfusions pose the risk of life-threatening haemolysis; therefore, Mi(a) blood group typing is necessary in ethnicities where the Mi(a) blood group is prevalent. One hundred and forty-three blood samples from Thai blood donors were used in the study. The samples included 50 Mi(a+) samples and 93 Mi(a-) samples, which were defined by serology. The samples were typed by Mi(a) typing qPCR, and 50 Mi(a+) samples were sequenced to identify the Mi(a) subtypes. Mi(a) subtyping qPCR was performed to define GP.Mur. Both Mi(a) typing and Mi(a) subtyping were tested on a conventional PCR platform. The results of Mi(a) typing qPCR were all concordant with serology. Sequencing of the 50 Mi(a+) samples revealed 47 GP.Mur samples and 3 GP.Hop or Bun samples. Mi(a) subtyping qPCR was the supplementary test used to further define GP.Mur from other Mi(a) subtypes. Both Mi(a) typing and Mi(a) subtyping performed well using a conventional PCR platform. Mi(a) typing qPCR correctly identified Mi(a) blood groups in a Thai population with the feasibility of Mi(a) subtype discrimination, and Mi(a) subtyping qPCR was able to further define GP.Mur from other Mi(a) subtypes. © 2015 British Blood Transfusion Society.

  8. Analysis of variation in virulence of Beauveria bassiana against insect pests of pigeonpea using qPCR.

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    Senthilraja, Govindasamy; Anand, Theerthagiri; Mohankumar, Subbarayalu; Raguchander, Thiruvengadam; Samiyappan, Ramasamy

    2018-03-01

    Beauveria bassiana is a broad spectrum microbial bioagent used for the control of agriculturally important insect pests. However, in our experiments, two virulent isolates of B. bassiana (B2 and B10) showed specific preference toward Maruca vitrata and Helicoverpa armigera of pigeonpea. To better understand this feature, we developed a qPCR assay to quantify the chitinase (virulence factor) of B. bassiana during the infection process. Isolates of B. bassiana were grown on insect cuticle amended medium and minimal medium (without insect cuticle) to assess the induction of chitinase. Our results revealed a positive correlation between expression of chitinase by B. bassiana and the substrates (with or without cuticles of M. vitrata and H. armigera) used. This study showcases the methodology to quantify the chitinase and analysis of variation in virulence of B. bassiana (B2 and B10) against M. vitrata and H. armigera. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. The utility of optical detection system (qPCR) and bioinformatics methods in reference gene expression analysis

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    Skarzyńska, Agnieszka; Pawełkowicz, Magdalena; PlÄ der, Wojciech; Przybecki, Zbigniew

    2016-09-01

    Real-time quantitative polymerase chain reaction is consider as the most reliable method for gene expression studies. However, the expression of target gene could be misinterpreted due to improper normalization. Therefore, the crucial step for analysing of qPCR data is selection of suitable reference genes, which should be validated experimentally. In order to choice the gene with stable expression in the designed experiment, we performed reference gene expression analysis. In this study genes described in the literature and novel genes predicted as control genes, based on the in silico analysis of transcriptome data were used. Analysis with geNorm and NormFinder algorithms allow to create the ranking of candidate genes and indicate the best reference for flower morphogenesis study. According to the results, genes CACS and CYCL were characterised the most stable expression, but the least suitable genes were TUA and EF.

  10. Isolation and Identification of Lactic Acid Bacteria from Traditional Dairy Products in Baotou and Bayannur of Midwestern Inner Mongolia and q-PCR Analysis of Predominant Species

    Science.gov (United States)

    2016-01-01

    In this study, traditional culture method and 16S rRNA gene analysis were applied to reveal the composition and diversity of lactic acid bacteria (LAB) of fermented cow milk, huruud and urum from Baotou and Bayannur of midwestern Inner Mongolia. Also, the quantitative results of dominant LAB species in three different types of dairy products from Baotou and Bayannur were gained by quantitative polymerase chain reaction (q-PCR) technology. Two hundred and two LAB strains isolated from sixty-six samples were identified and classified into four genera, namely Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, and twenty-one species and subspecies. From these isolates, Lactococcus lactis subsp. lactis (32.18%), Lactobacillus plantarum (12.38%) and Leuconosto mesenteroides (11.39%) were considered as the dominated LAB species under the condition of cultivating in MRS and M17 medium. And the q-PCR results revealed that the number of dominant species varied from samples to samples and from region to region. This study clearly shows the composition and diversity of LAB existing in fermented cow milk, huruud and urum, which could be considered as valuable resources for LAB isolation and further probiotic selection. PMID:27621691

  11. A method for quantitative analysis of standard and high-throughput qPCR expression data based on input sample quantity.

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    Mateusz G Adamski

    Full Text Available Over the past decade rapid advances have occurred in the understanding of RNA expression and its regulation. Quantitative polymerase chain reactions (qPCR have become the gold standard for quantifying gene expression. Microfluidic next generation, high throughput qPCR now permits the detection of transcript copy number in thousands of reactions simultaneously, dramatically increasing the sensitivity over standard qPCR. Here we present a gene expression analysis method applicable to both standard polymerase chain reactions (qPCR and high throughput qPCR. This technique is adjusted to the input sample quantity (e.g., the number of cells and is independent of control gene expression. It is efficiency-corrected and with the use of a universal reference sample (commercial complementary DNA (cDNA permits the normalization of results between different batches and between different instruments--regardless of potential differences in transcript amplification efficiency. Modifications of the input quantity method include (1 the achievement of absolute quantification and (2 a non-efficiency corrected analysis. When compared to other commonly used algorithms the input quantity method proved to be valid. This method is of particular value for clinical studies of whole blood and circulating leukocytes where cell counts are readily available.

  12. Evaluation by latent class analysis of a magnetic capture based DNA extraction followed by real-time qPCR as a new diagnostic method for detection of Echinococcus multilocularis in definitive hosts.

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    Maas, Miriam; van Roon, Annika; Dam-Deisz, Cecile; Opsteegh, Marieke; Massolo, Alessandro; Deksne, Gunita; Teunis, Peter; van der Giessen, Joke

    2016-10-30

    A new method, based on a magnetic capture based DNA extraction followed by qPCR, was developed for the detection of the zoonotic parasite Echinococcus multilocularis in definitive hosts. Latent class analysis was used to compare this new method with the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. In total, 60 red foxes and coyotes from three different locations were tested with both molecular methods and the sedimentation and counting technique (SCT) or intestinal scraping technique (IST). Though based on a limited number of samples, it could be established that the magnetic capture based DNA extraction followed by qPCR showed similar sensitivity and specificity as the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. All methods have a high specificity as shown by Bayesian latent class analysis. Both molecular assays have higher sensitivities than the combined SCT and IST, though the uncertainties in sensitivity estimates were wide for all assays tested. The magnetic capture based DNA extraction followed by qPCR has the advantage of not requiring hazardous chemicals like the phenol-chloroform DNA extraction followed by single tube nested PCR. This supports the replacement of the phenol-chloroform DNA extraction followed by single tube nested PCR by the magnetic capture based DNA extraction followed by qPCR for molecular detection of E. multilocularis in definitive hosts. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Latent class analysis of real time qPCR and bacteriological culturing for the diagnosis of Streptococcus agalactiae in cow composite milk samples.

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    Holmøy, Ingrid H; Toft, Nils; Jørgensen, Hannah J; Mørk, Tormod; Sølverød, Liv; Nødtvedt, Ane

    2018-06-01

    Streptococcus agalactiae (S. agalactiae) has re-emerged as a mastitis pathogen among Norwegian dairy cows. The Norwegian cattle health services recommend that infected herds implement measures to eradicate S. agalactiae, this includes a screening of milk samples from all lactating cows. The performance of the qPCR-test currently in use for this purpose has not been evaluated under field conditions. The objective of this study was to estimate the sensitivity and specificity of the real-time qPCR assay in use in Norway (Mastitis 4 qPCR, DNA Diagnostics A/S, Risskov, Denmark) and compare it to conventional bacteriological culturing for detection of S. agalactiae in milk samples. Because none of these tests are considered a perfect reference test, the evaluation was performed using latent class models in a Bayesian analysis. Aseptically collected cow-composite milk samples from 578 cows belonging to 6 herds were cultured and tested by qPCR. While 37 (6.4%) samples were positive for S. agalactiae by bacteriological culture, 66 (11.4%) samples were positive by qPCR. The within-herd prevalence in the six herds, as estimated by the latent class models ranged from 7.7 to 50.8%. At the recommended cut-off (cycle threshold 37), the sensitivity of the qPCR was significantly higher at 95.3 (95% posterior probability interval [PPI] [84.2; 99.6]) than that of bacteriological culture at 58.2 (95% PPI [43.8; 74.4]). However, bacterial culture had a higher specificity of 99.7 (95% PPI [98.5; 100.0]) compared to the qPCR at 98.5 (95% PPI [94.6; 99.9]). The median estimated negative predictive values of qPCR was consistently higher than those of the BC at all estimated prevalences, and the superiority of the qPCR increased with increasing within-herd prevalence. The median positive predictive values of BC was in general higher than the estimates for the qPCR, however, at the highest prevalence the predictive ability of both tests were similar. Copyright © 2018 Elsevier B.V. All

  14. Data Analysis of Sequences and qPCR for Microbial Communities during Algal Blooms

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    A training opportunity is open to a highly microbial-research-motivated student to conduct sequence analysis, explore novel genes and metabolic pathways, validate resultant findings using qPCR/RT-qPCR and summarize the findings

  15. Latent class analysis of real time qPCR and bacteriological culturing for the diagnosis of Streptococcus agalactiae in cow composite milk samples

    DEFF Research Database (Denmark)

    Holmøy, Ingrid H.; Toft, Nils; Jørgensen, Hannah J.

    2018-01-01

    Streptococcus agalactiae (S. agalactiae) has re-emerged as a mastitis pathogen among Norwegian dairy cows. The Norwegian cattle health services recommend that infected herds implement measures to eradicate S. agalactiae, this includes a screening of milk samples from all lactating cows....... The performance of the qPCR-test currently in use for this purpose has not been evaluated under field conditions. The objective of this study was to estimate the sensitivity and specificity of the real-time qPCR assay in use in Norway (Mastitis 4 qPCR, DNA Diagnostics A/S, Risskov, Denmark) and compare...... it to conventional bacteriological culturing for detection of S. agalactiae in milk samples. Because none of these tests are considered a perfect reference test, the evaluation was performed using latent class models in a Bayesian analysis. Aseptically collected cow-composite milk samples from 578 cows belonging...

  16. Validating Internal Control Genes for the Accurate Normalization of qPCR Expression Analysis of the Novel Model Plant Setaria viridis.

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    Julia Lambret-Frotté

    Full Text Available Employing reference genes to normalize the data generated with quantitative PCR (qPCR can increase the accuracy and reliability of this method. Previous results have shown that no single housekeeping gene can be universally applied to all experiments. Thus, the identification of a suitable reference gene represents a critical step of any qPCR analysis. Setaria viridis has recently been proposed as a model system for the study of Panicoid grasses, a crop family of major agronomic importance. Therefore, this paper aims to identify suitable S. viridis reference genes that can enhance the analysis of gene expression in this novel model plant. The first aim of this study was the identification of a suitable RNA extraction method that could retrieve a high quality and yield of RNA. After this, two distinct algorithms were used to assess the gene expression of fifteen different candidate genes in eighteen different samples, which were divided into two major datasets, the developmental and the leaf gradient. The best-ranked pair of reference genes from the developmental dataset included genes that encoded a phosphoglucomutase and a folylpolyglutamate synthase; genes that encoded a cullin and the same phosphoglucomutase as above were the most stable genes in the leaf gradient dataset. Additionally, the expression pattern of two target genes, a SvAP3/PI MADS-box transcription factor and the carbon-fixation enzyme PEPC, were assessed to illustrate the reliability of the chosen reference genes. This study has shown that novel reference genes may perform better than traditional housekeeping genes, a phenomenon which has been previously reported. These results illustrate the importance of carefully validating reference gene candidates for each experimental set before employing them as universal standards. Additionally, the robustness of the expression of the target genes may increase the utility of S. viridis as a model for Panicoid grasses.

  17. Monitoring of the Parasite Load in the Digestive Tract of Rhodnius prolixus by Combined qPCR Analysis and Imaging Techniques Provides New Insights into the Trypanosome Life Cycle.

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    Felipe de Almeida Dias

    Full Text Available Here we report the monitoring of the digestive tract colonization of Rhodnius prolixus by Trypanosoma cruzi using an accurate determination of the parasite load by qPCR coupled with fluorescence and bioluminescence imaging (BLI. These complementary methods revealed critical steps necessary for the parasite population to colonize the insect gut and establish vector infection.qPCR analysis of the parasite load in the insect gut showed several limitations due mainly to the presence of digestive-derived products that are thought to degrade DNA and inhibit further the PCR reaction. We developed a real-time PCR strategy targeting the T. cruzi repetitive satellite DNA sequence using as internal standard for normalization, an exogenous heterologous DNA spiked into insect samples extract, to precisely quantify the parasite load in each segment of the insect gut (anterior midgut, AM, posterior midgut, PM, and hindgut, H. Using combined fluorescence microscopy and BLI imaging as well as qPCR analysis, we showed that during their journey through the insect digestive tract, most of the parasites are lysed in the AM during the first 24 hours independently of the gut microbiota. During this short period, live parasites move through the PM to establish the onset of infection. At days 3-4 post-infection (p.i., the parasite population begins to colonize the H to reach a climax at day 7 p.i., which is maintained during the next two weeks. Remarkably, the fluctuation of the parasite number in H remains relatively stable over the two weeks after refeeding, while the populations residing in the AM and PM increases slightly and probably constitutes the reservoirs of dividing epimastigotes.These data show that a tuned dynamic control of the population operates in the insect gut to maintain an equilibrium between non-dividing infective trypomastigote forms and dividing epimastigote forms of the parasite, which is crucial for vector competence.

  18. Multi-laboratory survey of qPCR enterococci analysis method performance in U.S. coastal and inland surface waters

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    U.S. Environmental Protection Agency — Quantitative polymerase chain reaction (qPCR) has become a frequently used technique for quantifying enterococci in recreational surface waters, but there are...

  19. qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms

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    Michelle de Campos Soriani Azevedo

    2017-01-01

    Full Text Available Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR, a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV and negative (NPV predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H, the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

  20. qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms.

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    Azevedo, Michelle de Campos Soriani; Ramuno, Natália Mortari; Fachin, Luciana Raquel Vincenzi; Tassa, Mônica; Rosa, Patrícia Sammarco; Belone, Andrea de Faria Fernandes; Diório, Suzana Madeira; Soares, Cleverson Teixeira; Garlet, Gustavo Pompermaier; Trombone, Ana Paula Favaro

    Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease. Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

  1. Direct comparison of flow-FISH and qPCR as diagnostic tests for telomere length measurement in humans.

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    Fernanda Gutierrez-Rodrigues

    Full Text Available Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH, and quantitative PCR (qPCR. Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R(2 = 0.60; p<0.0001 and patients (R(2 = 0.51; p<0.0001. In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R(2 = 0.35; p<0.0001 and low correlation for patients (R(2 = 0.20; p = 0.001; Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R(2 = 0.33; p<0.0001 and did not correlate in the comparison of patients' samples (R(2 = 0.1, p = 0.08. Intra-assay coefficient of variation (CV was similar for flow-FISH (10.8 ± 7.1% and qPCR (9.5 ± 7.4%; p = 0.35, but the inter-assay CV was lower for flow-FISH (9.6 ± 7.6% vs. 16 ± 19.5%; p = 0.02. Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100% and specific (93% and 89%, respectively to distinguish very short telomeres. However, qPCR sensitivity (40% and specificity (63% to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity. In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the measurement of human

  2. Meta-Analysis of Quantification Methods Shows that Archaea and Bacteria Have Similar Abundances in the Subseafloor

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    May, Megan K.; Kevorkian, Richard T.; Steen, Andrew D.

    2013-01-01

    There is no universally accepted method to quantify bacteria and archaea in seawater and marine sediments, and different methods have produced conflicting results with the same samples. To identify best practices, we compiled data from 65 studies, plus our own measurements, in which bacteria and archaea were quantified with fluorescent in situ hybridization (FISH), catalyzed reporter deposition FISH (CARD-FISH), polyribonucleotide FISH, or quantitative PCR (qPCR). To estimate efficiency, we defined “yield” to be the sum of bacteria and archaea counted by these techniques divided by the total number of cells. In seawater, the yield was high (median, 71%) and was similar for FISH, CARD-FISH, and polyribonucleotide FISH. In sediments, only measurements by CARD-FISH in which archaeal cells were permeabilized with proteinase K showed high yields (median, 84%). Therefore, the majority of cells in both environments appear to be alive, since they contain intact ribosomes. In sediments, the sum of bacterial and archaeal 16S rRNA gene qPCR counts was not closely related to cell counts, even after accounting for variations in copy numbers per genome. However, qPCR measurements were precise relative to other qPCR measurements made on the same samples. qPCR is therefore a reliable relative quantification method. Inconsistent results for the relative abundance of bacteria versus archaea in deep subsurface sediments were resolved by the removal of CARD-FISH measurements in which lysozyme was used to permeabilize archaeal cells and qPCR measurements which used ARCH516 as an archaeal primer or TaqMan probe. Data from best-practice methods showed that archaea and bacteria decreased as the depth in seawater and marine sediments increased, although archaea decreased more slowly. PMID:24096423

  3. Fat stigmatization in television shows and movies: a content analysis.

    Science.gov (United States)

    Himes, Susan M; Thompson, J Kevin

    2007-03-01

    To examine the phenomenon of fat stigmatization messages presented in television shows and movies, a content analysis was used to quantify and categorize fat-specific commentary and humor. Fat stigmatization vignettes were identified using a targeted sampling procedure, and 135 scenes were excised from movies and television shows. The material was coded by trained raters. Reliability indices were uniformly high for the seven categories (percentage agreement ranged from 0.90 to 0.98; kappas ranged from 0.66 to 0.94). Results indicated that fat stigmatization commentary and fat humor were often verbal, directed toward another person, and often presented directly in the presence of the overweight target. Results also indicated that male characters were three times more likely to engage in fat stigmatization commentary or fat humor than female characters. To our knowledge, these findings provide the first information regarding the specific gender, age, and types of fat stigmatization that occur frequently in movies and television shows. The stimuli should prove useful in future research examining the role of individual difference factors (e.g., BMI) in the reaction to viewing such vignettes.

  4. Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR

    Directory of Open Access Journals (Sweden)

    Adrián Ruiz-Villalba

    2017-12-01

    Full Text Available Quantitative PCR allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, a survey with 93 validated assays for genes in the Wnt-pathway showed that the amplification of nonspecific products occurs frequently and is unrelated to Cq or PCR efficiency values. Titration experiments showed that the occurrence of low and high melting temperature artifacts was shown to be determined by annealing temperature, primer concentration and cDNA input. To explore the range of input variations that occur in the normal use of the Cre assay these conditions were mimicked in a complete two-way design of template −plasmid DNA- and non-template −mouse cDNA- concentrations. These experiments showed that the frequency of the amplification of the correct product and the artifact, as well as the valid quantification of the correct product, depended on the concentration of the non-template cDNA. This finding questions the interpretation of dilution series in which template as well as non-template concentrations are simultaneously decreasing. Repetition of this cDNA concentration experiment with other templates revealed that exact reproduction qPCR experiments was affected by the time it takes to complete the pipetting of a qPCR plate. Long bench times were observed to lead to significantly more artifacts. However, the measurement of artifact-associated fluorescence can be avoided by inclusion of a small heating step after the elongation phase in the amplification protocol. Taken together, this trouble-shooting journey showed that reliability and reproducibility of qPCR experiments not only depends on standardization and reporting of the biochemistry and technical aspects but also on hitherto neglected factors as sample dilution and waiting times in the laboratory work flow. Keywords: RT-qPCR, Melting curve analysis, Reaction parameters, Artifacts

  5. Advertising Discourse Analysis of FES stores: Killing Love, Cowards Show

    Directory of Open Access Journals (Sweden)

    Cristian Venegas Ahumada

    2013-08-01

    Full Text Available The objective is to analyze the structural and photographic discourse of the Autumn-Winter campaign 2008 of FES stores for young people. This was done by a semiotic theory and a critical-structural methodology of discourse. An analysis of 4 advertising photographs was done, and at once an analysis of the discourse “FES says no to violence against Women”, which explains the campaign’s target. The result is: The discourse was subjected to production condition (society of control and makes advertising a way to homogenize subjectivity of masses to consume. Recognition conditions demonstrate that this advertising discourse of symbolic violence means a type of violation of Men and Women Rights. An action like this requires commitment of Psychology in order to promote the social humanizing change, by means of university teaching and professional tasks.

  6. Bayesian analysis of repairable systems showing a bounded failure intensity

    International Nuclear Information System (INIS)

    Guida, Maurizio; Pulcini, Gianpaolo

    2006-01-01

    The failure pattern of repairable mechanical equipment subject to deterioration phenomena sometimes shows a finite bound for the increasing failure intensity. A non-homogeneous Poisson process with bounded increasing failure intensity is then illustrated and its characteristics are discussed. A Bayesian procedure, based on prior information on model-free quantities, is developed in order to allow technical information on the failure process to be incorporated into the inferential procedure and to improve the inference accuracy. Posterior estimation of the model-free quantities and of other quantities of interest (such as the optimal replacement interval) is provided, as well as prediction on the waiting time to the next failure and on the number of failures in a future time interval is given. Finally, numerical examples are given to illustrate the proposed inferential procedure

  7. Technical aspects and recommendations for single-cell qPCR.

    Science.gov (United States)

    Ståhlberg, Anders; Kubista, Mikael

    2018-02-01

    Single cells are basic physiological and biological units that can function individually as well as in groups in tissues and organs. It is central to identify, characterize and profile single cells at molecular level to be able to distinguish different kinds, to understand their functions and determine how they interact with each other. During the last decade several technologies for single-cell profiling have been developed and used in various applications, revealing many novel findings. Quantitative PCR (qPCR) is one of the most developed methods for single-cell profiling that can be used to interrogate several analytes, including DNA, RNA and protein. Single-cell qPCR has the potential to become routine methodology but the technique is still challenging, as it involves several experimental steps and few molecules are handled. Here, we discuss technical aspects and provide recommendation for single-cell qPCR analysis. The workflow includes experimental design, sample preparation, single-cell collection, direct lysis, reverse transcription, preamplification, qPCR and data analysis. Detailed reporting and sharing of experimental details and data will promote further development and make validation studies possible. Efforts aiming to standardize single-cell qPCR open up means to move single-cell analysis from specialized research settings to standard research laboratories. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Detection of foodborne pathogens by qPCR: A practical approach for food industry applications

    Directory of Open Access Journals (Sweden)

    María-José Chapela

    2015-12-01

    Full Text Available Microbiological analysis of food is an integrated part of microbial safety management in the food chain. Monitoring and controlling foodborne pathogens are traditionally carried out by conventional microbiological methods based on culture-dependent approaches in control laboratories and private companies. However, polymerase chain reaction (PCR has revolutionized microbiological analysis allowing detection of pathogenic microorganisms in food, without the necessity of classical isolation and identification. However, at present, PCR and quantitative polymerase chain reaction (qPCR are essential analytical tools for researchers working in the field of foodborne pathogens. This manuscript reviews recently described qPCR methods applied for foodborne bacteria detection, serving as economical, safe, and reliable alternatives for application in the food industry and control laboratories. Multiplex qPCR, which allows the simultaneous detection of more than one pathogen in one single reaction, saving considerable effort, time, and money, is emphasized in the article.

  9. Sampling and Pooling Methods for Capturing Herd Level Antibiotic Resistance in Swine Feces using qPCR and CFU Approaches

    DEFF Research Database (Denmark)

    Schmidt, Gunilla Veslemøy; Mellerup, Anders; Christiansen, Lasse Engbo

    2015-01-01

    The aim of this article was to define the sampling level and method combination that captures antibiotic resistance at pig herd level utilizing qPCR antibiotic resistance gene quantification and culture-based quantification of antibiotic resistant coliform indicator bacteria. Fourteen qPCR assays...... for commonly detected antibiotic resistance genes were developed, and used to quantify antibiotic resistance genes in total DNA from swine fecal samples that were obtained using different sampling and pooling methods. In parallel, the number of antibiotic resistant coliform indicator bacteria was determined...... in the same swine fecal samples. The results showed that the qPCR assays were capable of detecting differences in antibiotic resistance levels in individual animals that the coliform bacteria colony forming units (CFU) could not. Also, the qPCR assays more accurately quantified antibiotic resistance genes...

  10. Sources of Blood Meals of Sylvatic Triatoma guasayana near Zurima, Bolivia, Assayed with qPCR and 12S Cloning

    Science.gov (United States)

    Lucero, David E.; Ribera, Wilma; Pizarro, Juan Carlos; Plaza, Carlos; Gordon, Levi W.; Peña, Reynaldo; Morrissey, Leslie A.; Rizzo, Donna M.; Stevens, Lori

    2014-01-01

    Background In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. Methodology/Principal Findings We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). Conclusions/Significance We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors. PMID:25474154

  11. Sources of blood meals of sylvatic Triatoma guasayana near Zurima, Bolivia, assayed with qPCR and 12S cloning.

    Directory of Open Access Journals (Sweden)

    David E Lucero

    2014-12-01

    Full Text Available In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps. Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia.We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens, five for chicken (Gallus gallus and unicolored blackbird (Agelasticus cyanopus, and one for opossum (Monodelphis domestica. Using the qPCR assay we detected chicken (13 vectors, and human (14 vectors blood meals as well as an additional blood meal source, Canis sp. (4 vectors.We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

  12. Why the need for qPCR publication guidelines?--The case for MIQE.

    Science.gov (United States)

    Bustin, Stephen A

    2010-04-01

    The polymerase chain reaction (PCR) has matured from a labour- and time-intensive, low throughput qualitative gel-based technique to an easily automated, rapid, high throughput quantitative technology. Real-time quantitative PCR (qPCR) has become the benchmark technology for the detection and quantification of nucleic acids in a research, diagnostic, forensic and biotechnology setting. However, ill-assorted pre-assay conditions, poor assay design and inappropriate data analysis methodologies have resulted in the recurrent publication of data that are at best inconsistent and at worst irrelevant and even misleading. Furthermore, there is a lamentable lack of transparency of reporting, with the "Materials and Methods" sections of many publications, especially those with high impact factors, not fit for the purpose of evaluating the quality of any reported qPCR data. This poses a challenge to the integrity of the scientific literature, with serious consequences not just for basic research, but potentially calamitous implications for drug development and disease monitoring. These issues are being addressed by a set of guidelines that propose a minimum standard for the provision of information for qPCR experiments ("MIQE"). MIQE aims to restructure to-day's free-for-all qPCR methods into a more consistent format that will encourage detailed auditing of experimental detail, data analysis and reporting principles. General implementation of these guidelines is an important requisite for the maturing of qPCR into a robust, accurate and reliable nucleic acid quantification technology. Copyright 2009 Elsevier Inc. All rights reserved.

  13. Evaluation of Suitable Reference Genes for Normalization of qPCR Gene Expression Studies in Brinjal (Solanum melongena L.) During Fruit Developmental Stages.

    Science.gov (United States)

    Kanakachari, Mogilicherla; Solanke, Amolkumar U; Prabhakaran, Narayanasamy; Ahmad, Israr; Dhandapani, Gurusamy; Jayabalan, Narayanasamy; Kumar, Polumetla Ananda

    2016-02-01

    Brinjal/eggplant/aubergine is one of the major solanaceous vegetable crops. Recent availability of genome information greatly facilitates the fundamental research on brinjal. Gene expression patterns during different stages of fruit development can provide clues towards the understanding of its biological functions. Quantitative real-time PCR (qPCR) has become one of the most widely used methods for rapid and accurate quantification of gene expression. However, its success depends on the use of a suitable reference gene for data normalization. For qPCR analysis, a single reference gene is not universally suitable for all experiments. Therefore, reference gene validation is a crucial step. Suitable reference genes for qPCR analysis of brinjal fruit development have not been investigated so far. In this study, we have selected 21 candidate reference genes from the Brinjal (Solanum melongena) Plant Gene Indices database (compbio.dfci.harvard.edu/tgi/plant.html) and studied their expression profiles by qPCR during six different fruit developmental stages (0, 5, 10, 20, 30, and 50 days post anthesis) along with leaf samples of the Pusa Purple Long (PPL) variety. To evaluate the stability of gene expression, geNorm and NormFinder analytical softwares were used. geNorm identified SAND (SAND family protein) and TBP (TATA binding protein) as the best pairs of reference genes in brinjal fruit development. The results showed that for brinjal fruit development, individual or a combination of reference genes should be selected for data normalization. NormFinder identified Expressed gene (expressed sequence) as the best single reference gene in brinjal fruit development. In this study, we have identified and validated for the first time reference genes to provide accurate transcript normalization and quantification at various fruit developmental stages of brinjal which can also be useful for gene expression studies in other Solanaceae plant species.

  14. Importance Performance Analysis as a Trade Show Performance Evaluation and Benchmarking Tool

    OpenAIRE

    Tafesse, Wondwesen; Skallerud, Kåre; Korneliussen, Tor

    2010-01-01

    Author's accepted version (post-print). The purpose of this study is to introduce importance performance analysis as a trade show performance evaluation and benchmarking tool. Importance performance analysis considers exhibitors’ performance expectation and perceived performance in unison to evaluate and benchmark trade show performance. The present study uses data obtained from exhibitors of an international trade show to demonstrate how importance performance analysis can be used to eval...

  15. A multimarker qPCR platform for the characterisation of endometrial cancer.

    Science.gov (United States)

    Supernat, Anna; Łapińska-Szumczyk, Sylwia; Majewska, Hanna; Gulczyński, Jacek; Biernat, Wojciech; Wydra, Dariusz; Zaczek, Anna J

    2014-02-01

    The molecular background of endometrial cancer (EC) has not been fully elucidated. In the present study, we developed a quantitative PCR (qPCR) platform to examine the gene dosages of the potential molecular markers MGB1, TOP2A, ERBB1-4, MYC, CCND1, ESR1 and PI3K. The platform was applied in samples collected from 157 EC patients (stage I-IV) to verify its clinical utility and to examine the diagnostic and prognostic significance of the analysed biomarkers. The gene dosage pattern of the ERBB family and its downstream effectors PI3K and MYC showed particularly strong correlations with clinicopathological data. The ERBB PI3K/Akt pathway was upregulated in 31 (20%) of 156 cases. Activation of the ERBB PI3K/Akt pathway was positively correlated with a higher stage (p=0.001), higher grade (p=0.001), histological type II disease (p=0.0003) and metastases (p=0.02). The implemented hierarchical clustering revealed that cluster 2 was characterised by high copy numbers of the studied genes. Cluster 2 was associated with shorter overall survival (p=0.05). The platform was found to be a fast and simple method for direct analysis of the genes involved in uterine carcinogenesis, making it feasible for EC biology characterisation.

  16. Development and optimization of an efficient qPCR system for olive authentication in edible oils.

    Science.gov (United States)

    Alonso-Rebollo, Alba; Ramos-Gómez, Sonia; Busto, María D; Ortega, Natividad

    2017-10-01

    The applicability of qPCR in olive-oil authentication depends on the DNA obtained from the oils and the amplification primers. Therefore, four olive-specific amplification systems based on the trnL gene were designed (A-, B-, C- and D-trnL systems). The qPCR conditions, primer concentration and annealing temperature, were optimized. The systems were tested for efficiency and sensitivity to select the most suitable for olive oil authentication. The selected system (D-trnL) demonstrated specificity toward olive in contrast to other oleaginous species (canola, soybean, sunflower, maize, peanut and coconut) and showed high sensitivity in a broad linear dynamic range (LOD and LOQ: 500ng - 0.0625pg). This qPCR system enabled detection, with high sensitivity and specificity, of olive DNA isolated from oils processed in different ways, establishing it as an efficient method for the authentication of olive oil regardless of its category. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells.

    Science.gov (United States)

    Hussain, Musaddeq

    2015-11-10

    Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing of monoclonal antibody (mAb) drugs in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and must be controlled and monitored in order to ensure drug purity and safety. A conventional method for quantification of host residual DNA in drug requires extraction of DNA from the mAb drug substance with subsequent quantification of the extracted DNA using real-time PCR (qPCR). Here we report a method where the DNA extraction step is eliminated prior to qPCR. In this method, which we have named 'direct resDNA qPCR', the mAb drug substance is digested with a protease called KAPA in a 96-well PCR plate, the protease in the digest is then denatured at high temperature, qPCR reagents are added to the resultant reaction wells in the plate along with standards and controls in other wells of the same plate, and the plate subjected to qPCR for analysis of residual host DNA in the samples. This direct resDNA qPCR method for CHO is sensitive to 5.0fg of DNA with high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with four mAbs drug, two IgG1 and two IgG4. Both the purified drug substance as well as a number of process intermediate samples, e.g., bioreactor harvest, Protein A column eluate and ion-exchange column eluates were tested. This method simplifies the residual DNA quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Could the domestic cat play a significant role in the transmission of Echinococcus multilocularis? A study based on qPCR analysis of cat feces in a rural area in France

    Science.gov (United States)

    Knapp, Jenny; Combes, Benoît; Umhang, Gérald; Aknouche, Soufiane; Millon, Laurence

    2016-01-01

    Echinococcus multilocularis, a cestode parasite responsible for alveolar echinococcosis in humans, is often reported in Europe. It involves red foxes, domestic dogs, and domestic and wild cats as definitive hosts. The parasite infects small mammals and accidentally humans as intermediate hosts and develops in a similar way to a tumor, usually in the liver. Domestic animals are suspected of playing a role in parasite transmission, but this is rarely proven. Moreover, the role of domestic cats is thought to be small, because of experimental studies showing incomplete development of the parasite observed in their intestines. In the present study, we investigated copro-sampling performed in a rural and highly endemic area in Eastern France, on carnivore feces (n = 150). From these samples, the parasite was detected and identified by DNA analysis using quantitative PCR targeting part of a mitochondrial gene (Em-qPCR). Taeniid eggs were isolated from positive-Em-qPCR samples by flotation, and species identification was confirmed by sequencing on DNA extracts. From a total of 43 copro-samples from cats, four tested positive for E. multilocularis by the Em-qPCR. In two of these, we found parasite eggs that were identified as E. multilocularis. This finding was confirmed by sequencing, while one dog stool out of 61 collected was found to be positive, no egg was detectable. At the same time, 34% of fox stools tested positive for the parasite. The present study challenges the current idea that cats are only of minor significance in the E. multilocularis life cycle. PMID:27739398

  19. A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs.

    Science.gov (United States)

    Rampazzo, Rita de Cássia Pontello; Solcà, Manuela da Silva; Santos, Liliane Celestino Sales; Pereira, Lais de Novaes; Guedes, José Carlos Oliveira; Veras, Patrícia Sampaio Tavares; Fraga, Deborah Bittencourt Mothé; Krieger, Marco Aurélio; Costa, Alexandre Dias Tavares

    2017-11-15

    Canine visceral leishmaniasis (CVL) is a systemic disease caused by Leishmania infantum. A precise CVL diagnosis would allow for a faster and more specific treatment. Quantitative PCR (qPCR) is a sensitive and specific technique that can diagnose CVL and also monitor parasite load in the animal during the course of the infection or treatment. The aim of this study was to develop a ready-to-use (gelified and freezer-free) duplex qPCR for the identification of infected animals. We combined a new qPCR protocol that detects the canine 18S rRNA gene with an existing protocol for L. infantum kDNA detection, creating a duplex qPCR. This duplex method was then developed into a ready-to-use format. The performance of the duplex and singleplex reactions were compared in the traditional format (liquid and freezer-stored). Furthermore, the duplex qPCR performance was compared between the ready-to-use and traditional formats. The singleplex and new duplex qPCR exhibited the same detection limit in the traditional format (0.1 parasites/reaction). The ready-to-use format showed a detection limit of 1 parasite/reaction without affecting the reaction efficiency. The performance of the new qPCR protocol in the two formats was assessed using canine tissue samples from 82 dogs in an endemic CVL area that were previously characterized by standard serological and parasitological protocols. Splenic aspirates provided a higher rate of positivity (92.9%) followed by skin (50%) and blood (35.7%). The reported detection limits were observed for all tissues studied. Our results show that the amplification of L. infantum kDNA and canine DNA in a single tube, using either the traditional or ready-to-use format, exhibited the same diagnostic performance as amplification of the parasite kDNA alone. The detection of the host gene strengthens the qPCR results by confirming the presence and quality of DNA in the samples and the absence of polymerase inhibitors. The ready-to-use duplex qPCR format

  20. Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis.

    Science.gov (United States)

    Rojas, Alicia; Segev, Gilad; Markovics, Alex; Aroch, Itamar; Baneth, Gad

    2017-09-19

    Spirocerca lupi, the dog oesophageal nematode, causes a potentially fatal disease in domestic dogs, and is currently clinically diagnosed by coproscopy and oesophagoscopy. To date, a single molecular method, a semi-nested PCR, targeting the cox1 gene, has been developed to aid in the diagnosis of spirocercosis. The present study describes three novel high-resolution melt (HRM) quantitative PCR (qPCR) assays targeting fragments of the ITS1, 18S and cytb loci of S. lupi. The performance of these molecular assays in feces was compared to fecal flotation and to the previously described cox1 gene semi-nested PCR in 18 fecal samples from dogs with clinical oesophageal spirocercosis diagnosed by oesophagoscopy. The HRM qPCR for ITS1 and 18S were both able to detect 0.2 S. lupi eggs per gram (epg), while the HRM qPCR for the cytb and the semi-nested PCR for the cox1 detected 6 epg and 526 epg, respectively. Spirocerca lupi was detected in 61.1%, 44.4%, 27.8%, 11.1% and 5.6% of the fecal samples of dogs diagnosed with spirocercosis by using the ITS1 and 18S HRM qPCR assays, fecal flotation, cytb HRM qPCR and cox1 semi-nested PCR, respectively. All dogs positive by fecal flotation were also positive by ITS1 and 18S HRM qPCRs. Quantification of S. lupi eggs was successfully achieved in the HRM qPCRs and compared to the fecal flotation with no significant difference in the calculated concentrations between the HRM qPCRs that detected the 18S and ITS1 loci and the fecal flotation. The HRM qPCR for the 18S cross-amplified DNA from Toxocara canis and Toxascaris leonina. In contrast, the HRM qPCR for ITS1 did not cross-amplify DNA from other canine gastrointestinal parasites. This study presents two new molecular assays with significantly increased sensitivity for confirming and quantifying fecal S. lupi eggs. Of these, the HRM qPCR for ITS1 showed the best performance in terms of the limit of detection and absence of cross-amplification with other parasites. These assays will be

  1. Apparent polyploidization after gamma irradiation: pitfalls in the use of quantitative polymerase chain reaction (qPCR) for the estimation of mitochondrial and nuclear DNA gene copy numbers.

    Science.gov (United States)

    Kam, Winnie W Y; Lake, Vanessa; Banos, Connie; Davies, Justin; Banati, Richard

    2013-05-30

    Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization.

  2. MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS

    Directory of Open Access Journals (Sweden)

    Rodrigo Staggemeier

    2014-04-01

    Full Text Available A novel SYBR® green-real time polymerase chain reaction (qPCR was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

  3. Determination of the Physical Status (Episomal/Integral of HPV by qPCR in Esophageal Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Fariborz Soheili

    2017-03-01

    Full Text Available Background: In cervical cancer, the carcinogenic mechanism of human papillomavirus (HPV occurs through the integration of viral DNA into the host genome. This process initiates with a disruption in the E2 open reading frame (ORF of the viral genome. Disruption of E2 ORF results in an increased expression of the viral oncoproteins, E6 and E7, by removal of E2 suppression effect on their promoters. E6 and E7 interfere with the normal cell cycle by degrading the p53 and pRb tumor suppressor proteins, respectively. Objectives: The objective of this study was to determine the physical status (episomal/integral of HPV genome in esophageal squamous cell carcinoma (ESCC. Materials and Methods: The rate of copy numbers of E2 and E6 genes in HPV-18 and HPV-16 positive samples were analyzed by quantitative polymerase chain reaction (qPCR in order to assess the physical status (episomal/integral of HPV. DNA extracts from HeLa cell line were used as the positive control. Results: The E2 gene was detected in 1 sample, co-infected with HPV-16 and HPV-18. While, E6 gene was detected in all 11 HPV positive samples. The qPCR analysis showed the presence of integrated form of viral DNA in all HPV positive samples and only 1 mixed episomal-integrated form was detected. Conclusion: The presence of integrated forms of high risk HPV-16 and HPV-18 genomes might reflect a crucial process towards malignant transformation of ESCC.

  4. Proteome Analysis of the Plant Pathogenic Fungus Monilinia laxa Showing Host Specificity

    Directory of Open Access Journals (Sweden)

    Olja Bregar

    2012-01-01

    Full Text Available Brown rot fungus Monilinia laxa (Aderh. & Ruhl. Honey is an important plant pathogen in stone and pome fruits in Europe. We applied a proteomic approach in a study of M. laxa isolates obtained from apples and apricots in order to show the host specifity of the isolates and to analyse differentially expressed proteins in terms of host specifity, fungal pathogenicity and identification of candidate proteins for diagnostic marker development. Extracted mycelium proteins were separated by 2-D electrophoresis (2-DE and visualized by Coomassie staining in a non-linear pH range of 3–11 and Mr of 14–116 kDa. We set up a 2-DE reference map of M. laxa, resolving up to 800 protein spots, and used it for image analysis. The average technical coefficient of variance (13 % demonstrated a high reproducibility of protein extraction and 2-D polyacrylamide gel electrophoresis (2-DE PAGE, and the average biological coefficient of variance (23 % enabled differential proteomic analysis of the isolates. Multivariate statistical analysis (principal component analysis discriminated isolates from two different hosts, providing new data that support the existence of a M. laxa specialized form f. sp. mali, which infects only apples. A total of 50 differentially expressed proteins were further analyzed by LC-MS/MS, yielding 41 positive identifications. The identified mycelial proteins were functionally classified into 6 groups: amino acid and protein metabolism, energy production, carbohydrate metabolism, stress response, fatty acid metabolism and other proteins. Some proteins expressed only in apple isolates have been described as virulence factors in other fungi. The acetolactate synthase was almost 11-fold more abundant in apple-specific isolates than in apricot isolates and it might be implicated in M. laxa host specificity. Ten proteins identified only in apple isolates are potential candidates for the development of M. laxa host-specific diagnostic markers.

  5. Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue

    Directory of Open Access Journals (Sweden)

    Ravid Rivka

    2008-05-01

    Full Text Available Abstract Background Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD, Parkinson's disease (PD and dementia with Lewy bodies (DLB. Quantitative real-time PCR (RT qPCR is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB. Results The present study investigated the expression stability of 8 candidate reference genes, (ubiquitin C [UBC], tyrosine-3-monooxygenase [YWHAZ], RNA polymerase II polypeptide [RP II], hydroxymethylbilane synthase [HMBS], TATA box binding protein [TBP], β-2-microglobulin [B2M], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and succinate dehydrogenase complex-subunit A, [SDHA] in cerebellum and medial temporal gyrus of 6 AD, 6 PD, 6 DLB subjects, along with 5 matched controls using RT qPCR (TaqMan® Gene Expression Assays. Gene expression stability was analysed using geNorm to rank the candidate genes in order of decreasing stability in each disease group. The optimal number of genes recommended for accurate data normalization in each disease state was determined by pairwise variation analysis. Conclusion This study identified validated sets of mRNAs which would be appropriate for the normalization of RT qPCR data when studying gene expression in brain tissue of AD, PD, DLB and control subjects.

  6. Urine proteome analysis in Dent's disease shows high selective changes potentially involved in chronic renal damage.

    Science.gov (United States)

    Santucci, Laura; Candiano, Giovanni; Anglani, Franca; Bruschi, Maurizio; Tosetto, Enrica; Cremasco, Daniela; Murer, Luisa; D'Ambrosio, Chiara; Scaloni, Andrea; Petretto, Andrea; Caridi, Gianluca; Rossi, Roberta; Bonanni, Alice; Ghiggeri, Gian Marco

    2016-01-01

    Definition of the urinary protein composition would represent a potential tool for diagnosis in many clinical conditions. The use of new proteomic technologies allows detection of genetic and post-trasductional variants that increase sensitivity of the approach but complicates comparison within a heterogeneous patient population. Overall, this limits research of urinary biomarkers. Studying monogenic diseases are useful models to address this issue since genetic variability is reduced among first- and second-degree relatives of the same family. We applied this concept to Dent's disease, a monogenic condition characterised by low-molecular-weight proteinuria that is inherited following an X-linked trait. Results are presented here on a combined proteomic approach (LC-mass spectrometry, Western blot and zymograms for proteases and inhibitors) to characterise urine proteins in a large family (18 members, 6 hemizygous patients, 6 carrier females, and 6 normals) with Dent's diseases due to the 1070G>T mutation of the CLCN5. Gene ontology analysis on more than 1000 proteins showed that several clusters of proteins characterised urine of affected patients compared to carrier females and normal subjects: proteins involved in extracellular matrix remodelling were the major group. Specific analysis on metalloproteases and their inhibitors underscored unexpected mechanisms potentially involved in renal fibrosis. Studying with new-generation techniques for proteomic analysis of the members of a large family with Dent's disease sharing the same molecular defect allowed highly repetitive results that justify conclusions. Identification in urine of proteins actively involved in interstitial matrix remodelling poses the question of active anti-fibrotic drugs in Dent's patients. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. HIV-1 phylogenetic analysis shows HIV-1 transits through the meninges to brain and peripheral tissues.

    Science.gov (United States)

    Lamers, Susanna L; Gray, Rebecca R; Salemi, Marco; Huysentruyt, Leanne C; McGrath, Michael S

    2011-01-01

    Brain infection by the human immunodeficiency virus type 1 (HIV-1) has been investigated in many reports with a variety of conclusions concerning the time of entry and degree of viral compartmentalization. To address these diverse findings, we sequenced HIV-1 gp120 clones from a wide range of brain, peripheral and meningeal tissues from five patients who died from several HIV-1 associated disease pathologies. High-resolution phylogenetic analysis confirmed previous studies that showed a significant degree of compartmentalization in brain and peripheral tissue subpopulations. Some intermixing between the HIV-1 subpopulations was evident, especially in patients that died from pathologies other than HIV-associated dementia. Interestingly, the major tissue harboring virus from both the brain and peripheral tissues was the meninges. These results show that (1) HIV-1 is clearly capable of migrating out of the brain, (2) the meninges are the most likely primary transport tissues, and (3) infected brain macrophages comprise an important HIV reservoir during highly active antiretroviral therapy. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Retrospective Analysis of Patient Presentations at the Sydney (Australia) Royal Easter Show from 2012 to 2014.

    Science.gov (United States)

    Crabtree, Nathan; Mo, Shirley; Ong, Leon; Jegathees, Thuvarahan; Wei, Daniel; Fahey, David; Liu, Jia Jenny

    2017-04-01

    Introduction Comprehensive studies on the relationship between patient demographics and subsequent treatment and disposition at a single mass-gathering event are lacking. The Sydney Royal Easter Show (SRES; Sydney Olympic Park, New South Wales, Australia) is an annual, 14-day, agricultural mass-gathering event occurring around the Easter weekend, attracting more than 800,000 patrons per year. In this study, patient records from the SRES were analyzed to examine relationships between weather, crowd size, day of week, and demographics on treatment and disposition. This information would help to predict factors affecting patient treatment and disposition to guide ongoing training of first responders and to evaluate the appropriateness of staffing skills mix at future events. Hypothesis Patient demographics, environmental factors, and attendance would influence the nature and severity of presentations at the SRES, which would influence staffing requirements. A retrospective analysis of 4,141 patient record forms was performed for patients who presented to St John Ambulance (Australian Capital Territory, Australia) at the SRES between 2012 and 2014 inclusive. Presentation type was classified using a previously published minimum data set. Data on weather and crowd size were obtained from the Australian Bureau of Meteorology (Melbourne, Victoria, Australia) and the SRES, respectively. Statistical analyses were performed using SPSS v22 (IBM; Armonk, New York USA). Between 2012 to 2014, over 2.5 million people attended the SRES with 4,141 patients treated onsite. As expected, the majority of presentations were injuries (49%) and illnesses (46%). Although patient demographics and presentation types did not change over time, the duration of treatment increased. A higher proportion of patients were discharged to hospital or home compared to the proportion of patients discharged back to the event. Patients from rural/regional locations (accounting for 15% of all patients) were

  9. Quantification of B16 Melanoma Cells in Lungs Using Triplex Q-PCR - A New Approach to Evaluate Melanoma Cell Metastasis and Tumor Control

    DEFF Research Database (Denmark)

    Sorensen, Maria R; Pedersen, Sara R; Lindkvist, Annika

    2014-01-01

    of survival once the tumor has metastasized. In the present study, we have developed a new assay for quantitative analysis of B16 melanoma metastasis in the lungs. We have used a triplex Q-PCR to determine the expression of the melanoma genes GP100/Pmel and tyrosinase-related protein 2 (TRP-2), and found...... that B16.F10gp cells were detectable in the lungs as early as 2 hours after intravenous challenge with ≥10(4) tumor cells. When investigating the gene expression as a function of time, we observed a gradual decrease from 2-24 hours post tumor challenge followed by an increase of approximately 2 log10...... the outgrowth of subcutaneous melanomas. Results obtained using Q-PCR were compared to conventional counting of metastatic foci under a dissection microscope. A marked reduction in gene expression was observed in the lungs after vaccination with both vectors; however, Ad-Ii-GP showed the highest protection...

  10. A novel genotoxin-specific qPCR array based on the metabolically competent human HepaRG™ cell line as a rapid and reliable tool for improved in vitro hazard assessment.

    Science.gov (United States)

    Ates, Gamze; Mertens, Birgit; Heymans, Anja; Verschaeve, Luc; Milushev, Dimiter; Vanparys, Philippe; Roosens, Nancy H C; De Keersmaecker, Sigrid C J; Rogiers, Vera; Doktorova, Tatyana Y

    2018-04-01

    Although the value of the regulatory accepted batteries for in vitro genotoxicity testing is recognized, they result in a high number of false positives. This has a major impact on society and industries developing novel compounds for pharmaceutical, chemical, and consumer products, as afflicted compounds have to be (prematurely) abandoned or further tested on animals. Using the metabolically competent human HepaRG ™ cell line and toxicogenomics approaches, we have developed an upgraded, innovative, and proprietary gene classifier. This gene classifier is based on transcriptomic changes induced by 12 genotoxic and 12 non-genotoxic reference compounds tested at sub-cytotoxic concentrations, i.e., IC10 concentrations as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The resulting gene classifier was translated into an easy-to-handle qPCR array that, as shown by pathway analysis, covers several different cellular processes related to genotoxicity. To further assess the predictivity of the tool, a set of 5 known positive and 5 known negative test compounds for genotoxicity was evaluated. In addition, 2 compounds with debatable genotoxicity data were tested to explore how the qPCR array would classify these. With an accuracy of 100%, when equivocal results were considered positive, the results showed that combining HepaRG ™ cells with a genotoxin-specific qPCR array can improve (geno)toxicological hazard assessment. In addition, the developed qPCR array was able to provide additional information on compounds for which so far debatable genotoxicity data are available. The results indicate that the new in vitro tool can improve human safety assessment of chemicals in general by basing predictions on mechanistic toxicogenomics information.

  11. Dioscorea alata tuber proteome analysis shows over thirty dioscorin isoforms and novel tuber proteins.

    Science.gov (United States)

    Sharma, Shruti; Gupta, Ravi; Deswal, Renu

    2017-05-01

    In Dioscorea, dioscorin (31 kDa) is the major storage protein constituting 85% of the total tuber proteins. An integrated proteomic and biochemical approach was used to understand the physiological role of dioscorin in the two contrasting growth stages (germinating and mature tuber). HPLC analysis showed 3 fold reduction in mannitol and 12.88 and 1.24 fold increase in sucrose and maltose in the germinating tuber. A 1.8 and 3 fold increase in sucrose phosphate synthase and mannitol dehydrogenase activity respectively was observed in the germinating tuber while a 2 fold higher invertase probably lowers the sucrose accumulation in the mature tuber. SDS-PAGE and 2-D maps of the mature and germinating tubers confirmed depletion (more than 50%) of dioscorin on germination. Dioscorin was purified using ion exchange and gel filtration chromatography with 43.32 fold purification and 38.16 yield. Out of a trail of 35 spots at 31 kDa only 12 spots (identified as dioscorin isoforms) were present in the 2D gel of the purified fraction. To search for other tuber proteins besides dioscorin, the unbound fractions of DEAE column were analysed by 2DGE. DREB 1A, caffeic acid 3-O-methyltransferase and Rab-1 small GTP binding protein were identified perhaps for the first time in the Dioscorea proteome. The interactome analysis revealed these to be involved in oxidative stress, carotenoid synthesis and vesicular transport. This is perhaps the first attempt to identify tuber proteome (although limited) and to understand the physiological significance of these proteins. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  12. Genome-wide analysis of adolescent psychotic-like experiences shows genetic overlap with psychiatric disorders.

    Science.gov (United States)

    Pain, Oliver; Dudbridge, Frank; Cardno, Alastair G; Freeman, Daniel; Lu, Yi; Lundstrom, Sebastian; Lichtenstein, Paul; Ronald, Angelica

    2018-03-31

    This study aimed to test for overlap in genetic influences between psychotic-like experience traits shown by adolescents in the community, and clinically-recognized psychiatric disorders in adulthood, specifically schizophrenia, bipolar disorder, and major depression. The full spectra of psychotic-like experience domains, both in terms of their severity and type (positive, cognitive, and negative), were assessed using self- and parent-ratings in three European community samples aged 15-19 years (Final N incl. siblings = 6,297-10,098). A mega-genome-wide association study (mega-GWAS) for each psychotic-like experience domain was performed. Single nucleotide polymorphism (SNP)-heritability of each psychotic-like experience domain was estimated using genomic-relatedness-based restricted maximum-likelihood (GREML) and linkage disequilibrium- (LD-) score regression. Genetic overlap between specific psychotic-like experience domains and schizophrenia, bipolar disorder, and major depression was assessed using polygenic risk score (PRS) and LD-score regression. GREML returned SNP-heritability estimates of 3-9% for psychotic-like experience trait domains, with higher estimates for less skewed traits (Anhedonia, Cognitive Disorganization) than for more skewed traits (Paranoia and Hallucinations, Parent-rated Negative Symptoms). Mega-GWAS analysis identified one genome-wide significant association for Anhedonia within IDO2 but which did not replicate in an independent sample. PRS analysis revealed that the schizophrenia PRS significantly predicted all adolescent psychotic-like experience trait domains (Paranoia and Hallucinations only in non-zero scorers). The major depression PRS significantly predicted Anhedonia and Parent-rated Negative Symptoms in adolescence. Psychotic-like experiences during adolescence in the community show additive genetic effects and partly share genetic influences with clinically-recognized psychiatric disorders, specifically schizophrenia and

  13. Rapid quantification of plant-powdery mildew interactions by qPCR and conidiospore counts.

    Science.gov (United States)

    Weßling, Ralf; Panstruga, Ralph

    2012-08-31

    The powdery mildew disease represents a valuable patho-system to study the interaction between plant hosts and obligate biotrophic fungal pathogens. Numerous discoveries have been made on the basis of the quantitative evaluation of plant-powdery mildew interactions, especially in the context of hyper-susceptible and/or resistant plant mutants. However, the presently available methods to score the pathogenic success of powdery mildew fungi are laborious and thus not well suited for medium- to high-throughput analysis. Here we present two new protocols that allow the rapid quantitative assessment of powdery mildew disease development. One procedure depends on quantitative polymerase chain reaction (qPCR)-based evaluation of fungal biomass, while the other relies on the quantification of fungal conidiospores. We validated both techniques using the powdery mildew pathogen Golovinomyces orontii on a set of hyper-susceptible and resistant Arabidopsis thaliana mutants and found that both cover a wide dynamic range of one to two (qPCR) and four to five (quantification of conidia) orders of magnitude, respectively. The two approaches yield reproducible results and are easy to perform without specialized equipment. The qPCR and spore count assays rapidly and reproducibly quantify powdery mildew pathogenesis. Our methods are performed at later stages of infection and discern mutant phenotypes accurately. The assays therefore complement currently used procedures of powdery mildew quantification and can overcome some of their limitations. In addition, they can easily be adapted to other plant-powdery mildew patho-systems.

  14. Gut Transcriptome Analysis Shows Different Food Utilization Efficiency by the Grasshopper Oedaleous asiaticus (Orthoptera: Acrididae).

    Science.gov (United States)

    Huang, Xunbing; McNeill, Mark Richard; Ma, Jingchuan; Qin, Xinghu; Tu, Xiongbing; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

    2017-08-01

    Oedaleus asiaticus B. Bienko is a persistent pest occurring in north Asian grasslands. We found that O. asiaticus feeding on Stipa krylovii Roshev. had higher approximate digestibility (AD), efficiency of conversion of ingested food (ECI), and efficiency of conversion of digested food (ECD), compared with cohorts feeding on Leymus chinensis (Trin.) Tzvel, Artemisia frigida Willd., or Cleistogenes squarrosa (Trin.) Keng. Although this indicated high food utilization efficiency for S. krylovii, the physiological processes and molecular mechanisms underlying these biological observations are not well understood. Transcriptome analysis was used to examine how gene expression levels in O. asiaticus gut are altered by feeding on the four plant species. Nymphs (fifth-instar female) that fed on S. krylovii had the largest variation in gene expression profiles, with a total of 88 genes significantly upregulated compared with those feeding on the other three plants, mainly including nutrition digestive genes of protein, carbohydrate, and lipid digestion. GO and KEGG enrichment also showed that feeding S. krylovii could upregulate the nutrition digestion-related molecular function, biological process, and pathways. These changes in transcripts levels indicate that the physiological processes of activating nutrition digestive enzymes and metabolism pathways can well explain the high food utilization of S. krylovii by O. asiaticus. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. Factor analysis shows association between family activity environment and children's health behaviour.

    Science.gov (United States)

    Hendrie, Gilly A; Coveney, John; Cox, David N

    2011-12-01

    To characterise the family activity environment in a questionnaire format, assess the questionnaire's reliability and describe its predictive ability by examining the relationships between the family activity environment and children's health behaviours - physical activity, screen time and fruit and vegetable intake. This paper describes the creation of a tool, based on previously validated scales, adapted from the food domain. Data are from 106 children and their parents (Adelaide, South Australia). Factor analysis was used to characterise factors within the family activity environment. Pearson-Product Moment correlations between the family environment and child outcomes, controlling for demographic variation, were examined. Three factors described the family activity environment - parental activity involvement, opportunity for role modelling and parental support for physical activity - and explained 37.6% of the variance. Controlling for demographic factors, the scale was significantly correlated with children's health behaviour - physical activity (r=0.27), screen time (r=-0.24) and fruit and vegetable intake (r=0.34). The family activity environment questionnaire shows high internal consistency and moderate predictive ability. This study has built on previous research by taking a more comprehensive approach to measuring the family activity environment. This research suggests the family activity environment should be considered in family-based health promotion interventions. © 2011 The Authors. ANZJPH © 2011 Public Health Association of Australia.

  16. Counting mycobacteria in infected human cells and mouse tissue: a comparison between qPCR and CFU.

    Directory of Open Access Journals (Sweden)

    Sharad Pathak

    Full Text Available Due to the slow growth rate and pathogenicity of mycobacteria, enumeration by traditional reference methods like colony counting is notoriously time-consuming, inconvenient and biohazardous. Thus, novel methods that rapidly and reliably quantify mycobacteria are warranted in experimental models to facilitate basic research, development of vaccines and anti-mycobacterial drugs. In this study we have developed quantitative polymerase chain reaction (qPCR assays for simultaneous quantification of mycobacterial and host DNA in infected human macrophage cultures and in mouse tissues. The qPCR method cannot discriminate live from dead bacteria and found a 10- to 100-fold excess of mycobacterial genomes, relative to colony formation. However, good linear correlations were observed between viable colony counts and qPCR results from infected macrophage cultures (Pearson correlation coefficient [r] for M. tuberculosis = 0.82; M. a. avium = 0.95; M. a. paratuberculosis = 0.91. Regression models that predict colony counts from qPCR data in infected macrophages were validated empirically and showed a high degree of agreement with observed counts. Similar correlation results were also obtained in liver and spleen homogenates of M. a. avium infected mice, although the correlations were distinct for the early phase (< day 9 post-infection and later phase (≥ day 20 post-infection liver r = 0.94 and r = 0.91; spleen r = 0.91 and r = 0.87, respectively. Interestingly, in the mouse model the number of live bacteria as determined by colony counts constituted a much higher proportion of the total genomic qPCR count in the early phase (geometric mean ratio of 0.37 and 0.34 in spleen and liver, respectively, as compared to later phase of infection (geometric mean ratio of 0.01 in both spleen and liver. Overall, qPCR methods offer advantages in biosafety, time-saving, assay range and reproducibility compared to colony counting. Additionally, the duplex format allows

  17. Selection of reference genes for qPCR in hairy root cultures of peanut

    Directory of Open Access Journals (Sweden)

    Medrano Giuliana

    2011-10-01

    Full Text Available Abstract Background Hairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments. Results A total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from A. rhizogenes. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in Arabidopsis. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA treated root cultures than those treated with sodium acetate (NaOAc. Conclusions This work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (TBP2 and a gene encoding a ribosomal protein (RPL8C. A

  18. BIOMETRICAL ANALYSIS OF ARABIAN FOALS AND THEIR LATER SUCCESSES IN SHOWS AND ON RACE TRACK

    Directory of Open Access Journals (Sweden)

    M. PIESZKA

    2007-10-01

    Full Text Available Arabian horses are one of the most important products of Polish horse breeding.Many of them are International and World champions in shows; others are very wellknown as courageous race horses. To obtain such champions it is necessary to takeunder consideration many factors affecting the final results. The objective of thisstudy was to evaluate the effect of biometrical measurements of the foals at birthaccording to their future successes in shows and on racetrack. The study was carriedout on 143 horses winning in shows and in races. Body weight, height at withers,girth and canon circumferences taken at birth of these horses were analysed.Additionally coat colour was studied. All studied animals were divided into threegroups according to each measurement and the differences between such groupswere evaluated according points obtained for particular place at shows and place inraces. It was stated that horses heavier at birth and with higher girth circumferencegot more successes both at shows and on racetrack. Horses with higher height atwithers at birth were more successful in shows while animals with higher canoncircumference won oftener at race track. It was observed that the most courageousrace horses were bay while most champions were grey.

  19. Economic impact and market analysis of a special event: The Great New England Air Show

    Science.gov (United States)

    Rodney B. Warnick; David C. Bojanic; Atul Sheel; Apurv Mather; Deepak Ninan

    2010-01-01

    We conducted a post-event evaluation for the Great New England Air Show to assess its general economic impact and to refine economic estimates where possible. In addition to the standard economic impact variables, we examined travel distance, purchase decision involvement, event satisfaction, and frequency of attendance. Graphic mapping of event visitors' home ZIP...

  20. Radiographic analysis of odontogenic cysts showing displacement of the mandibular canal

    International Nuclear Information System (INIS)

    Cho, Bong Hae

    2003-01-01

    To assess the radiographic findings of odontogenic cysts showing displacement of the mandibular canal using computed tomographic (CT) and panoramic images. CT and panoramic images of 63 odontogenic cysts (27 dentigerous, 16 odontogenic keratocysts, and 20 radicular cysts) were analyzed to evaluate the following parameters: the dimension and shape of the cysts, and the effect of the cysts on the mandibular canal and cortical plates. Of the 63 cysts examined in the study, 35 (55.6%) showed inferior displacement of the mandibular canal and 46 (73.0%) showed perforation of the canal. There were statistically significant differenced between CT and panoramic images in depicting displacement and perforation of the mandibular canal. Cortical expansion was seen in 46 cases (73.0%) and cortical perforation in 23 cases (36.5%). The radicular cysts showed cortical expansion and perforation less frequently than the other cyst groups. Large cysts of mandible should be evaluated by multiplanar CT images in order to detect the mandibular canal and cortical bone involvement.

  1. Validation and comparison of reference genes for qPCR normalization of celery (Apium graveolens at different development stages

    Directory of Open Access Journals (Sweden)

    Meng-Yao eLi

    2016-03-01

    Full Text Available A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in qPCR analysis. Celery is one of the representative vegetable in Apiaceae and is widely cultivated and consumed in the world. However, no reports have been previously published concerning reference genes in celery. In this study, the expression stabilities of nine candidate reference genes in leaf blade and petiole at different development stages were evaluated using three statistics algorithms geNorm, NormFinder, and BestKeeper. Our results showed that TUB-B, TUB-A, and UBC were the most reference genes among all tested samples. GAPDH represented the maximum stability for most individual sample, while the UBQ displayed the minimum stability. To further validate the stability of reference genes, the expression pattern of AgAP2-2 was calculated by using the selected genes for normalization. In addition, the expression patterns of several development-related genes were studied using the selected reference gene. Our results will be beneficial for further studies on gene transcription in celery.

  2. Social network analysis shows direct evidence for social transmission of tool use in wild chimpanzees.

    Directory of Open Access Journals (Sweden)

    Catherine Hobaiter

    2014-09-01

    Full Text Available Social network analysis methods have made it possible to test whether novel behaviors in animals spread through individual or social learning. To date, however, social network analysis of wild populations has been limited to static models that cannot precisely reflect the dynamics of learning, for instance, the impact of multiple observations across time. Here, we present a novel dynamic version of network analysis that is capable of capturing temporal aspects of acquisition--that is, how successive observations by an individual influence its acquisition of the novel behavior. We apply this model to studying the spread of two novel tool-use variants, "moss-sponging" and "leaf-sponge re-use," in the Sonso chimpanzee community of Budongo Forest, Uganda. Chimpanzees are widely considered the most "cultural" of all animal species, with 39 behaviors suspected as socially acquired, most of them in the domain of tool-use. The cultural hypothesis is supported by experimental data from captive chimpanzees and a range of observational data. However, for wild groups, there is still no direct experimental evidence for social learning, nor has there been any direct observation of social diffusion of behavioral innovations. Here, we tested both a static and a dynamic network model and found strong evidence that diffusion patterns of moss-sponging, but not leaf-sponge re-use, were significantly better explained by social than individual learning. The most conservative estimate of social transmission accounted for 85% of observed events, with an estimated 15-fold increase in learning rate for each time a novice observed an informed individual moss-sponging. We conclude that group-specific behavioral variants in wild chimpanzees can be socially learned, adding to the evidence that this prerequisite for culture originated in a common ancestor of great apes and humans, long before the advent of modern humans.

  3. Social network analysis shows direct evidence for social transmission of tool use in wild chimpanzees.

    Science.gov (United States)

    Hobaiter, Catherine; Poisot, Timothée; Zuberbühler, Klaus; Hoppitt, William; Gruber, Thibaud

    2014-09-01

    Social network analysis methods have made it possible to test whether novel behaviors in animals spread through individual or social learning. To date, however, social network analysis of wild populations has been limited to static models that cannot precisely reflect the dynamics of learning, for instance, the impact of multiple observations across time. Here, we present a novel dynamic version of network analysis that is capable of capturing temporal aspects of acquisition--that is, how successive observations by an individual influence its acquisition of the novel behavior. We apply this model to studying the spread of two novel tool-use variants, "moss-sponging" and "leaf-sponge re-use," in the Sonso chimpanzee community of Budongo Forest, Uganda. Chimpanzees are widely considered the most "cultural" of all animal species, with 39 behaviors suspected as socially acquired, most of them in the domain of tool-use. The cultural hypothesis is supported by experimental data from captive chimpanzees and a range of observational data. However, for wild groups, there is still no direct experimental evidence for social learning, nor has there been any direct observation of social diffusion of behavioral innovations. Here, we tested both a static and a dynamic network model and found strong evidence that diffusion patterns of moss-sponging, but not leaf-sponge re-use, were significantly better explained by social than individual learning. The most conservative estimate of social transmission accounted for 85% of observed events, with an estimated 15-fold increase in learning rate for each time a novice observed an informed individual moss-sponging. We conclude that group-specific behavioral variants in wild chimpanzees can be socially learned, adding to the evidence that this prerequisite for culture originated in a common ancestor of great apes and humans, long before the advent of modern humans.

  4. Can Automated Facial Expression Analysis Show Differences Between Autism and Typical Functioning?

    Science.gov (United States)

    Borsos, Zsófia; Gyori, Miklos

    2017-01-01

    Exploratory analyses of emotional expressions using a commercially available facial expression recognition software are reported, from the context of a serious game for screening purposes. Our results are based on a comparative analysis of two matched groups of kindergarten-age children (high-functioning children with autism spectrum condition: n=13; typically developing children: n=13). Results indicate that this technology has the potential to identify autism-specific emotion expression features, and may play a role in affective diagnostic and assistive technologies.

  5. A micro-epidemiological analysis of febrile malaria in Coastal Kenya showing hotspots within hotspots.

    Science.gov (United States)

    Bejon, Philip; Williams, Thomas N; Nyundo, Christopher; Hay, Simon I; Benz, David; Gething, Peter W; Otiende, Mark; Peshu, Judy; Bashraheil, Mahfudh; Greenhouse, Bryan; Bousema, Teun; Bauni, Evasius; Marsh, Kevin; Smith, David L; Borrmann, Steffen

    2014-04-24

    Malaria transmission is spatially heterogeneous. This reduces the efficacy of control strategies, but focusing control strategies on clusters or 'hotspots' of transmission may be highly effective. Among 1500 homesteads in coastal Kenya we calculated (a) the fraction of febrile children with positive malaria smears per homestead, and (b) the mean age of children with malaria per homestead. These two measures were inversely correlated, indicating that children in homesteads at higher transmission acquire immunity more rapidly. This inverse correlation increased gradually with increasing spatial scale of analysis, and hotspots of febrile malaria were identified at every scale. We found hotspots within hotspots, down to the level of an individual homestead. Febrile malaria hotspots were temporally unstable, but 4 km radius hotspots could be targeted for 1 month following 1 month periods of surveillance.DOI: http://dx.doi.org/10.7554/eLife.02130.001. Copyright © 2014, Bejon et al.

  6. Analysis of allergen immunotherapy studies shows increased clinical efficacy in highly symptomatic patients

    DEFF Research Database (Denmark)

    Howarth, P; Malling, Hans-Jørgen; Molimard, M

    2011-01-01

    them. Thus, clinical studies of AIT can neither establish baseline symptom levels nor limit the enrolment of patients to those with the most severe symptoms. Allergen immunotherapy treatment effects are therefore diluted by patients with low symptoms for a particular pollen season. The objective...... tertiles). The difference observed in the average score in each tertile in active vs placebo-treated patients was assessed. This allowed an estimation of the efficacy that could be achieved in patients from sites where symptoms were high during the pollen season. Results:  An increased treatment effect...... of this analysis was to assess the effect possible to achieve with AIT in the groups of patients presenting the most severe allergic symptoms. Methods:  Study centres were grouped into tertiles categorized according to symptom severity scores observed in the placebo patients in each centre (low, middle and high...

  7. Meta-analysis of grain yield QTL identified during agricultural drought in grasses showed consensus.

    Science.gov (United States)

    Swamy, B P Mallikarjuna; Vikram, Prashant; Dixit, Shalabh; Ahmed, H U; Kumar, Arvind

    2011-06-16

    In the last few years, efforts have been made to identify large effect QTL for grain yield under drought in rice. However, identification of most precise and consistent QTL across the environments and genetics backgrounds is essential for their successful use in Marker-assisted Selection. In this study, an attempt was made to locate consistent QTL regions associated with yield increase under drought by applying a genome-wide QTL meta-analysis approach. The integration of 15 maps resulted in a consensus map with 531 markers and a total map length of 1821 cM. Fifty-three yield QTL reported in 15 studies were projected on a consensus map and meta-analysis was performed. Fourteen meta-QTL were obtained on seven chromosomes. MQTL1.2, MQTL1.3, MQTL1.4, and MQTL12.1 were around 700 kb and corresponded to a reasonably small genetic distance of 1.8 to 5 cM and they are suitable for use in marker-assisted selection (MAS). The meta-QTL for grain yield under drought coincided with at least one of the meta-QTL identified for root and leaf morphology traits under drought in earlier reports. Validation of major-effect QTL on a panel of random drought-tolerant lines revealed the presence of at least one major QTL in each line. DTY12.1 was present in 85% of the lines, followed by DTY4.1 in 79% and DTY1.1 in 64% of the lines. Comparative genomics of meta-QTL with other cereals revealed that the homologous regions of MQTL1.4 and MQTL3.2 had QTL for grain yield under drought in maize, wheat, and barley respectively. The genes in the meta-QTL regions were analyzed by a comparative genomics approach and candidate genes were deduced for grain yield under drought. Three groups of genes such as stress-inducible genes, growth and development-related genes, and sugar transport-related genes were found in clusters in most of the meta-QTL. Meta-QTL with small genetic and physical intervals could be useful in Marker-assisted selection individually and in combinations. Validation and comparative

  8. Meta-analysis of field experiments shows no change in racial discrimination in hiring over time.

    Science.gov (United States)

    Quillian, Lincoln; Pager, Devah; Hexel, Ole; Midtbøen, Arnfinn H

    2017-10-10

    This study investigates change over time in the level of hiring discrimination in US labor markets. We perform a meta-analysis of every available field experiment of hiring discrimination against African Americans or Latinos ( n = 28). Together, these studies represent 55,842 applications submitted for 26,326 positions. We focus on trends since 1989 ( n = 24 studies), when field experiments became more common and improved methodologically. Since 1989, whites receive on average 36% more callbacks than African Americans, and 24% more callbacks than Latinos. We observe no change in the level of hiring discrimination against African Americans over the past 25 years, although we find modest evidence of a decline in discrimination against Latinos. Accounting for applicant education, applicant gender, study method, occupational groups, and local labor market conditions does little to alter this result. Contrary to claims of declining discrimination in American society, our estimates suggest that levels of discrimination remain largely unchanged, at least at the point of hire.

  9. Global Trend Analysis of Multi-decade Soil Temperature Records Show Soils Resistant to Warming

    Science.gov (United States)

    Frey, S. D.; Jennings, K.

    2017-12-01

    Soil temperature is an important determinant of many subterranean ecological processes including plant growth, nutrient cycling, and carbon sequestration. Soils are expected to warm in response to increasing global surface temperatures; however, despite the importance of soil temperature to ecosystem processes, less attention has been given to examining changes in soil temperature over time. We collected long-term (> 20 years) soil temperature records from approximately 50 sites globally, many with multiple depths (5 - 100 cm), and examined temperature trends over the last few decades. For each site and depth we calculated annual summer means and conducted non-parametric Mann Kendall trend and Sen slope analysis to assess changes in summer soil temperature over the length of each time series. The mean summer soil temperature trend across all sites and depths was not significantly different than zero (mean = 0.004 °C year-1 ± 0.033 SD), suggesting that soils have not warmed over the observation period. Of the subset of sites that exhibit significant increases in temperature over time, site location, depth of measurement, time series length, and neither start nor end date seem to be related to trend strength. These results provide evidence that the thermal regime of soils may have a stronger buffering capacity than expected, having important implications for the global carbon cycle and feedbacks to climate change.

  10. Analysis of nonlocal phonon thermal conductivity simulations showing the ballistic to diffusive crossover

    Science.gov (United States)

    Allen, Philip B.

    2018-04-01

    Simulations [e.g., X. W. Zhou et al., Phys. Rev. B 79, 115201 (2009), 10.1103/PhysRevB.79.115201] show nonlocal effects of the ballistic/diffusive crossover. The local temperature has nonlinear spatial variation not contained in the local Fourier law j ⃗(r ⃗) =-κ ∇ ⃗T (r ⃗) . The heat current j ⃗(r ⃗) depends not just on the local temperature gradient ∇ ⃗T (r ⃗) but also on temperatures at points r⃗' within phonon mean free paths, which can be micrometers long. This paper uses the Peierls-Boltzmann transport theory in nonlocal form to analyze the spatial variation Δ T (r ⃗) . The relaxation-time approximation (RTA) is used because the full solution is very challenging. Improved methods of extrapolation to obtain the bulk thermal conductivity κ are proposed. Callaway invented an approximate method of correcting RTA for the q ⃗ (phonon wave vector or crystal momentum) conservation of N (Normal as opposed to Umklapp) anharmonic collisions. This method is generalized to the nonlocal case where κ (k ⃗) depends on the wave vector of the current j ⃗(k ⃗) and temperature gradient i k ⃗Δ T (k ⃗) .

  11. Metachronous metastasis- and survival-analysis show prognostic importance of lymphadenectomy for colon carcinomas

    Directory of Open Access Journals (Sweden)

    Laubert Tilman

    2012-03-01

    Full Text Available Abstract Background Lymphadenectomy is performed to assess patient prognosis and to prevent metastasizing. Recently, it was questioned whether lymph node metastases were capable of metastasizing and therefore, if lymphadenectomy was still adequate. We evaluated whether the nodal status impacts on the occurrence of distant metastases by analyzing a highly selected cohort of colon cancer patients. Methods 1,395 patients underwent surgery exclusively for colon cancer at the University of Lübeck between 01/1993 and 12/2008. The following exclusion criteria were applied: synchronous metastasis, R1-resection, prior/synchronous second carcinoma, age Results Five-year survival rates for TM + and TM- were 21% and 73%, respectively (p Conclusions Besides a higher T-category, a positive N-stage independently implies a higher probability to develop distant metastases and correlates with poor survival. Our data thus show a prognostic relevance of lymphadenectomy which should therefore be retained until conclusive studies suggest the unimportance of lmyphadenectomy.

  12. Rapid Identification and Quantification of Aureococcus anophagefferens by qPCR Method (Taqman) in the Qinhuangdao Coastal Area: A Region for Recurrent Brown Tide Breakout in China.

    Science.gov (United States)

    Wang, Li-Ping; Lei, Kun

    2016-12-01

    Since 2009, Aureococcus anophagefferens has caused brown tide to occur recurrently in Qinhuangdao coastal area, China. Because the algal cells of A. anophagefferens are so tiny (~3 µm) that it is very hard to identify exactly under a microscope for natural water samples, it is very urgent to develop a method for efficient and continuous monitoring. Here specific primers and Taqman probe are designed to develop a real-time quantitative PCR (qPCR) method for identification and quantification continually. The algal community and cell abundance of A. anophagefferens in the study area (E 119°20'-119°50' and N 39°30'-39°50') from April to October in 2013 are detected by pyrosequencing, and are used to validate the specification and precision of qPCR method for natural samples. Both pyrosequencing and qPCR shows that the targeted cells are present only in May, June and July, and the cell abundance are July > June > May. Although there are various algal species including dinoflagellata, diatom, Cryptomonadales, Chrysophyceae and Chlorophyta living in the natural seawater simultaneously, no disturbance happens to qPCR method. This qPCR method could detect as few as 10 targeted cells, indicating it is able to detect the algal cells at pre-bloom levels. Therefore, qPCR with Taqman probe provides a powerful and sensitive method to monitor the brown tide continually in Qinhuangdao coastal area, China. The results provide a necessary technology support for forecasting the brown tide initiation, in China.

  13. A comparative study of digital PCR and real-time qPCR for the detection and quantification of HPV mRNA in sentinel lymph nodes of cervical cancer patients.

    Science.gov (United States)

    Carow, Katrin; Read, Christina; Häfner, Norman; Runnebaum, Ingo B; Corner, Adam; Dürst, Matthias

    2017-10-30

    Qualitative analyses showed that the presence of HPV mRNA in sentinel lymph nodes of cervical cancer patients with pN0 status is associated with significantly decreased recurrence free survival. To further address the clinical potential of the strategy and to define prognostic threshold levels it is necessary to use a quantitative assay. Here, we compare two methods of quantification: digital PCR and standard quantitative PCR. Serial dilutions of 5 ng-5 pg RNA (≙ 500-0.5 cells) of the cervical cancer cell line SiHa were prepared in 5 µg RNA of the HPV-negative human keratinocyte cell line HaCaT. Clinical samples consisted of 10 sentinel lymph nodes with varying HPV transcript levels. Reverse transcription of total RNA (5 µg RNA each) was performed in 100 µl and cDNA aliquots were analyzed by qPCR and dPCR. Digital PCR was run in the RainDrop ® Digital PCR system (RainDance Technologies) using a probe-based detection of HPV E6/E7 cDNA PCR products with 11 µl template. qPCR was done using a Rotor Gene Q 5plex HRM (Qiagen) amplifying HPV E6/E7 cDNA in a SYBR Green format with 1 µl template. For the analysis of both, clinical samples and serial dilution samples, dPCR and qPCR showed comparable sensitivity. With regard to reproducibility, both methods differed considerably, especially for low template samples. Here, we found with qPCR a mean variation coefficient of 126% whereas dPCR enabled a significantly lower mean variation coefficient of 40% (p = 0.01). Generally, we saw with dPCR a substantial reduction of subsampling errors, which most likely reflects the large cDNA amounts available for analysis. Compared to real-time PCR, dPCR shows higher reliability. Thus, our HPV mRNA dPCR assay holds promise for the clinical evaluation of occult tumor cells in histologically tumor-free lymph nodes in future studies.

  14. HF183/BFDrev and HumM2 qPCR data

    Data.gov (United States)

    U.S. Environmental Protection Agency — Concentration estimates for HF183/BFDrev and HumM2 qPCR genetic markers in raw sewage collected from 54 geographic locations across the United States. This dataset...

  15. Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

    Science.gov (United States)

    Ogrean, Christy; Jackson, Ben; Covino, James

    2010-01-01

    The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts. Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process. Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method. PMID:20567213

  16. Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies.

    Science.gov (United States)

    Arvia, Rosaria; Sollai, Mauro; Pierucci, Federica; Urso, Carmelo; Massi, Daniela; Zakrzewska, Krystyna

    2017-08-01

    Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma and high viral load in the skin was proposed as a risk factor for the occurrence of this tumour. MCPyV DNA was detected, with lower frequency, in different skin cancers but since the viral load was usually low, the real prevalence of viral DNA could be underestimated. To evaluate the performance of two assays (qPCR and ddPCR) for MCPyV detection and quantification in formalin fixed paraffin embedded (FFPE) tissue samples. Both assays were designed to simultaneous detection and quantification of both MCPyV as well as house-keeping DNA in clinical samples. The performance of MCPyV quantification was investigated using serial dilutions of cloned target DNA. We also evaluated the applicability of both tests for the analysis of 76 FFPE cutaneous biopsies. The two approaches resulted equivalent with regard to the reproducibility and repeatability and showed a high degree of linearity in the dynamic range tested in the present study. Moreover, qPCR was able to quantify ≥10 5 copies per reaction, while the upper limit of ddPCR was 10 4 copies. There was not significant difference between viral load measured by the two methods The detection limit of both tests was 0,15 copies per reaction, however, the number of positive samples obtained by ddPCR was higher than that obtained by qPCR (45% and 37% respectively). The ddPCR represents a better method for detection of MCPyV in FFPE biopsies, mostly these containing low copies number of viral genome. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Application of real-time PCR (qPCR) for characterization of microbial populations and type of milk in dairy food products.

    Science.gov (United States)

    Agrimonti, Caterina; Bottari, Benedetta; Sardaro, Maria Luisa Savo; Marmiroli, Nelson

    2017-09-08

    Dairy foods represent an important sector of the food market for their nutritional qualities and their organoleptic characteristics, which are often linked to tradition and to region. These products are typically protected by labels such as PDO (Protected Designation of Origin) and PGI (Protected Geographical Indication). Real-time PCR (qPCR) is a fundamental tool in "Food Genomics;" a discipline concerned with the residual DNA in food, which, alongside traditional physical and chemical methods, is frequently used to determine product safety, quality and authenticity. Compared to conventional or "end-point" PCR, qPCR incorporates continuous monitoring of reaction progress, thereby enabling quantification of target DNA. This review describes qPCR applications to the analysis of microbiota, and to the identification of the animal species source of milk from which dairy products have been made. These are important aspects for ensuring safety and authenticity. The various applications of qPCR are discussed, as well as advantages and disadvantages in comparison with other analytical methods.

  18. A performance study on three qPCR quantification kits and their compatibilities with the 6-dye DNA profiling systems.

    Science.gov (United States)

    Lin, Sze-Wah; Li, Christina; Ip, Stephen C Y

    2018-03-01

    DNA quantification plays an integral role in forensic DNA profiling. Not only does it estimate the total amount of amplifiable human autosomal and male DNA to ensure optimal amplification of target DNA for subsequent analysis, but also assesses the extraction efficiency and purity of the DNA extract. Latest DNA quantification systems even offer an estimate for the degree of DNA degradation in a sample. Here, we report the performance of three new generation qPCR kits, namely Investigator ® Quantiplex HYres Kit from QIAGEN, Quantifiler ® Trio DNA Quantification Kit from Applied Biosystems™, and PowerQuant ® System from Promega, and their compatibilities with three 6-dye DNA profiling systems. Our results have demonstrated that all three kits generate standard curves with satisfactory consistency and reproducibility, and are capable of screening out traces of male DNA in the presence of 30-fold excess of female DNA. They also exhibit a higher tolerance to PCR inhibition than Quantifiler ® Human DNA Quantification Kit from Applied Biosystems™ in autosomal DNA quantification. PowerQuant ® , as compared to Quantiplex HYres and Quantifiler ® Trio, shows a better precision for both autosomal and male DNA quantifications. Quantifiler ® Trio and PowerQuant ® in contrast to Quantiplex HYres offer better correlations with lower discrepancies between autosomal and male DNA quantification, and their additional degradation index features provide a detection platform for inhibited and/or degraded DNA template. Regarding the compatibility between these quantification and profiling systems: (1) both Quantifiler ® Trio and PowerQuant ® work well with GlobalFiler and Fusion 6C, allowing a fairly accurate prediction of their DNA typing results based on the quantification values; (2) Quantiplex HYres offers a fairly reliable IPC system for detecting any potential inhibitions on Investigator 24plex, whereas Quantifiler ® Trio and PowerQuant ® suit better for Global

  19. Cryptosporidium diagnosis by qPCR in cats at Rio de Janeiro state, Brazil

    Directory of Open Access Journals (Sweden)

    Lara Patrícia Santos Carrasco

    2016-11-01

    Full Text Available ABSTRACT. Carrasco L.P.S., Oliveira R.L.S., Moreira C.M.R., Santos C.R.G.R., Corgozinho K.B. & Souza H.J.M. [Cryptosporidium diagnosis by qPCR in cats at Rio de Janeiro state, Brazil.] Diagnóstico de Cryptosporidium spp. pela técnica de qPCR em gatos no estado do Rio de Janeiro, Brasil. Revista Brasileira de Medicina Veterinária, 38(Supl.:22-26, 2016. Programa de Pós-Graduação em MedicinaVeteriná- ria, Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, BR-465, Km 7, Seropédica, RJ 23890-000, Brasil. E-mail: carrasco.lara@gmail.com Cryptosporidium spp. is recognized as an important etiologic agent of diarrhea in many countries. The aim of this study was to detect the presence of DNA of the parasite Cryptosporidium spp. in feces of cats with history of chronic diarrhea attended in the Feline Medicine Sector of the Veterinary Hospital of the Federal Rural University of Rio de Janeiro, by the polymerase chain reaction technique in real time (RT-PCR. In this study, 100 animals were admitted, of any breed or sex and from 8 weeks of age. As inclusion criteria, patients had to have diarrhea history for more than three weeks, with little success of clinical response to previously established therapies. From the samples obtained by collecting via washing the animal colon and spontaneous defecation, methods of direct examination of the feces, centrifugal flotation technique and real-time PCR were carried out. Of all cats selected for this study, 10% showed infection by Cryptosporidium spp. Most positive animals were aged over one year (70% and only 30% had up to one year old. Cats were 50% purebred and 50% were domestic short hair cats. The clinical signs presented by these cats at the time of consultation were diarrhea (60% and prolapsed rectum (40%. Four animals had co-infections with other enteropathogens (40%, such as Giardia, Toxocara sp. or Tritrichomonas fetus alone or combined. We concluded that infection by

  20. Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR

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    Roman Wölfel

    2012-08-01

    Full Text Available Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

  1. Legionella detection by culture and qPCR: Comparing apples and oranges.

    Science.gov (United States)

    Whiley, Harriet; Taylor, Michael

    2016-01-01

    Legionella spp. are the causative agent of Legionnaire's disease and an opportunistic pathogen of significant public health concern. Identification and quantification from environmental sources is crucial for identifying outbreak origins and providing sufficient information for risk assessment and disease prevention. Currently there are a range of methods for Legionella spp. quantification from environmental sources, but the two most widely used and accepted are culture and real-time polymerase chain reaction (qPCR). This paper provides a review of these two methods and outlines their advantages and limitations. Studies from the last 10 years which have concurrently used culture and qPCR to quantify Legionella spp. from environmental sources have been compiled. 26/28 studies detected Legionella at a higher rate using qPCR compared to culture, whilst only one study detected equivalent levels of Legionella spp. using both qPCR and culture. Aggregating the environmental samples from all 28 studies, 2856/3967 (72%) tested positive for the presence of Legionella spp. using qPCR and 1331/3967 (34%) using culture. The lack of correlation between methods highlights the need to develop an acceptable standardized method for quantification that is sufficient for risk assessment and management of this human pathogen.

  2. Proteomic Analysis Shows Constitutive Secretion of MIF and p53-associated Activity of COX-2−/− Lung Fibroblasts

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    Mandar Dave

    2017-12-01

    Full Text Available The differential expression of two closelyassociated cyclooxygenase isozymes, COX-1 and COX-2, exhibited functions beyond eicosanoid metabolism. We hypothesized that COX-1 or COX-2 knockout lung fibroblasts may display altered protein profiles which may allow us to further differentiate the functional roles of these isozymes at the molecular level. Proteomic analysis shows constitutive production of macrophage migration inhibitory factor (MIF in lung fibroblasts derived from COX-2−/− but not wild-type (WT or COX-1−/− mice. MIF was spontaneously released in high levels into the extracellular milieu of COX2−/− fibroblasts seemingly from the preformed intracellular stores, with no change in the basal gene expression of MIF. The secretion and regulation of MIF in COX-2−/− was “prostaglandin-independent.” GO analysis showed that concurrent with upregulation of MIF, there is a significant surge in expression of genes related to fibroblast growth, FK506 binding proteins, and isomerase activity in COX-2−/− cells. Furthermore, COX-2−/− fibroblasts also exhibit a significant increase in transcriptional activity of various regulators, antagonists, and co-modulators of p53, as well as in the expression of oncogenes and related transcripts. Integrative Oncogenomics Cancer Browser (IntroGen analysis shows downregulation of COX-2 and amplification of MIF and/or p53 activity during development of glioblastomas, ependymoma, and colon adenomas. These data indicate the functional role of the MIF-COX-p53 axis in inflammation and cancer at the genomic and proteomic levels in COX-2-ablated cells. This systematic analysis not only shows the proinflammatory state but also unveils a molecular signature of a pro-oncogenic state of COX-1 in COX-2 ablated cells.

  3. Genomic analysis of codon usage shows influence of mutation pressure, natural selection, and host features on Marburg virus evolution.

    Science.gov (United States)

    Nasrullah, Izza; Butt, Azeem M; Tahir, Shifa; Idrees, Muhammad; Tong, Yigang

    2015-08-26

    The Marburg virus (MARV) has a negative-sense single-stranded RNA genome, belongs to the family Filoviridae, and is responsible for several outbreaks of highly fatal hemorrhagic fever. Codon usage patterns of viruses reflect a series of evolutionary changes that enable viruses to shape their survival rates and fitness toward the external environment and, most importantly, their hosts. To understand the evolution of MARV at the codon level, we report a comprehensive analysis of synonymous codon usage patterns in MARV genomes. Multiple codon analysis approaches and statistical methods were performed to determine overall codon usage patterns, biases in codon usage, and influence of various factors, including mutation pressure, natural selection, and its two hosts, Homo sapiens and Rousettus aegyptiacus. Nucleotide composition and relative synonymous codon usage (RSCU) analysis revealed that MARV shows mutation bias and prefers U- and A-ended codons to code amino acids. Effective number of codons analysis indicated that overall codon usage among MARV genomes is slightly biased. The Parity Rule 2 plot analysis showed that GC and AU nucleotides were not used proportionally which accounts for the presence of natural selection. Codon usage patterns of MARV were also found to be influenced by its hosts. This indicates that MARV have evolved codon usage patterns that are specific to both of its hosts. Moreover, selection pressure from R. aegyptiacus on the MARV RSCU patterns was found to be dominant compared with that from H. sapiens. Overall, mutation pressure was found to be the most important and dominant force that shapes codon usage patterns in MARV. To our knowledge, this is the first detailed codon usage analysis of MARV and extends our understanding of the mechanisms that contribute to codon usage and evolution of MARV.

  4. A reliable and feasible qPCR strategy for titrating AAV vectors.

    Science.gov (United States)

    Wang, Feng; Cui, Xiuling; Wang, Mingxi; Xiao, Weidong; Xu, Ruian

    2013-07-05

    Previous studies have revealed that traditional real-time quantitative PCR (qPCR) underestimates adeno-associated virus (AAV) titer. Because the inverted terminal repeat (ITR) exists in all AAV vectors, the only remaining element from the wild genome could form special configurations to interfere with qPCR titration. To solve this problem, a modified and universal qPCR method was tested and established. In this work, there was a great variation in titration of ssAAV2-EGFP (Enhanced Green Fluorescence Protein) and scAAV2-EGFP genome by traditional qPCR. For ssAAV2-EGFP, the highest titer was found by using the targeting EGFP primers and the lowest titer was measured by those targeting bovine growth hormone polyA element (pBGH) primers. Experimental data were reverse for ssAAV2-EGFP and scAAV2-EGFP. Here we report an improved and universal SmaI qPCR method, based on cleaving all ITRs in AAV2 genome by SmaI with several advantages: (1) impact of all ITRs in ssAAV2 and scAAV2 was dismissed; (2) titers increased remarkably, up to 7-fold, especially for scAAV2; (3) the variation of titers was reduced when different primers were applied. A similar phenomenon was also observed in other ssAAV2 and scAAV2 products when the range of titration was at 3×107 to 7×109 V.G/µl in this study. This modified qPCR strategy can increase rAAV' titer and reduce titration variance, possibly become a universal method for titrating AAV vectors.

  5. Recruitment Methods and Show Rates to a Prostate Cancer Early Detection Program for High-Risk Men: A Comprehensive Analysis

    Science.gov (United States)

    Giri, Veda N.; Coups, Elliot J.; Ruth, Karen; Goplerud, Julia; Raysor, Susan; Kim, Taylor Y.; Bagden, Loretta; Mastalski, Kathleen; Zakrzewski, Debra; Leimkuhler, Suzanne; Watkins-Bruner, Deborah

    2009-01-01

    Purpose Men with a family history (FH) of prostate cancer (PCA) and African American (AA) men are at higher risk for PCA. Recruitment and retention of these high-risk men into early detection programs has been challenging. We report a comprehensive analysis on recruitment methods, show rates, and participant factors from the Prostate Cancer Risk Assessment Program (PRAP), which is a prospective, longitudinal PCA screening study. Materials and Methods Men 35–69 years are eligible if they have a FH of PCA, are AA, or have a BRCA1/2 mutation. Recruitment methods were analyzed with respect to participant demographics and show to the first PRAP appointment using standard statistical methods Results Out of 707 men recruited, 64.9% showed to the initial PRAP appointment. More individuals were recruited via radio than from referral or other methods (χ2 = 298.13, p < .0001). Men recruited via radio were more likely to be AA (p<0.001), less educated (p=0.003), not married or partnered (p=0.007), and have no FH of PCA (p<0.001). Men recruited via referrals had higher incomes (p=0.007). Men recruited via referral were more likely to attend their initial PRAP visit than those recruited by radio or other methods (χ2 = 27.08, p < .0001). Conclusions This comprehensive analysis finds that radio leads to higher recruitment of AA men with lower socioeconomic status. However, these are the high-risk men that have lower show rates for PCA screening. Targeted motivational measures need to be studied to improve show rates for PCA risk assessment for these high-risk men. PMID:19758657

  6. Elasmobranch qPCR reference genes: a case study of hypoxia preconditioned epaulette sharks

    Directory of Open Access Journals (Sweden)

    Ashton Kevin J

    2010-04-01

    Full Text Available Abstract Background Elasmobranch fishes are an ancient group of vertebrates which have high potential as model species for research into evolutionary physiology and genomics. However, no comparative studies have established suitable reference genes for quantitative PCR (qPCR in elasmobranchs for any physiological conditions. Oxygen availability has been a major force shaping the physiological evolution of vertebrates, especially fishes. Here we examined the suitability of 9 reference candidates from various functional categories after a single hypoxic insult or after hypoxia preconditioning in epaulette shark (Hemiscyllium ocellatum. Results Epaulette sharks were caught and exposed to hypoxia. Tissues were collected from 10 controls, 10 individuals with single hypoxic insult and 10 individuals with hypoxia preconditioning (8 hypoxic insults, 12 hours apart. We produced sequence information for reference gene candidates and monitored mRNA expression levels in four tissues: cerebellum, heart, gill and eye. The stability of the genes was examined with analysis of variance, geNorm and NormFinder. The best ranking genes in our study were eukaryotic translation elongation factor 1 beta (eef1b, ubiquitin (ubq and polymerase (RNA II (DNA directed polypeptide F (polr2f. The performance of the ribosomal protein L6 (rpl6 was tissue-dependent. Notably, in one tissue the analysis of variance indicated statistically significant differences between treatments for genes that were ranked as the most stable candidates by reference gene software. Conclusions Our results indicate that eef1b and ubq are generally the most suitable reference genes for the conditions and tissues in the present epaulette shark studies. These genes could also be potential reference gene candidates for other physiological studies examining stress in elasmobranchs. The results emphasise the importance of inter-group variation in reference gene evaluation.

  7. Chitinase mRNA Levels Determined by QPCR in Crab-Eating Monkey (Macaca fascicularis) Tissues: Species-Specific Expression of Acidic Mammalian Chitinase and Chitotriosidase.

    Science.gov (United States)

    Uehara, Maiko; Tabata, Eri; Ishii, Kazuhiro; Sawa, Akira; Ohno, Misa; Sakaguchi, Masayoshi; Matoska, Vaclav; Bauer, Peter O; Oyama, Fumitaka

    2018-05-09

    Mice and humans express two active chitinases: acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Both chitinases are thought to play important roles in specific pathophysiological conditions. The crab-eating monkey ( Macaca fascicularis ) is one of the most frequently used nonhuman primate models in basic and applied biomedical research. Here, we performed gene expression analysis of two chitinases in normal crab-eating monkey tissues by way of quantitative real-time polymerase chain reaction (qPCR) using a single standard DNA molecule. Levels of AMCase and CHIT1 messenger RNAs (mRNAs) were highest in the stomach and the lung, respectively, when compared to other tissues. Comparative gene expression analysis of mouse, monkey, and human using monkey⁻mouse⁻human hybrid standard DNA showed that the AMCase mRNA levels were exceptionally high in mouse and monkey stomachs while very low in the human stomach. As for the CHIT1 mRNA, we detected higher levels in the monkey lung when compared with those of mouse and human. The differences of mRNA expression between the species in the stomach tissues were basically reflecting the levels of the chitinolytic activities. These results indicate that gene expression of AMCase and CHIT1 differs between mammalian species and requiring special attention in handling data in chitinase-related studies in particular organisms.

  8. From root to fruit: RNA-Seq analysis shows that arbuscular mycorrhizal symbiosis may affect tomato fruit metabolism.

    Science.gov (United States)

    Zouari, Inès; Salvioli, Alessandra; Chialva, Matteo; Novero, Mara; Miozzi, Laura; Tenore, Gian Carlo; Bagnaresi, Paolo; Bonfante, Paola

    2014-03-21

    Tomato (Solanum lycopersicum) establishes a beneficial symbiosis with arbuscular mycorrhizal (AM) fungi. The formation of the mycorrhizal association in the roots leads to plant-wide modulation of gene expression. To understand the systemic effect of the fungal symbiosis on the tomato fruit, we used RNA-Seq to perform global transcriptome profiling on Moneymaker tomato fruits at the turning ripening stage. Fruits were collected at 55 days after flowering, from plants colonized with Funneliformis mosseae and from control plants, which were fertilized to avoid responses related to nutrient deficiency. Transcriptome analysis identified 712 genes that are differentially expressed in fruits from mycorrhizal and control plants. Gene Ontology (GO) enrichment analysis of these genes showed 81 overrepresented functional GO classes. Up-regulated GO classes include photosynthesis, stress response, transport, amino acid synthesis and carbohydrate metabolism functions, suggesting a general impact of fungal symbiosis on primary metabolisms and, particularly, on mineral nutrition. Down-regulated GO classes include cell wall, metabolism and ethylene response pathways. Quantitative RT-PCR validated the RNA-Seq results for 12 genes out of 14 when tested at three fruit ripening stages, mature green, breaker and turning. Quantification of fruit nutraceutical and mineral contents produced values consistent with the expression changes observed by RNA-Seq analysis. This RNA-Seq profiling produced a novel data set that explores the intersection of mycorrhization and fruit development. We found that the fruits of mycorrhizal plants show two transcriptomic "signatures": genes characteristic of a climacteric fleshy fruit, and genes characteristic of mycorrhizal status, like phosphate and sulphate transporters. Moreover, mycorrhizal plants under low nutrient conditions produce fruits with a nutrient content similar to those from non-mycorrhizal plants under high nutrient conditions

  9. Systems level analysis of systemic sclerosis shows a network of immune and profibrotic pathways connected with genetic polymorphisms.

    Directory of Open Access Journals (Sweden)

    J Matthew Mahoney

    2015-01-01

    Full Text Available Systemic sclerosis (SSc is a rare systemic autoimmune disease characterized by skin and organ fibrosis. The pathogenesis of SSc and its progression are poorly understood. The SSc intrinsic gene expression subsets (inflammatory, fibroproliferative, normal-like, and limited are observed in multiple clinical cohorts of patients with SSc. Analysis of longitudinal skin biopsies suggests that a patient's subset assignment is stable over 6-12 months. Genetically, SSc is multi-factorial with many genetic risk loci for SSc generally and for specific clinical manifestations. Here we identify the genes consistently associated with the intrinsic subsets across three independent cohorts, show the relationship between these genes using a gene-gene interaction network, and place the genetic risk loci in the context of the intrinsic subsets. To identify gene expression modules common to three independent datasets from three different clinical centers, we developed a consensus clustering procedure based on mutual information of partitions, an information theory concept, and performed a meta-analysis of these genome-wide gene expression datasets. We created a gene-gene interaction network of the conserved molecular features across the intrinsic subsets and analyzed their connections with SSc-associated genetic polymorphisms. The network is composed of distinct, but interconnected, components related to interferon activation, M2 macrophages, adaptive immunity, extracellular matrix remodeling, and cell proliferation. The network shows extensive connections between the inflammatory- and fibroproliferative-specific genes. The network also shows connections between these subset-specific genes and 30 SSc-associated polymorphic genes including STAT4, BLK, IRF7, NOTCH4, PLAUR, CSK, IRAK1, and several human leukocyte antigen (HLA genes. Our analyses suggest that the gene expression changes underlying the SSc subsets may be long-lived, but mechanistically interconnected

  10. Longitudinal monitoring of bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

    Science.gov (United States)

    Both veterinarians caring for bottlenose dolphins (Tursiops truncatus) in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin health. Quantitative PCR (qPCR) can be used to assess cytokine expression patterns of peripheral blood m...

  11. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  12. The need for transparency and good practices in the qPCR literature

    DEFF Research Database (Denmark)

    Bustin, Stephen A; Benes, Vladimir; Garson, Jeremy

    2013-01-01

    Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication...

  13. Expression analysis of genes associated with human osteosarcoma tumors shows correlation of RUNX2 overexpression with poor response to chemotherapy

    International Nuclear Information System (INIS)

    Sadikovic, Bekim; Thorner, Paul; Chilton-MacNeill, Susan; Martin, Jeff W; Cervigne, Nilva K; Squire, Jeremy; Zielenska, Maria

    2010-01-01

    Human osteosarcoma is the most common pediatric bone tumor. There is limited understanding of the molecular mechanisms underlying osteosarcoma oncogenesis, and a lack of good diagnostic as well as prognostic clinical markers for this disease. Recent discoveries have highlighted a potential role of a number of genes including: RECQL4, DOCK5, SPP1, RUNX2, RB1, CDKN1A, P53, IBSP, LSAMP, MYC, TNFRSF1B, BMP2, HISTH2BE, FOS, CCNB1, and CDC5L. Our objective was to assess relative expression levels of these 16 genes as potential biomarkers of osteosarcoma oncogenesis and chemotherapy response in human tumors. We performed quantitative expression analysis in a panel of 22 human osteosarcoma tumors with differential response to chemotherapy, and 5 normal human osteoblasts. RECQL4, SPP1, RUNX2, and IBSP were significantly overexpressed, and DOCK5, CDKN1A, RB1, P53, and LSAMP showed significant loss of expression relative to normal osteoblasts. In addition to being overexpressed in osteosarcoma tumor samples relative to normal osteoblasts, RUNX2 was the only gene of the 16 to show significant overexpression in tumors that had a poor response to chemotherapy relative to good responders. These data underscore the loss of tumor suppressive pathways and activation of specific oncogenic mechanisms associated with osteosarcoma oncogenesis, while drawing attention to the role of RUNX2 expression as a potential biomarker of chemotherapy failure in osteosarcoma

  14. Global morphological analysis of marine viruses shows minimal regional variation and dominance of non-tailed viruses.

    Science.gov (United States)

    Brum, Jennifer R; Schenck, Ryan O; Sullivan, Matthew B

    2013-09-01

    Viruses influence oceanic ecosystems by causing mortality of microorganisms, altering nutrient and organic matter flux via lysis and auxiliary metabolic gene expression and changing the trajectory of microbial evolution through horizontal gene transfer. Limited host range and differing genetic potential of individual virus types mean that investigations into the types of viruses that exist in the ocean and their spatial distribution throughout the world's oceans are critical to understanding the global impacts of marine viruses. Here we evaluate viral morphological characteristics (morphotype, capsid diameter and tail length) using a quantitative transmission electron microscopy (qTEM) method across six of the world's oceans and seas sampled through the Tara Oceans Expedition. Extensive experimental validation of the qTEM method shows that neither sample preservation nor preparation significantly alters natural viral morphological characteristics. The global sampling analysis demonstrated that morphological characteristics did not vary consistently with depth (surface versus deep chlorophyll maximum waters) or oceanic region. Instead, temperature, salinity and oxygen concentration, but not chlorophyll a concentration, were more explanatory in evaluating differences in viral assemblage morphological characteristics. Surprisingly, given that the majority of cultivated bacterial viruses are tailed, non-tailed viruses appear to numerically dominate the upper oceans as they comprised 51-92% of the viral particles observed. Together, these results document global marine viral morphological characteristics, show that their minimal variability is more explained by environmental conditions than geography and suggest that non-tailed viruses might represent the most ecologically important targets for future research.

  15. Marmosets, Raree shows and Pulcinelle: an Analysis and Edition of a Hitherto-Unpublished Carnival Play by Antonio de Zamora

    Directory of Open Access Journals (Sweden)

    Fernando Plata

    2013-05-01

    Full Text Available This paper is an analyisis, annotation and edition of the Mojiganga del mundinovo (‘The raree show, a carnival play’ by Antonio de Zamora. The play was performed in 1698 Madrid by the troupe of Carlos Vallejo, along with the sacramental one-act play El templo vivo de Dios (‘The living temple of God’. The hitherto unpublished text is based on the only two extant manuscripts, located in archives in Madrid. Despite the play’s title, my analysis argues that the novelty in this play is not so much the raree show, a contraption popularized four decades earlier in Golden Age theater, as the marmosets. The death of two marmosets and the ensuing desolation of their owner, Ms. Estupenda, both trigger the play and provide it with a plot. The marmosets, too, point to a changing mentality in late 17th-century society, regarding the possession among ladies of marmosets and other monkeys as pets.

  16. Use of real-time qPCR to quantify members of the unculturable heterotrophic bacterial community in a deep sea marine sponge, Vetulina sp.

    Science.gov (United States)

    Cassler, M; Peterson, C L; Ledger, A; Pomponi, S A; Wright, A E; Winegar, R; McCarthy, P J; Lopez, J V

    2008-04-01

    In this report, real-time quantitative PCR (TaqMan qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable, microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean Lithistid sponges have shown a wide diversity of microbes that included at least six major subdivisions; however, very little overlap was observed between the culturable and unculturable microbial communities. Based on sequence data of three culture-independent Lithistid-derived representative bacteria, we designed probe/primer sets for TaqMan qPCR to quantitatively characterize selected microbial residents in a Lithistid sponge, Vetulina, metagenome. TaqMan assays included specificity testing, DNA limit of detection analysis, and quantification of specific microbial rRNA sequences such as Nitrospira-like microbes and Actinobacteria up to 172 million copies per microgram per Lithistid sponge metagenome. By contrast, qPCR amplification with probes designed for common previously cultured sponge-associated bacteria in the genera Rheinheimera and Marinomonas and a representative of the CFB group resulted in only minimal detection of the Rheiheimera in total DNA extracted from the sponge. These data verify that a large portion of the microbial community within Lithistid sponges may consist of currently unculturable microorganisms.

  17. A Java program for LRE-based real-time qPCR that enables large-scale absolute quantification.

    Science.gov (United States)

    Rutledge, Robert G

    2011-03-02

    Linear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis. Utilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification. The LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples.

  18. A Java program for LRE-based real-time qPCR that enables large-scale absolute quantification.

    Directory of Open Access Journals (Sweden)

    Robert G Rutledge

    Full Text Available BACKGROUND: Linear regression of efficiency (LRE introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis. FINDINGS: Utilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification. CONCLUSIONS: The LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples.

  19. Comparative shotgun proteomic analysis of wild and domesticated Opuntia spp. species shows a metabolic adaptation through domestication.

    Science.gov (United States)

    Pichereaux, Carole; Hernández-Domínguez, Eric E; Santos-Diaz, Maria Del Socorro; Reyes-Agüero, Antonio; Astello-García, Marizel; Guéraud, Françoise; Negre-Salvayre, Anne; Schiltz, Odile; Rossignol, Michel; Barba de la Rosa, Ana Paulina

    2016-06-30

    The Opuntia genus is widely distributed in America, but the highest richness of wild species are found in Mexico, as well as the most domesticated Opuntia ficus-indica, which is the most domesticated species and an important crop in agricultural economies of arid and semiarid areas worldwide. During domestication process, the Opuntia morphological characteristics were favored, such as less and smaller spines in cladodes and less seeds in fruits, but changes at molecular level are almost unknown. To obtain more insights about the Opuntia molecular changes through domestication, a shotgun proteomic analysis and database-dependent searches by homology was carried out. >1000 protein species were identified and by using a label-free quantitation method, the Opuntia proteomes were compared in order to identify differentially accumulated proteins among wild and domesticated species. Most of the changes were observed in glucose, secondary, and 1C metabolism, which correlate with the observed protein, fiber and phenolic compounds accumulation in Opuntia cladodes. Regulatory proteins, ribosomal proteins, and proteins related with response to stress were also observed in differential accumulation. These results provide new valuable data that will help to the understanding of the molecular changes of Opuntia species through domestication. Opuntia species are well adapted to dry and warm conditions in arid and semiarid regions worldwide, and they are highly productive plants showing considerable promises as an alternative food source. However, there is a gap regarding Opuntia molecular mechanisms that enable them to grow in extreme environmental conditions and how the domestication processes has changed them. In the present study, a shotgun analysis was carried out to characterize the proteomes of five Opuntia species selected by its domestication degree. Our results will help to a better understanding of proteomic features underlying the selection and specialization under

  20. Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR

    Science.gov (United States)

    Stokdyk, Joel P.; Firnstahl, Aaron; Spencer, Susan K.; Burch, Tucker R; Borchardt, Mark A.

    2016-01-01

    The limit of detection (LOD) for qPCR-based analyses is not consistently defined or determined in studies on waterborne pathogens. Moreover, the LODs reported often reflect the qPCR assay alone rather than the entire sample process. Our objective was to develop an approach to determine the 95% LOD (lowest concentration at which 95% of positive samples are detected) for the entire process of waterborne pathogen detection. We began by spiking the lowest concentration that was consistently positive at the qPCR step (based on its standard curve) into each procedural step working backwards (i.e., extraction, secondary concentration, primary concentration), which established a concentration that was detectable following losses of the pathogen from processing. Using the fraction of positive replicates (n = 10) at this concentration, we selected and analyzed a second, and then third, concentration. If the fraction of positive replicates equaled 1 or 0 for two concentrations, we selected another. We calculated the LOD using probit analysis. To demonstrate our approach we determined the 95% LOD for Salmonella enterica serovar Typhimurium, adenovirus 41, and vaccine-derived poliovirus Sabin 3, which were 11, 12, and 6 genomic copies (gc) per reaction (rxn), respectively (equivalent to 1.3, 1.5, and 4.0 gc L−1 assuming the 1500 L tap-water sample volume prescribed in EPA Method 1615). This approach limited the number of analyses required and was amenable to testing multiple genetic targets simultaneously (i.e., spiking a single sample with multiple microorganisms). An LOD determined this way can facilitate study design, guide the number of required technical replicates, aid method evaluation, and inform data interpretation.

  1. Evolution Analysis of the Aux/IAA Gene Family in Plants Shows Dual Origins and Variable Nuclear Localization Signals

    Directory of Open Access Journals (Sweden)

    Wentao Wu

    2017-10-01

    Full Text Available The plant hormone auxin plays pivotal roles in many aspects of plant growth and development. The auxin/indole-3-acetic acid (Aux/IAA gene family encodes short-lived nuclear proteins acting on auxin perception and signaling, but the evolutionary history of this gene family remains to be elucidated. In this study, the Aux/IAA gene family in 17 plant species covering all major lineages of plants is identified and analyzed by using multiple bioinformatics methods. A total of 434 Aux/IAA genes was found among these plant species, and the gene copy number ranges from three (Physcomitrella patens to 63 (Glycine max. The phylogenetic analysis shows that the canonical Aux/IAA proteins can be generally divided into five major clades, and the origin of Aux/IAA proteins could be traced back to the common ancestor of land plants and green algae. Many truncated Aux/IAA proteins were found, and some of these truncated Aux/IAA proteins may be generated from the C-terminal truncation of auxin response factor (ARF proteins. Our results indicate that tandem and segmental duplications play dominant roles for the expansion of the Aux/IAA gene family mainly under purifying selection. The putative nuclear localization signals (NLSs in Aux/IAA proteins are conservative, and two kinds of new primordial bipartite NLSs in P. patens and Selaginella moellendorffii were discovered. Our findings not only give insights into the origin and expansion of the Aux/IAA gene family, but also provide a basis for understanding their functions during the course of evolution.

  2. Evolution Analysis of the Aux/IAA Gene Family in Plants Shows Dual Origins and Variable Nuclear Localization Signals.

    Science.gov (United States)

    Wu, Wentao; Liu, Yaxue; Wang, Yuqian; Li, Huimin; Liu, Jiaxi; Tan, Jiaxin; He, Jiadai; Bai, Jingwen; Ma, Haoli

    2017-10-08

    The plant hormone auxin plays pivotal roles in many aspects of plant growth and development. The auxin/indole-3-acetic acid (Aux/IAA) gene family encodes short-lived nuclear proteins acting on auxin perception and signaling, but the evolutionary history of this gene family remains to be elucidated. In this study, the Aux/IAA gene family in 17 plant species covering all major lineages of plants is identified and analyzed by using multiple bioinformatics methods. A total of 434 Aux/IAA genes was found among these plant species, and the gene copy number ranges from three ( Physcomitrella patens ) to 63 ( Glycine max ). The phylogenetic analysis shows that the canonical Aux/IAA proteins can be generally divided into five major clades, and the origin of Aux/IAA proteins could be traced back to the common ancestor of land plants and green algae. Many truncated Aux/IAA proteins were found, and some of these truncated Aux/IAA proteins may be generated from the C-terminal truncation of auxin response factor (ARF) proteins. Our results indicate that tandem and segmental duplications play dominant roles for the expansion of the Aux/IAA gene family mainly under purifying selection. The putative nuclear localization signals (NLSs) in Aux/IAA proteins are conservative, and two kinds of new primordial bipartite NLSs in P. patens and Selaginella moellendorffii were discovered. Our findings not only give insights into the origin and expansion of the Aux/IAA gene family, but also provide a basis for understanding their functions during the course of evolution.

  3. Multiplex real-time quantitative PCR, microscopy and rapid diagnostic immuno-chromatographic tests for the detection of Plasmodium spp: performance, limit of detection analysis and quality assurance

    Directory of Open Access Journals (Sweden)

    Ralevski Filip

    2009-12-01

    Full Text Available Abstract Background Accurate laboratory diagnosis of malaria species in returning travelers is paramount in the treatment of this potentially fatal infectious disease. Materials and methods A total of 466 blood specimens from returning travelers to Africa, Asia, and South/Central America with suspected malaria infection were collected between 2007 and 2009 at the reference public health laboratory. These specimens were assessed by reference microscopy, multipex real-time quantitative polymerase chain reaction (QPCR, and two rapid diagnostic immuno-chromatographic tests (ICT in a blinded manner. Key clinical laboratory parameters such as limit of detection (LOD analysis on clinical specimens by parasite stage, inter-reader variability of ICTs, staffing implications, quality assurance and cost analysis were evaluated. Results QPCR is the most analytically sensitive method (sensitivity 99.41%, followed by CARESTART (sensitivity 88.24%, and BINAXNOW (sensitivity 86.47% for the diagnosis of malaria in returning travelers when compared to reference microscopy. However, microscopy was unable to specifically identify Plasmodia spp. in 18 out of 170 positive samples by QPCR. Moreover, the 17 samples that were negative by microscopy and positive by QPCR were also positive by ICTs. Quality assurance was achieved for QPCR by exchanging a blinded proficiency panel with another reference laboratory. The Kappa value of inter-reader variability among three readers for BINAXNOW and CARESTART was calculated to be 0.872 and 0.898 respectively. Serial dilution studies demonstrated that the QPCR cycle threshold correlates linearly with parasitemia (R2 = 0.9746 in a clinically relevant dynamic range and retains a LOD of 11 rDNA copies/μl for P. falciparum, which was several log lower than reference microscopy and ICTs. LOD for QPCR is affected not only by parasitemia but the parasite stage distribution of each clinical specimen. QPCR was approximately 6-fold more

  4. Construction of Genetically Engineered Streptococcus gordonii Strains to Provide Control in QPCR Assays for Assessing Microbiological Quality in Recreational Water.

    Science.gov (United States)

    Quantitative PCR (QPCR) methods for beach monitoring by estimating abundance of Enterococcus spp. in recreational waters use internal, positive controls which address only the amplification of target DNA. In this study two internal, positive controls were developed to control for...

  5. Global gene analysis of oocytes from early stages in human folliculogenesis shows high expression of novel genes in reproduction

    DEFF Research Database (Denmark)

    Markholt, Sara; Grøndahl, M L; Ernst, Erik

    2012-01-01

    The pool of primordial follicles in humans is laid down during embryonic development and follicles can remain dormant for prolonged intervals, often decades, until individual follicles resume growth. The mechanisms that induce growth and maturation of primordial follicles are poorly understood...... but follicles once activated either continue growth or undergo atresia. We have isolated pure populations of oocytes from human primordial, intermediate and primary follicles using laser capture micro-dissection microscopy and evaluated the global gene expression profiles by whole-genome microarray analysis......) and the mitochondrial-encoded ATPase6 (ATP6). Thus, the present study provides not only a technique to capture and perform transcriptome analysis of the sparse material of human oocytes from the earliest follicle stages but further includes a comprehensive basis for our understanding of the regulatory factors...

  6. Comprehensive Analysis of Oculocutaneous Albinism among Non-Hispanic Caucasians Shows that OCA1 Is the Most Prevalent OCA Type

    OpenAIRE

    Hutton, Saunie M.; Spritz, Richard A.

    2008-01-01

    Oculocutaneous albinism (OCA) is a genetically heterogeneous group of disorders characterized by absent or reduced pigmentation of the skin, hair, and eyes. In humans, four genes have been associated with “classical” OCA and another 12 genes with syndromic forms of OCA. To assess the prevalence of different forms of OCA and different gene mutations among non-Hispanic Caucasian patients, we performed DNA sequence analysis of the four genes associated with “classical” OCA (TYR, OCA2, TYRP1, SLC...

  7. Individual patient data meta-analysis shows no association between the SNP rs1800469 in TGFB and late radiotherapy toxicity

    International Nuclear Information System (INIS)

    Barnett, Gillian C.; Elliott, Rebecca M.; Alsner, Jan; Andreassen, Christian N.; Abdelhay, Osama; Burnet, Neil G.; Chang-Claude, Jenny; Coles, Charlotte E.; Gutiérrez-Enríquez, Sara; Fuentes-Raspall, Maria J.; Alonso-Muñoz, Maria C.; Kerns, Sarah; Raabe, Annette; Symonds, R. Paul; Seibold, Petra; Talbot, Chris J.; Wenz, Frederik; Wilkinson, Jennifer; Yarnold, John; Dunning, Alison M.

    2012-01-01

    Background and purpose: Reported associations between risk of radiation-induced normal tissue injury and single nucleotide polymorphisms (SNPs) in TGFB1, encoding the pro-fibrotic cytokine transforming growth factor-beta 1 (TGF-β1), remain controversial. To overcome publication bias, the international Radiogenomics Consortium collected and analysed individual patient level data from both published and unpublished studies. Materials and methods: TGFB1 SNP rs1800469 c.-1347T>C (previously known as C-509T) genotype, treatment-related data, and clinically-assessed fibrosis (measured at least 2 years after therapy) were available in 2782 participants from 11 cohorts. All received adjuvant breast radiotherapy. Associations between late fibrosis or overall toxicity, reported by STAT (Standardised Total Average Toxicity) score, and rs1800469 genotype were assessed. Results: No statistically significant associations between either fibrosis or overall toxicity and rs1800469 genotype were observed with univariate or multivariate regression analysis. The multivariate odds ratio (OR), obtained from meta-analysis, for an increase in late fibrosis grade with each additional rare allele of rs1800469 was 0.98 (95% Confidence Interval (CI) 0.85–1.11). This CI is sufficiently narrow to rule out any clinically relevant effect on toxicity risk in carriers vs. non-carriers with a high probability. Conclusion: This meta-analysis has not confirmed previous reports of association between fibrosis or overall toxicity and rs1800469 genotype in breast cancer patients. It has demonstrated successful collaboration within the Radiogenomics Consortium.

  8. Comparative Analysis of Clinical Samples Showing Weak Serum Reaction on AutoVue System Causing ABO Blood Typing Discrepancies.

    Science.gov (United States)

    Jo, Su Yeon; Lee, Ju Mi; Kim, Hye Lim; Sin, Kyeong Hwa; Lee, Hyeon Ji; Chang, Chulhun Ludgerus; Kim, Hyung Hoi

    2017-03-01

    ABO blood typing in pre-transfusion testing is a major component of the high workload in blood banks that therefore requires automation. We often experienced discrepant results from an automated system, especially weak serum reactions. We evaluated the discrepant results by the reference manual method to confirm ABO blood typing. In total, 13,113 blood samples were tested with the AutoVue system; all samples were run in parallel with the reference manual method according to the laboratory protocol. The AutoVue system confirmed ABO blood typing of 12,816 samples (97.7%), and these results were concordant with those of the manual method. The remaining 297 samples (2.3%) showed discrepant results in the AutoVue system and were confirmed by the manual method. The discrepant results involved weak serum reactions (serum reactions, samples from patients who had received stem cell transplants, ABO subgroups, and specific system error messages. Among the 98 samples showing ≤1+ reaction grade in the AutoVue system, 70 samples (71.4%) showed a normal serum reaction (≥2+ reaction grade) with the manual method, and 28 samples (28.6%) showed weak serum reaction in both methods. ABO blood tying of 97.7% samples could be confirmed by the AutoVue system and a small proportion (2.3%) needed to be re-evaluated by the manual method. Samples with a 2+ reaction grade in serum typing do not need to be evaluated manually, while those with ≤1+ reaction grade do.

  9. Isolation and expression analysis of EcbZIP17 from different finger millet genotypes shows conserved nature of the gene.

    Science.gov (United States)

    Chopperla, Ramakrishna; Singh, Sonam; Mohanty, Sasmita; Reddy, Nanja; Padaria, Jasdeep C; Solanke, Amolkumar U

    2017-10-01

    Basic leucine zipper (bZIP) transcription factors comprise one of the largest gene families in plants. They play a key role in almost every aspect of plant growth and development and also in biotic and abiotic stress tolerance. In this study, we report isolation and characterization of EcbZIP17 , a group B bZIP transcription factor from a climate smart cereal, finger millet ( Eleusine coracana L.). The genomic sequence of EcbZIP17 is 2662 bp long encompassing two exons and one intron with ORF of 1722 bp and peptide length of 573 aa. This gene is homologous to AtbZIP17 ( Arabidopsis ), ZmbZIP17 (maize) and OsbZIP60 (rice) which play a key role in endoplasmic reticulum (ER) stress pathway. In silico analysis confirmed the presence of basic leucine zipper (bZIP) and transmembrane (TM) domains in the EcbZIP17 protein. Allele mining of this gene in 16 different genotypes by Sanger sequencing revealed no variation in nucleotide sequence, including the 618 bp long intron. Expression analysis of EcbZIP17 under heat stress exhibited similar pattern of expression in all the genotypes across time intervals with highest upregulation after 4 h. The present study established the conserved nature of EcbZIP17 at nucleotide and expression level.

  10. Transcriptomic and metabolomic analysis of Yukon Thellungiella plants grown in cabinets and their natural habitat show phenotypic plasticity

    Directory of Open Access Journals (Sweden)

    Guevara David R

    2012-10-01

    Full Text Available Abstract Background Thellungiella salsuginea is an important model plant due to its natural tolerance to abiotic stresses including salt, cold, and water deficits. Microarray and metabolite profiling have shown that Thellungiella undergoes stress-responsive changes in transcript and organic solute abundance when grown under controlled environmental conditions. However, few reports assess the capacity of plants to display stress-responsive traits in natural habitats where concurrent stresses are the norm. Results To determine whether stress-responsive changes observed in cabinet-grown plants are recapitulated in the field, we analyzed leaf transcript and metabolic profiles of Thellungiella growing in its native Yukon habitat during two years of contrasting meteorological conditions. We found 673 genes showing differential expression between field and unstressed, chamber-grown plants. There were comparatively few overlaps between genes expressed under field and cabinet treatment-specific conditions. Only 20 of 99 drought-responsive genes were expressed both in the field during a year of low precipitation and in plants subjected to drought treatments in cabinets. There was also a general pattern of lower abundance among metabolites found in field plants relative to control or stress-treated plants in growth cabinets. Nutrient availability may explain some of the observed differences. For example, proline accumulated to high levels in cold and salt-stressed cabinet-grown plants but proline content was, by comparison, negligible in plants at a saline Yukon field site. We show that proline accumulated in a stress-responsive manner in Thellungiella plants salinized in growth cabinets and in salt-stressed seedlings when nitrogen was provided at 1.0 mM. In seedlings grown on 0.1 mM nitrogen medium, the proline content was low while carbohydrates increased. The relatively higher content of sugar-like compounds in field plants and seedlings on low nitrogen

  11. Transcriptomic and metabolomic analysis of Yukon Thellungiella plants grown in cabinets and their natural habitat show phenotypic plasticity.

    Science.gov (United States)

    Guevara, David R; Champigny, Marc J; Tattersall, Ashley; Dedrick, Jeff; Wong, Chui E; Li, Yong; Labbe, Aurelie; Ping, Chien-Lu; Wang, Yanxiang; Nuin, Paulo; Golding, G Brian; McCarry, Brian E; Summers, Peter S; Moffatt, Barbara A; Weretilnyk, Elizabeth A

    2012-10-01

    Thellungiella salsuginea is an important model plant due to its natural tolerance to abiotic stresses including salt, cold, and water deficits. Microarray and metabolite profiling have shown that Thellungiella undergoes stress-responsive changes in transcript and organic solute abundance when grown under controlled environmental conditions. However, few reports assess the capacity of plants to display stress-responsive traits in natural habitats where concurrent stresses are the norm. To determine whether stress-responsive changes observed in cabinet-grown plants are recapitulated in the field, we analyzed leaf transcript and metabolic profiles of Thellungiella growing in its native Yukon habitat during two years of contrasting meteorological conditions. We found 673 genes showing differential expression between field and unstressed, chamber-grown plants. There were comparatively few overlaps between genes expressed under field and cabinet treatment-specific conditions. Only 20 of 99 drought-responsive genes were expressed both in the field during a year of low precipitation and in plants subjected to drought treatments in cabinets. There was also a general pattern of lower abundance among metabolites found in field plants relative to control or stress-treated plants in growth cabinets. Nutrient availability may explain some of the observed differences. For example, proline accumulated to high levels in cold and salt-stressed cabinet-grown plants but proline content was, by comparison, negligible in plants at a saline Yukon field site. We show that proline accumulated in a stress-responsive manner in Thellungiella plants salinized in growth cabinets and in salt-stressed seedlings when nitrogen was provided at 1.0 mM. In seedlings grown on 0.1 mM nitrogen medium, the proline content was low while carbohydrates increased. The relatively higher content of sugar-like compounds in field plants and seedlings on low nitrogen media suggests that Thellungiella shows

  12. Transcriptomic and metabolomic analysis of Yukon Thellungiella plants grown in cabinets and their natural habitat show phenotypic plasticity

    Science.gov (United States)

    2012-01-01

    Background Thellungiella salsuginea is an important model plant due to its natural tolerance to abiotic stresses including salt, cold, and water deficits. Microarray and metabolite profiling have shown that Thellungiella undergoes stress-responsive changes in transcript and organic solute abundance when grown under controlled environmental conditions. However, few reports assess the capacity of plants to display stress-responsive traits in natural habitats where concurrent stresses are the norm. Results To determine whether stress-responsive changes observed in cabinet-grown plants are recapitulated in the field, we analyzed leaf transcript and metabolic profiles of Thellungiella growing in its native Yukon habitat during two years of contrasting meteorological conditions. We found 673 genes showing differential expression between field and unstressed, chamber-grown plants. There were comparatively few overlaps between genes expressed under field and cabinet treatment-specific conditions. Only 20 of 99 drought-responsive genes were expressed both in the field during a year of low precipitation and in plants subjected to drought treatments in cabinets. There was also a general pattern of lower abundance among metabolites found in field plants relative to control or stress-treated plants in growth cabinets. Nutrient availability may explain some of the observed differences. For example, proline accumulated to high levels in cold and salt-stressed cabinet-grown plants but proline content was, by comparison, negligible in plants at a saline Yukon field site. We show that proline accumulated in a stress-responsive manner in Thellungiella plants salinized in growth cabinets and in salt-stressed seedlings when nitrogen was provided at 1.0 mM. In seedlings grown on 0.1 mM nitrogen medium, the proline content was low while carbohydrates increased. The relatively higher content of sugar-like compounds in field plants and seedlings on low nitrogen media suggests that

  13. Quantitative Analysis of the Contributing Factors Affecting Specialty Care No-Show Rates at Brooke Army Medical Center

    Science.gov (United States)

    2007-03-30

    PHYSICAL THERAPY ,TMC 53 9 14.52% AUDIOLOGY,BAMC 133 22 14.19% OPHTH PEDS,BAMC 28 4 12.50% OCCUPATIONAL THERAPY ,BAMC 135 18 11.76% ALLERGY CLINIC,BAMC...for physical therapy and have a total of 4 appointments set up with that clinic. For this study, only the first referral was counted and not the follow...Nephrology. Some of the other low no-show rate clinics were: Pain Management (1.27%), Endocrinology (1.52%), General Surgery (1.83%), Rheumatology (1.89

  14. Pre-amplification in the context of high-throughput qPCR gene expression experiment

    Czech Academy of Sciences Publication Activity Database

    Korenková, Vlasta; Scott, J.; Novosadová, Vendula; Jindřichová, Marie; Langerová, Lucie; Švec, David; Šídová, Monika; Sjoback, R.

    2015-01-01

    Roč. 16, č. 5 (2015) ISSN 1471-2199 R&D Projects: GA ČR(CZ) GAP304/12/1585; GA ČR(CZ) GA15-08239S; GA ČR GA13-02154S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : High-throughput qPCR * Gene expression * Exponential pre-amplification Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.500, year: 2015

  15. Comprehensive analysis of oculocutaneous albinism among non-Hispanic caucasians shows that OCA1 is the most prevalent OCA type.

    Science.gov (United States)

    Hutton, Saunie M; Spritz, Richard A

    2008-10-01

    Oculocutaneous albinism (OCA) is a genetically heterogeneous group of disorders characterized by absent or reduced pigmentation of the skin, hair, and eyes. In humans, four genes have been associated with "classical" OCA and another 12 genes with syndromic forms of OCA. To assess the prevalence of different forms of OCA and different gene mutations among non-Hispanic Caucasian patients, we performed DNA sequence analysis of the four genes associated with "classical" OCA (TYR, OCA2, TYRP1, SLC45A2), the two principal genes associated with syndromic OCA (HPS1, HPS4), and a candidate OCA gene (SILV), in 121 unrelated, unselected non-Hispanic/Latino Caucasian patients carrying the clinical diagnosis of OCA. We identified apparent pathologic TYR gene mutations in 69% of patients, OCA2 mutations in 18%, SLC45A2 mutations in 6%, and no apparent pathological mutations in 7% of patients. We found no mutations of TYRP1, HPS1, HPS4, or SILV in any patients. Although we observed a diversity of mutations for each gene, a relatively small number of different mutant alleles account for a majority of the total. This study demonstrates that, contrary to long-held clinical lore, OCA1, not OCA2, is by far the most frequent cause of OCA among Caucasian patients.

  16. Anatomical Network Analysis Shows Decoupling of Modular Lability and Complexity in the Evolution of the Primate Skull

    Science.gov (United States)

    Esteve-Altava, Borja; Boughner, Julia C.; Diogo, Rui; Villmoare, Brian A.; Rasskin-Gutman, Diego

    2015-01-01

    Modularity and complexity go hand in hand in the evolution of the skull of primates. Because analyses of these two parameters often use different approaches, we do not know yet how modularity evolves within, or as a consequence of, an also-evolving complex organization. Here we use a novel network theory-based approach (Anatomical Network Analysis) to assess how the organization of skull bones constrains the co-evolution of modularity and complexity among primates. We used the pattern of bone contacts modeled as networks to identify connectivity modules and quantify morphological complexity. We analyzed whether modularity and complexity evolved coordinately in the skull of primates. Specifically, we tested Herbert Simon’s general theory of near-decomposability, which states that modularity promotes the evolution of complexity. We found that the skulls of extant primates divide into one conserved cranial module and up to three labile facial modules, whose composition varies among primates. Despite changes in modularity, statistical analyses reject a positive feedback between modularity and complexity. Our results suggest a decoupling of complexity and modularity that translates to varying levels of constraint on the morphological evolvability of the primate skull. This study has methodological and conceptual implications for grasping the constraints that underlie the developmental and functional integration of the skull of humans and other primates. PMID:25992690

  17. Morphometric analysis of molars in a Middle Pleistocene population shows a mosaic of 'modern' and Neanderthal features.

    Science.gov (United States)

    Martinón-Torres, María; Spěváčková, Petra; Gracia-Téllez, Ana; Martínez, Ignacio; Bruner, Emiliano; Arsuaga, Juan Luis; Bermúdez de Castro, José María

    2013-10-01

    Previous studies of upper first molar (M1) crown shape have shown significant differences between Homo sapiens and Homo neanderthalensis that were already present in the European Middle Pleistocene populations, including the large dental sample from Atapuerca-Sima de los Huesos (SH). Analysis of other M1 features such as the total crown base area, cusp proportions, cusp angles and occlusal polygon have confirmed the differences between both lineages, becoming a useful tool for the taxonomic assignment of isolated teeth from Late Pleistocene sites. However, until now the pattern of expression of these variables has not been known for the SH sample. This fossil sample, the largest collection from the European Middle Pleistocene, is generally interpreted as being from the direct ancestors of Neanderthals, and thus is a reference sample for assessing the origin of the Neanderthal morphologies. Surprisingly, our study reveals that SH M(1) s present a unique mosaic of H. neanderthalensis and H. sapiens features. Regarding the cusp angles and the relative occlusal polygon area, SH matches the H. neanderthalensis pattern. However, regarding the total crown base area and relative cusps size, SH M(1) s are similar to H. sapiens, with a small crown area, a strong hypocone reduction and a protocone enlargement, although the protocone expansion in SH is significantly larger than in any other group studied. The SH dental sample calls into question the uniqueness of some so-called modern traits. Our study also sounds a note of caution on the use of M(1) occlusal morphology for the alpha taxonomy of isolated M(1) s. © 2013 Anatomical Society.

  18. Morphometric analysis of molars in a Middle Pleistocene population shows a mosaic of ‘modern’ and Neanderthal features

    Science.gov (United States)

    Martinón-Torres, María; Spěváčková, Petra; Gracia-Téllez, Ana; Martínez, Ignacio; Bruner, Emiliano; Arsuaga, Juan Luis; Bermúdez de Castro, José María

    2013-01-01

    Previous studies of upper first molar (M1) crown shape have shown significant differences between Homo sapiens and Homo neanderthalensis that were already present in the European Middle Pleistocene populations, including the large dental sample from Atapuerca-Sima de los Huesos (SH). Analysis of other M1 features such as the total crown base area, cusp proportions, cusp angles and occlusal polygon have confirmed the differences between both lineages, becoming a useful tool for the taxonomic assignment of isolated teeth from Late Pleistocene sites. However, until now the pattern of expression of these variables has not been known for the SH sample. This fossil sample, the largest collection from the European Middle Pleistocene, is generally interpreted as being from the direct ancestors of Neanderthals, and thus is a reference sample for assessing the origin of the Neanderthal morphologies. Surprisingly, our study reveals that SH M1s present a unique mosaic of H. neanderthalensis and H. sapiens features. Regarding the cusp angles and the relative occlusal polygon area, SH matches the H. neanderthalensis pattern. However, regarding the total crown base area and relative cusps size, SH M1s are similar to H. sapiens, with a small crown area, a strong hypocone reduction and a protocone enlargement, although the protocone expansion in SH is significantly larger than in any other group studied. The SH dental sample calls into question the uniqueness of some so-called modern traits. Our study also sounds a note of caution on the use of M1 occlusal morphology for the alpha taxonomy of isolated M1s. PMID:23914934

  19. Meta-analysis shows similar re-bleeding rates among Western and Eastern populations after index video capsule endoscopy.

    Science.gov (United States)

    Tziatzios, Georgios; Gkolfakis, Paraskevas; Hassan, Cesare; Toth, Ervin; Zullo, Angelo; Koulaouzidis, Anastasios; Dimitriadis, George D; Triantafyllou, Konstantinos

    2018-03-01

    Video capsule endoscopy (VCE) is the first-line diagnostic procedure for investigating obscure gastrointestinal bleeding (OGIB). Different re-bleeding rates following index VCE have been reported among Western and Eastern studies. We conducted a comprehensive literature search to identify studies examining re-bleeding rates after VCE for OGIB. Meta-analysis assessed the pooled proportion of re-bleeding events after VCE for OGIB according to study's origin (Western vs. Eastern) and according to the length of follow-up (≥24 months vs. Western and 16 Eastern) studies with 5796 patients. Significant heterogeneity was detected among meta-analyzed studies. Overall, the pooled re-bleeding rate was similar between Western (29%; 95% CI: 23-34) and Eastern (21%; 95% CI: 15-27) populations, irrespective of the length of follow-up. The odds of re-bleeding was significantly higher after positive as compared to negative index VCE in Eastern studies (OR: 1.77; 95% CI: 1.07-2.94). Application of specific treatment after positive index VCE was associated with lower re-bleeding odds in both Western (OR: 0.37; 95% CI: 0.16-0.87) and Eastern (OR: 0.39; 95% CI: 0.21-0.72) populations. Patients undergoing VCE for OGIB have similar re-bleeding rates in the East and the West, regardless of the length of follow-up. However, increased re-bleeding odds after positive index VCE is observed in Eastern studies. Copyright © 2018 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

  20. X-ray survival characteristics and genetic analysis for nine saccharomyces deletion mutants that show altered radiation sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Game, John C.; Williamson, Marsha S.; Baccari, Clelia

    2004-01-07

    The availability of a genome-wide set of Saccharomyces deletion mutants provides a chance to identify all the yeast genes involved in DNA repair. Using X-rays, we are screening these mutants to identify additional genes that show increased sensitivity to the lethal effects of ionizing radiation. For each mutant identified as sensitive, we are confirming that the sensitivity phenotype co-segregates with the deletion allele and are obtaining multipoint survival-versus-dose assays in at least two haploid and one homozygous diploid strains. We present data for deletion mutants involving the genes DOT1, MDM20, NAT3, SPT7, SPT20, GCN5, HFI1, DCC1 and VID21/EAF1, and discuss their potential roles in repair. Eight of these genes have a clear radiation-sensitive phenotype when deleted, but the ninth, GCN5, has at most a borderline phenotype. None of the deletions confer substantial sensitivity to ultra-violet radiation, although one or two may confer marginal sensitivity. The DOT1 gene is of interest because its only known function is to methylate one lysine residue in the core of the histone H3 protein. We find that histone H3 mutants (supplied by K. Struhl) in which this residue is replaced by other amino-acids are also X-ray sensitive, seeming to confirm that methylation of the lysine-79 residue is required for effective repair of radiation damage.

  1. Comprehensive behavioral analysis of Ox1r-/- mice showed implication of orexin receptor-1 in mood, anxiety and social behavior

    Directory of Open Access Journals (Sweden)

    Md Golam Abbas

    2015-12-01

    Full Text Available Neuropeptides orexin A and orexin B, which are exclusively produced by neurons in the lateral hypothalamic area, play an important role in the regulation of a wide range of behaviors and homeostatic processes, including regulation of sleep/wakefulness states and energy homeostasis. The orexin system has close anatomical and functional relationships with systems that regulate the autonomic nervous system, emotion, mood, the reward system and sleep/wakefulness states. Recent pharmacological studies using selective antagonists have suggested that orexin receptor-1 (OX1R is involved in physiological processes that regulate emotion, the reward system and autonomic nervous system. Here, we examined Ox1r-/- mice with a comprehensive behavioral test battery to screen additional OX1R functions. Ox1r-/- mice showed increased anxiety-like behavior, altered depression-like behavior, slightly decreased spontaneous locomotor activity, reduced social interaction, increased startle response and decreased prepulse inhibition. These results suggest that OX1R plays roles in social behaviour and sensory motor gating in addition to roles in mood and anxiety.

  2. An actuarial analysis shows that offering lung cancer screening as an insurance benefit would save lives at relatively low cost.

    Science.gov (United States)

    Pyenson, Bruce S; Sander, Marcia S; Jiang, Yiding; Kahn, Howard; Mulshine, James L

    2012-04-01

    Lung cancer screening is not established as a public health practice, yet the results of a recent large randomized controlled trial showed that screening with low-dose spiral computed tomography reduces lung cancer mortality. Using actuarial models, this study estimated the costs and benefits of annual lung cancer screening offered as a commercial insurance benefit in the high-risk US population ages 50-64. Assuming current commercial reimbursement rates for treatment, we found that screening would cost about $1 per insured member per month in 2012 dollars. The cost per life-year saved would be below $19,000, an amount that compares favorably with screening for cervical, breast, and colorectal cancers. Our results suggest that commercial insurers should consider lung cancer screening of high-risk individuals to be high-value coverage and provide it as a benefit to people who are at least fifty years old and have a smoking history of thirty pack-years or more. We also believe that payers and patients should demand screening from high-quality, low-cost providers, thus helping set an example of efficient system innovation.

  3. Transcriptome analysis shows activation of the arginine deiminase pathway in Lactococcus lactis as a response to ethanol stress.

    Science.gov (United States)

    Díez, Lorena; Solopova, Ana; Fernández-Pérez, Rocío; González, Miriam; Tenorio, Carmen; Kuipers, Oscar P; Ruiz-Larrea, Fernanda

    2017-09-18

    This paper describes the molecular response of Lactococcus lactis NZ9700 to ethanol. This strain is a well-known nisin producer and a lactic acid bacteria (LAB) model strain. Global transcriptome profiling using DNA microarrays demonstrated a bacterial adaptive response to the presence of 2% ethanol in the culture broth and differential expression of 67 genes. The highest up-regulation was detected for those genes involved in arginine degradation through the arginine deiminase (ADI) pathway (20-40 fold up-regulation). The metabolic responses to ethanol of wild type L. lactis strains were studied and compared to those of regulator-deletion mutants MG∆argR and MG∆ahrC. The results showed that in the presence of 2% ethanol those strains with an active ADI pathway reached higher growth rates when arginine was available in the culture broth than in absence of arginine. In a chemically defined medium strains with an active ADI pathway consumed arginine and produced ornithine in the presence of 2% ethanol, hence corroborating that arginine catabolism is involved in the bacterial response to ethanol. This is the first study of the L. lactis response to ethanol stress to demonstrate the relevance of arginine catabolism for bacterial adaptation and survival in an ethanol containing medium. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum.

    Science.gov (United States)

    Rubio, Marcela da Silva; Penha Filho, Rafael Antonio Casarin; Almeida, Adriana Maria de; Berchieri, Angelo

    2017-12-01

    Currently there are 2659 Salmonella serovars. The host-specific biovars Salmonella Pullorum and Salmonella Gallinarum cause systemic infections in food-producing and wild birds. Fast diagnosis is crucial to control the dissemination in avian environments. The present work describes the development of a multiplex qPCR in real time using a low-cost DNA dye (SYBr Green) to identify and quantify these biovars. Primers were chosen based on genomic regions of difference (RoD) and optimized to control dimers. Primers pSGP detect both host-specific biovars but not other serovars and pSG and pSP differentiate biovars. Three amplicons showed different melting temperatures (Tm), allowing differentiation. The pSGP amplicon (97 bp) showed Tm of 78°C for both biovars. The pSG amplicon (273 bp) showed a Tm of 86.2°C for S. Gallinarum and pSP amplicon (260 bp) dissociated at 84.8°C for S. Pullorum identification. The multiplex qPCR in real time showed high sensitivity and was capable of quantifying 10 8 -10 1 CFU of these biovars.

  5. Development of a qPCR method to rapidly assess the function of NKT cells.

    Science.gov (United States)

    Sohn, Silke; Tiper, Irina; Japp, Emily; Sun, Wenji; Tkaczuk, Katherine; Webb, Tonya J

    2014-05-01

    NKT cells comprise a rare, but important subset of T cells which account for ~0.2% of the total circulating T cell population. NKT cells are known to have anti-tumor functions and rapidly produce high levels of cytokines following activation. Several clinical trials have sought to exploit the effector functions of NKT cells. While some studies have shown promise, NKT cells are approximately 50% lower in cancer patients compared to healthy donors of the same age and gender, thus limiting their therapeutic efficacy. These studies indicate that baseline levels of activation should be assessed before initiating an NKT cell based immunotherapeutic strategy. The goal of this study was to develop a sensitive method to rapidly assess NKT cell function. We utilized artificial antigen presenting cells in combination with qPCR in order to determine NKT cell function in peripheral blood mononuclear cells from healthy donors and breast cancer patients. We found that NKT cell activation can be detected by qPCR, but not by ELISA, in healthy donors as well as in breast cancer patients following four hour stimulation. This method utilizing CD1d-expressing aAPCs will enhance our knowledge of NKT cell biology and could potentially be used as a novel tool in adoptive immunotherapeutic strategies. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Is qPCR a Reliable Indicator of Cyanotoxin Risk in Freshwater?

    Science.gov (United States)

    Pacheco, Ana Beatriz F.; Guedes, Iame A.; Azevedo, Sandra M.F.O.

    2016-01-01

    The wide distribution of cyanobacteria in aquatic environments leads to the risk of water contamination by cyanotoxins, which generate environmental and public health issues. Measurements of cell densities or pigment contents allow both the early detection of cellular growth and bloom monitoring, but these methods are not sufficiently accurate to predict actual cyanobacterial risk. To quantify cyanotoxins, analytical methods are considered the gold standards, but they are laborious, expensive, time-consuming and available in a limited number of laboratories. In cyanobacterial species with toxic potential, cyanotoxin production is restricted to some strains, and blooms can contain varying proportions of both toxic and non-toxic cells, which are morphologically indistinguishable. The sequencing of cyanobacterial genomes led to the description of gene clusters responsible for cyanotoxin production, which paved the way for the use of these genes as targets for PCR and then quantitative PCR (qPCR). Thus, the quantification of cyanotoxin genes appeared as a new method for estimating the potential toxicity of blooms. This raises a question concerning whether qPCR-based methods would be a reliable indicator of toxin concentration in the environment. Here, we review studies that report the parallel detection of microcystin genes and microcystin concentrations in natural populations and also a smaller number of studies dedicated to cylindrospermopsin and saxitoxin. We discuss the possible issues associated with the contradictory findings reported to date, present methodological limitations and consider the use of qPCR as an indicator of cyanotoxin risk. PMID:27338471

  7. Is qPCR a Reliable Indicator of Cyanotoxin Risk in Freshwater?

    Directory of Open Access Journals (Sweden)

    Ana Beatriz F. Pacheco

    2016-06-01

    Full Text Available The wide distribution of cyanobacteria in aquatic environments leads to the risk of water contamination by cyanotoxins, which generate environmental and public health issues. Measurements of cell densities or pigment contents allow both the early detection of cellular growth and bloom monitoring, but these methods are not sufficiently accurate to predict actual cyanobacterial risk. To quantify cyanotoxins, analytical methods are considered the gold standards, but they are laborious, expensive, time-consuming and available in a limited number of laboratories. In cyanobacterial species with toxic potential, cyanotoxin production is restricted to some strains, and blooms can contain varying proportions of both toxic and non-toxic cells, which are morphologically indistinguishable. The sequencing of cyanobacterial genomes led to the description of gene clusters responsible for cyanotoxin production, which paved the way for the use of these genes as targets for PCR and then quantitative PCR (qPCR. Thus, the quantification of cyanotoxin genes appeared as a new method for estimating the potential toxicity of blooms. This raises a question concerning whether qPCR-based methods would be a reliable indicator of toxin concentration in the environment. Here, we review studies that report the parallel detection of microcystin genes and microcystin concentrations in natural populations and also a smaller number of studies dedicated to cylindrospermopsin and saxitoxin. We discuss the possible issues associated with the contradictory findings reported to date, present methodological limitations and consider the use of qPCR as an indicator of cyanotoxin risk.

  8. Validation of qPCR Methods for the Detection of Mycobacterium in New World Animal Reservoirs.

    Directory of Open Access Journals (Sweden)

    Genevieve Housman

    2015-11-01

    Full Text Available Zoonotic pathogens that cause leprosy (Mycobacterium leprae and tuberculosis (Mycobacterium tuberculosis complex, MTBC continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect M. leprae as well as MTBC in wild marmosets. All marmosets were negative for M. leprae DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts.

  9. Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol

    Directory of Open Access Journals (Sweden)

    Haug Trude M

    2010-11-01

    Full Text Available Abstract Background The incidence of false positives is a potential problem in single-cell PCR experiments. This paper describes an optimized protocol for single-cell qPCR measurements in primary pituitary cell cultures following patch-clamp recordings. Two different cell harvesting methods were assessed using both the GH4 prolactin producing cell line from rat, and primary cell culture from fish pituitaries. Results Harvesting whole cells followed by cell lysis and qPCR performed satisfactory on the GH4 cell line. However, harvesting of whole cells from primary pituitary cultures regularly produced false positives, probably due to RNA leakage from cells ruptured during the dispersion of the pituitary cells. To reduce RNA contamination affecting the results, we optimized the conditions by harvesting only the cytosol through a patch pipette, subsequent to electrophysiological experiments. Two important factors proved crucial for reliable harvesting. First, silanizing the patch pipette glass prevented foreign extracellular RNA from attaching to charged residues on the glass surface. Second, substituting the commonly used perforating antibiotic amphotericin B with β-escin allowed efficient cytosol harvest without loosing the giga seal. Importantly, the two harvesting protocols revealed no difference in RNA isolation efficiency. Conclusion Depending on the cell type and preparation, validation of the harvesting technique is extremely important as contaminations may give false positives. Here we present an optimized protocol allowing secure harvesting of RNA from single cells in primary pituitary cell culture following perforated whole cell patch clamp experiments.

  10. Real-time PCR (qPCR) primer design using free online software.

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. Copyright © 2010 Wiley Periodicals, Inc.

  11. Comparison of nested PCR and qPCR for the detection and quantitation of BoHV6 DNA.

    Science.gov (United States)

    Kubiś, Piotr; Materniak, Magdalena; Kuźmak, Jacek

    2013-12-01

    Nested PCR and qPCR (quantitative PCR) tests based on glycoprotein B (gB) gene were designed for detecting Bovine herpesvirus 6 (BoHV6) in bovine whole blood samples and wild ruminant blood clots (deer and roe-deer). This virus, commonly known as BLHV (bovine lymphotropic herpesvirus) belongs to the Herpesviridae family, subfamily Gammaherpesvirinae and Macavirus genus. DNA isolated from 92 dairy cow blood samples and 69 wild ruminant clots were examined for the presence of BoHV6 using nested PCR and qPCR tests. Viral DNA was detected by using nested PCR in 59 out of 92 bovine blood samples (64.1%), and by qPCR in 68 out of 92 bovine blood samples (73.9%), but none out of 69 DNA samples isolated from wild ruminant blood clots, was positive in both assays. The specificity of nested PCR and qPCR was confirmed by using BoHV1, BoHV4, BoHV6, BFV, BIV, and BLV DNA. The sensitivity of nested PCR and qPCR was determined using a serially 10-fold diluted vector pCR2.1HgB (2 × 10(0)-2 × 10(6)copies/reaction). In this testing, qPCR was more sensitive than the nested PCR, detecting two copies of BoHV6 whilst the limit of detection for nested PCR was 20 copies. In all qPCR assays, the coefficients of determination (R(2)) ranged between 0.990 and 0.999, and the calculated amplification efficiencies (Eff%) within the range of 89.7-106.9. The intra- and inter-assay CV (coefficient of variation) values did not exceed 4%. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Babesia bovis and Babesia bigemina infection levels estimated by qPCR in Angus cattle from an endemic area of São Paulo state, Brazil.

    Science.gov (United States)

    Giglioti, R; Oliveira, H N; Santana, C H; Ibelli, A M G; Néo, T A; Bilhassi, T B; Rabelo, M D; Machado, R Z; Brito, L G; Oliveira, M C S

    2016-07-01

    The levels of infection by Babesia bovis and Babesia bigemina were estimated by absolute quantification through the quantitative PCR technique (qPCR). Fifty-one contemporaneous Angus cattle were evaluated on two occasions. The number of standard female Rhipicephalus microplus ticks present on the left side of the body was counted and blood samples were drawn from the tail vein into tubes containing the anticoagulant EDTA. The blood samples were submitted to DNA extraction and used to quantify the number of copies (NC) of DNA from B. bovis and B. bigemina by qPCR. The data on tick count and number of DNA copies were transformed for normalization and analyzed by a mixed model method. A multivariate model with repeated measures of the same animal, including the effects of collection, parasite species and their interaction, was used. The repeatability values were obtained from the matrix of (co)variances and were expressed for each species. The correlations between the counts of different species on the same animal, in the same collection or different collections, were also estimated. The results showed the qPCR could distinguish the two between infection by the two Babesia species. Infection levels by B. bovis and B. bigemina were detected in 100% and 98% of the animals, respectively. Significant differences were found (Phemoparasites did not depend on the tick infestation levels at the moment of each collection. The repeatability values estimated indicate that under the study conditions, the variations in the tick infestation levels and of parasitemia by B. bovis and B. bigemina depend more on factors related to each collection than on intrinsic factors of the animal. Copyright © 2016 Elsevier GmbH. All rights reserved.

  13. The Development of a Novel qPCR Assay-Set for Identifying Fecal Contamination Originating from Domestic Fowls and Waterfowl in Israel

    Directory of Open Access Journals (Sweden)

    Shoshanit eOhad

    2016-02-01

    Full Text Available The emerging Microbial Source Tracking (MST methodologies aim to identify fecal contamination originating from domestic and wild animals, and from humans. Avian MST is especially challenging, primarily because the Aves class includes both domesticated and wild species with highly diverse habitats and dietary characteristics. The quest for specific fecal bacterial MST markers can be difficult with respect to attaining sufficient assay sensitivity and specificity. The present study utilizes High Throughput Sequencing (HTS to screen bacterial 16S rRNA genes from fecal samples collected from both domestic and wild avian species. Operational taxonomic unit (OTU analysis was then performed, from which sequences were retained for downstream qPCR marker development. Identification of unique avian host DNA sequences, absent in non-avian hosts, was then carried out using a dedicated database of bacterial 16S rRNA gene taken from the Ribosomal Database Project. Six qPCR assays were developed targeting the 16S rRNA gene of Lactobacillus, Gallibacterium, Firmicutes, Fusobacteriaceae and other bacteria. Two assays (Av4143 and Av163 identified most of the avian fecal samples and demonstrated sensitivity values of 91% and 70%, respectively. The Av43 assay only identified droppings from battery hens and poultry, whereas each of the other three assays (Av24, Av13, and Av216 identified waterfowl species with lower sensitivities values. The development of an MST assay-panel, which includes both domestic and wild avian species, expands the currently known MST analysis capabilities for decoding fecal contamination.

  14. Relationship and variation of qPCR and culturable enterococci estimates in ambient surface waters are predictable

    Science.gov (United States)

    Whitman, Richard L.; Ge, Zhongfu; Nevers, Meredith B.; Boehm, Alexandria B.; Chern, Eunice C.; Haugland, Richard A.; Lukasik, Ashley M.; Molina, Marirosa; Przybyla-Kelly, Kasia; Shively, Dawn A.; White, Emily M.; Zepp, Richard G.; Byappanahalli, Muruleedhara N.

    2010-01-01

    The quantitative polymerase chain reaction (qPCR) method provides rapid estimates of fecal indicator bacteria densities that have been indicated to be useful in the assessment of water quality. Primarily because this method provides faster results than standard culture-based methods, the U.S. Environmental Protection Agency is currently considering its use as a basis for revised ambient water quality criteria. In anticipation of this possibility, we sought to examine the relationship between qPCR-based and culture-based estimates of enterococci in surface waters. Using data from several research groups, we compared enterococci estimates by the two methods in water samples collected from 37 sites across the United States. A consistent linear pattern in the relationship between cell equivalents (CCE), based on the qPCR method, and colony-forming units (CFU), based on the traditional culturable method, was significant (P 10CFU > 2.0/100 mL) while uncertainty increases at lower CFU values. It was further noted that the relative error in replicated qPCR estimates was generally higher than that in replicated culture counts even at relatively high target levels, suggesting a greater need for replicated analyses in the qPCR method to reduce relative error. Further studies evaluating the relationship between culture and qPCR should take into account analytical uncertainty as well as potential differences in results of these methods that may arise from sample variability, different sources of pollution, and environmental factors.

  15. Comparison of ELISA, nested PCR and sequencing and a novel qPCR for detection of Giardia isolates from Jordan.

    Science.gov (United States)

    Hijjawi, Nawal; Yang, Rongchang; Hatmal, Ma'mon; Yassin, Yasmeen; Mharib, Taghrid; Mukbel, Rami; Mahmoud, Sameer Alhaj; Al-Shudifat, Abdel-Ellah; Ryan, Una

    2018-02-01

    Little is known about the prevalence of Giardia duodenalis in human patients in Jordan and all previous studies have used direct microscopy, which lacks sensitivity. The present study developed a novel quantitative PCR (qPCR) assay at the β-giardin (bg) locus and evaluated its use as a frontline test for the diagnosis of giardiasis in comparison with a commercially available ELISA using nested PCR and sequencing of the glutamate dehydrogenase (gdh) locus (gdh nPCR) as the gold standard. A total of 96 human faecal samples were collected from 96 patients suffering from diarrhoea from 5 regions of Jordan and were screened using the ELISA and qPCR. The analytical specificity of the bg qPCR assay revealed no cross-reactions with other genera and detected all the Giardia isolates tested. Analytical sensitivity was 1 Giardia cyst per μl of DNA extract. The overall prevalence of Giardia was 64.6%. The clinical sensitivity and specificity of the bg qPCR was 89.9% and 82.9% respectively compared to 76.5 and 68.0% for the ELISA. This study is the first to compare three different methods (ELISA, bg qPCR, nested PCR and sequencing at the gdh locus) to diagnose Jordanian patients suffering from giardiasis and to analyze their demographic data. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Correlation between real-time qPCR and development of strongyle eggs from cattle

    DEFF Research Database (Denmark)

    Drag, Markus; Nejsum, Peter; Höglund, Johan

    2014-01-01

    Differentiation of veterinary important parasitic strongyle eggs is time-consuming, because morphologically distinct third-stage larvae (L3) must be cultured for species/genus identification. A recently published qPCR technique provides a non-labour intensive method for detection and quantification...... of the two most important nematode eggs in cattle faeces. However, as quantification correlates with DNA content, quantification of copy numbers of the second internal transcribed spacer (ITS2) region is problematic as DNA content increases during egg development. The aim of this study was to assess...... the impact of oxygen availability and temperature on the multiplication of ITS2 copy numbers in O. ostertagi eggs. Fresh eggs were recovered from cattle faeces by sieving, flotation and entrapment in nylon-mesh filters and subsequently deposited in aliquots (n=18) of 5 ml distilled water with air circulation...

  17. Real-time pcr (qpcr) assay for rhizoctonia solani anastomoses group ag2-2 iiib

    International Nuclear Information System (INIS)

    Abbas, S.J.; Ahmad, B.

    2014-01-01

    Rhizoctonia solani anastomosis group AG2-2 IIIB is a severe sugar beet and maize pathogen. It causes crown and root rot disease which leads to yield losses world-wide. The soil-borne pathogen is difficult to detect and quantify by conventional methods. We developed a real-time PCR (qPCR) assay for the quantification of genomic DNA of Rhizoctonia solani AG2-2 IIIB based on the ITS region of rDNA genes. The limit of quantification of the assay is 1.8 pg genomic DNA. The amplification efficiency was 96.4. The assay will be helpful in the diagnoses of Rhizoctonia solani infection of sugar beet and maize roots and in the quantification of R. solani AG2-2 IIIB inoculum in plant debris and soil. (author)

  18. Sampling and Pooling Methods for Capturing Herd Level Antibiotic Resistance in Swine Feces using qPCR and CFU Approaches

    DEFF Research Database (Denmark)

    Schmidt, Gunilla Veslemøy; Mellerup, Anders; Christiansen, Lasse Engbo

    2015-01-01

    The aim of this article was to define the sampling level and method combination that captures antibiotic resistance at pig herd level utilizing qPCR antibiotic resistance gene quantification and culture-based quantification of antibiotic resistant coliform indicator bacteria. Fourteen qPCR assays...... for commonly detected antibiotic resistance genes were developed, and used to quantify antibiotic resistance genes in total DNA from swine fecal samples that were obtained using different sampling and pooling methods. In parallel, the number of antibiotic resistant coliform indicator bacteria was determined...... when comparing individual sampling and pooling methods. qPCR on pooled samples was found to be a good representative for the general resistance level in a pig herd compared to the coliform CFU counts. It had significantly reduced relative standard deviations compared to coliform CFU counts in the same...

  19. Monitoring bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

    Science.gov (United States)

    Eberle, Kirsten C.; Venn-Watson, Stephanie K.; Jensen, Eric D.; Porter, Tracy J.; Waters, Theresa E.; Sacco, Randy E.

    2017-01-01

    Both veterinarians caring for dolphins in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin (Tursiops truncatus) health. Quantitative PCR (qPCR) can be used to assess cytokine transcription patterns of peripheral blood mononuclear cells (PBMC). This can supplement currently available blood tests with information on immune status. Full realization of this potential requires establishment of normal ranges of cytokine gene transcription levels in bottlenose dolphins. We surveyed four dolphins over the span of seven months by serial bleeds. PBMC were stimulated with phytohaemagglutinin (1, 5, and 10 μg/mL) and concanavalin A (1 μg/mL) for 48 H in vitro. RNA from these cultures was probed by qPCR using Tursiops truncatus-specific primers (IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-13, IL-18, IFN-γ and TNF-α). Two blood samples from an additional bottlenose dolphin diagnosed with acute pulmonary disease add further perspective to the data. We observed that mitogen choice made a significant difference in the magnitude of gene transcription observed. On the other hand, most cytokines tested exhibited limited intra-animal variation. However, IL-6 and IL-12p40 differed between older and younger dolphins. Furthermore, the magnitude of mitogenic response clusters the tested cytokines into three groups. The data provide a reference for the selection of target cytokine mRNAs and their expected range of mitogen-stimulated cytokine gene transcription for future studies. PMID:29272269

  20. Molecular Quantification of Zooplankton Gut Content: The Case For qPCR

    Science.gov (United States)

    Frischer, M. E.; Walters, T. L.; Gibson, D. M.; Nejstgaard, J. C.; Troedsson, C.

    2016-02-01

    The ability to obtain information about feeding selectivity and rates in situ for zooplankton is vital for understanding the mechanisms structuring marine ecosystems. However, directly estimating feeding selection and rates of zooplankton, without bias, associated with culturing conditions has been notoriously difficult. A potential approach for addressing this problem is to target prey-specific DNA as a marker for prey ingestion and selection. In this study we report the development of a differential length amplification quantitative PCR (dla-qPCR) assay targeting the 18S rRNA gene to validate the use of a DNA-based approach to quantify consumption of specific plankton prey by the pelagic tunicate (doliolid) Dolioletta gegenbauri. Compared to copepods and other marine animals, the digestion of prey genomic DNA inside the gut of doliolids is low. This method minimizes potential underestimations, and therefore allows prey DNA to be used as an effective indicator of prey consumption. We also present an initial application of a qPCR-assay to estimate consumption of specific prey species on the southeastern continental shelf of the U.S., where doliolids stochastically bloom in response to upwelling events. Estimated feeding rates, based on qPCR, were in the same range as those estimated from clearance rates in laboratory feeding studies. In the field, consumption of specific prey, including the centric diatom Thalassiosira spp. was detected in the gut of wild caught D. gegenbauri at the levels consistent with their abundance in the water column at the time of collection. Thus, both experimental and field investigations support the hypothesis that a qPCR approach will be useful for the quantitative investigation of the in situ diet of D. gegenbauri without introduced bias' associated with cultivation.

  1. Monitoring bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR.

    Directory of Open Access Journals (Sweden)

    Amelia Ruth Hofstetter

    Full Text Available Both veterinarians caring for dolphins in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin (Tursiops truncatus health. Quantitative PCR (qPCR can be used to assess cytokine transcription patterns of peripheral blood mononuclear cells (PBMC. This can supplement currently available blood tests with information on immune status. Full realization of this potential requires establishment of normal ranges of cytokine gene transcription levels in bottlenose dolphins. We surveyed four dolphins over the span of seven months by serial bleeds. PBMC were stimulated with phytohaemagglutinin (1, 5, and 10 μg/mL and concanavalin A (1 μg/mL for 48 H in vitro. RNA from these cultures was probed by qPCR using Tursiops truncatus-specific primers (IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-13, IL-18, IFN-γ and TNF-α. Two blood samples from an additional bottlenose dolphin diagnosed with acute pulmonary disease add further perspective to the data. We observed that mitogen choice made a significant difference in the magnitude of gene transcription observed. On the other hand, most cytokines tested exhibited limited intra-animal variation. However, IL-6 and IL-12p40 differed between older and younger dolphins. Furthermore, the magnitude of mitogenic response clusters the tested cytokines into three groups. The data provide a reference for the selection of target cytokine mRNAs and their expected range of mitogen-stimulated cytokine gene transcription for future studies.

  2. RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR.

    Science.gov (United States)

    Gentile, Adriana-Mariel; Lhamyani, Said; Coín-Aragüez, Leticia; Oliva-Olivera, Wilfredo; Zayed, Hatem; Vega-Rioja, Antonio; Monteseirin, Javier; Romero-Zerbo, Silvana-Yanina; Tinahones, Francisco-José; Bermúdez-Silva, Francisco-Javier; El Bekay, Rajaa

    2016-01-01

    Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR.

  3. Detection of Legionella by quantitative-polymerase chain reaction (qPCR) for monitoring and risk assessment

    DEFF Research Database (Denmark)

    Krøjgaard, Louise H.; Krogfelt, Karen A.; Albrechtsen, Hans-Jorgen

    2011-01-01

    Background: Culture and quantitative polymerase chain reaction (qPCR) assays for the detection of Legionella were compared on samples from a residential area before and after two interventions. A total of 84 samples were collected from shower hoses and taps as first flush samples and at constant...... temperature. Samples were grouped according to the origin of the sample, a) circulation water b) water from empty apartments c) water from shower hoses. The aims were to investigate the usefulness of qPCR compared to culture for monitoring remedial actions for elimination of Legionella bacteria and as a tool...

  4. Comparison of individual and pooled samples for quantification of antimicrobial resistance genes in swine feces by high-throughput qPCR

    DEFF Research Database (Denmark)

    Clasen, Julie; Mellerup, Anders; Olsen, J. E.

    2015-01-01

    There is a considerable societal interest in the careful monitoring of antimicrobial resistance (AMR) levels in human and animal populations. Sampling and data analysis can be both costly and time consuming. Optimization of sample pooling procedures is therefore important to reduce costs...... and analysis times. The objective of this study was to estimate how many individual fecal samples are needed to pool to get a representative sample for quantification of AMR-genes in a Danish pig herd. 20 individual fecal samples were collected from one section in a Danish pig herd. One to five rectal fecal...... samples were taken from each pen with respect to the number of pigs in the pen. A total of 48 pools were made of increasing number of individual samples. The levels of 9 different AMR-genes were quantified using dynamic qPCR arrays on the BioMark HD system(Fluidigm®).DNA was extracted using the Maxwell...

  5. Sizing up arthropod genomes: an evaluation of the impact of environmental variation on genome size estimates by flow cytometry and the use of qPCR as a method of estimation.

    Science.gov (United States)

    Gregory, T Ryan; Nathwani, Paula; Bonnett, Tiffany R; Huber, Dezene P W

    2013-09-01

    A study was undertaken to evaluate both a pre-existing method and a newly proposed approach for the estimation of nuclear genome sizes in arthropods. First, concerns regarding the reliability of the well-established method of flow cytometry relating to impacts of rearing conditions on genome size estimates were examined. Contrary to previous reports, a more carefully controlled test found negligible environmental effects on genome size estimates in the fly Drosophila melanogaster. Second, a more recently touted method based on quantitative real-time PCR (qPCR) was examined in terms of ease of use, efficiency, and (most importantly) accuracy using four test species: the flies Drosophila melanogaster and Musca domestica and the beetles Tribolium castaneum and Dendroctonus ponderosa. The results of this analysis demonstrated that qPCR has the tendency to produce substantially different genome size estimates from other established techniques while also being far less efficient than existing methods.

  6. Barcoded pyrosequencing analysis of the microbial community in a simulator of the human gastrointestinal tract showed a colon region-specific microbiota modulation for two plant-derived polysaccharide blends.

    Science.gov (United States)

    Marzorati, Massimo; Maignien, Lois; Verhelst, An; Luta, Gabriela; Sinnott, Robert; Kerckhof, Frederiek Maarten; Boon, Nico; Van de Wiele, Tom; Possemiers, Sam

    2013-02-01

    The combination of a Simulator of the Human Intestinal Microbial Ecosystem with ad hoc molecular techniques (i.e. pyrosequencing, denaturing gradient gel electrophoresis and quantitative PCR) allowed an evaluation of the extent to which two plant polysaccharide supplements could modify a complex gut microbial community. The presence of Aloe vera gel powder and algae extract in product B as compared to the standard blend (product A) improved its fermentation along the entire simulated colon. The potential extended effect of product B in the simulated distal colon, as compared to product A, was confirmed by: (i) the separate clustering of the samples before and after the treatment in the phylogenetic-based dendrogram and OTU-based PCoA plot only for product B; (ii) a higher richness estimator (+33 vs. -36 % of product A); and (iii) a higher dynamic parameter (21 vs. 13 %). These data show that the combination of well designed in vitro simulators with barcoded pyrosequencing is a powerful tool for characterizing changes occurring in the gut microbiota following a treatment. However, for the quantification of low-abundance species-of interest because of their relationship to potential positive health effects (i.e. bifidobacteria or lactobacilli)-conventional molecular ecological approaches, such as PCR-DGGE and qPCR, still remain a very useful complementary tool.

  7. SISH/CISH or qPCR as alternative techniques to FISH for determination of HER2 amplification status on breast tumors core needle biopsies: a multicenter experience based on 840 cases.

    Science.gov (United States)

    Jacquemier, Jocelyne; Spyratos, Frédérique; Esterni, Benjamin; Mozziconacci, Marie-Joëlle; Antoine, Martine; Arnould, Laurent; Lizard, Sarab; Bertheau, Philippe; Lehmann-Che, Jacqueline; Fournier, Cécile Blanc; Krieger, Sophie; Bibeau, Frédéric; Lamy, Pierre-Jean; Chenard, Marie Pierre; Legrain, Michèle; Guinebretière, Jean-Marc; Loussouarn, Delphine; Macgrogan, Gaëtan; Hostein, Isabelle; Mathieu, Marie Christine; Lacroix, Ludovic; Valent, Alexander; Robin, Yves Marie; Revillion, Françoise; Triki, Magali Lacroix; Seaume, Aline; Salomon, Anne Vincent; de Cremoux, Patricia; Portefaix, Geneviève; Xerri, Luc; Vacher, Sophie; Bièche, Ivan; Penault-Llorca, Frédérique

    2013-07-22

    Until now, FISH has been the gold standard technique to identify HER2 amplification status in ambiguous cases of breast cancer. Alternative techniques have been developed to increase the capacities of investigating HER2 amplification status. The aims of this multicenter study in a large series of breast cancer patients were to prospectively compare the level of performance of CISH, SISH, and qPCR alternative techniques on paraffin-embedded core biopsies with "gold standard FISH" for evaluation of HER2 amplification status. This study was performed on 840 cases scored by immunohistochemistry (IHC): 0=317 (38%), 1+=183 (22%), 2+=109 (13%), 3+=231 (27%). Each of the 15 French centers participating in the study analyzed 56 breast carcinoma cases diagnosed on fixed paraffin-embedded core biopsies. HER2 amplification status was determined by commercially available FISH used as the reference technique with determination of the HER2/CEN17 ratio or HER2 copy number status. The alternative techniques performed on the same cases were commercially available SISH or CISH and a common qPCR method especially designed for the study including a set of 10 primer pairs: 2 for HER2 (exons 8 and 26), 5 to evaluate chromosome 17 polysomy TAOK1, UTP6, MRM1, MKS1, SSTR2 and 3 for diploidy control TSN, LAP3 and ADAMTS16. The concordance between IHC and FISH was 96% to 95% based on the HER2/CEN17 ratio (n=766) or HER2 copy number (n=840), respectively. The concordance of the alternative techniques with FISH was excellent: 97% and 98% for SISH (498 and 587 cases), 98% and 75% for CISH (108 and 204 cases) and 95% and 93% (699 and 773 cases) for qPCR based on the HER2/CEN17 ratio or HER2 copy number, respectively. Similarly, sensitivity ranged from 99% to 95% for SISH, 100% to 99% for CISH and 89% to 80% for qPCR. The concordance with FISH (ratio) in the 2+ cases was 89% for SISH, 100% for CISH and 93% for qPCR. These alternative techniques showed an excellent concordance with FISH in core

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

    NARCIS (Netherlands)

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

    2016-01-01

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

  9. Detection and discrimination of five E. coli pathotypes using a combinatory SYBR® Green qPCR screening system.

    Science.gov (United States)

    Barbau-Piednoir, Elodie; Denayer, Sarah; Botteldoorn, Nadine; Dierick, Katelijne; De Keersmaecker, Sigrid C J; Roosens, Nancy H

    2018-04-01

    A detection and discrimination system for five Escherichia coli pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic E. coli (CoSYPS Path E. coli). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of E. coli: shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) E. coli. The SYBR® Green qPCR assays target the uidA, ipaH, eae, aggR, aaiC, stx1, and stx2 genes. uidA controls for E. coli presence and all the other genes are specific targets of E. coli pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs' design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path E. coli system was subsequently evaluated on four food matrices artificially contaminated with pathogenic E. coli. It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.

  10. Analytical and diagnostic performance of a qPCR assay for Ichthyophonus spp. compared to the tissue culture 'gold standard'.

    Science.gov (United States)

    Lowe, Vanessa C; Hershberger, Paul K; Friedman, Carolyn S

    2018-06-04

    Parasites of the genus Ichthyophonus infect many fish species and have a non-uniform distribution within host tissues. Due in part to this uneven distribution, the comparative sensitivity and accuracy of using molecular-based detection methods versus culture to estimate parasite prevalence is under debate. We evaluated the analytical and diagnostic performance of an existing qPCR assay in comparison to the 'gold standard' culture method using Pacific herring Clupea pallasii with known exposure history. We determined that the assay is suitable for use in this host, and diagnostic specificity was consistently high (>98%) in both heart and liver tissues. Diagnostic sensitivity could not be fully assessed due to low infection rates, but our results suggest that qPCR is not as sensitive as culture under all circumstances. Diagnostic sensitivity of qPCR relative to culture is likely affected by the amount of sample processed. The prevalence values estimated by the 2 methods were not significantly different when sample amounts were equal (heart tissue), but when the assayed sample amounts were unequal (liver tissue), the culture method detected a significantly higher prevalence of the parasite than qPCR. Further, culture of liver also detected significantly more Ichthyophonus infections than culture of heart, suggesting that the density and distribution of parasites in tissues also plays a role in assay sensitivity. This sensitivity issue would be most problematic for fish with light infections. Although qPCR does not detect the presence of a live organism, DNA-based pathogen detection methods provide the opportunity for alternate testing strategies when culture is not possible.

  11. #13ReasonsWhy Health Professionals and Educators are Tweeting: A Systematic Analysis of Uses and Perceptions of Show Content and Learning Outcomes.

    Science.gov (United States)

    Walker, Kimberly K; Burns, Kelli

    2018-04-27

    This study is a content analysis of health professionals' and educators' tweets about a popular Netflix show that depicts teen suicide: 13 Reasons Why. A content analysis of 740 tweets was conducted to determine the main themes associated with professionals' and educators' tweets about the show, as well as the valence of the tweets. Additionally, a thematic analysis of linked content in tweets (n = 178) was conducted to explore additional content shared about the show and modeling outcomes. Results indicated the largest percentage of tweets was related to social learning, particularly about outcomes that could occur from viewing the show. The valence of the tweets about outcomes was more positive than negative. However, linked materials commonly circulated in tweets signified greater concern with unintended learning outcomes. Some of the linked content included media guidelines for reporting on suicide with recommendations that entertainment producers follow the guidelines. This study emphasizes the importance of including social learning objectives in future typologies of Twitter uses and demonstrates the importance of examining linked content in Twitter studies.

  12. Detection of Replication Competent Lentivirus Using a qPCR Assay for VSV-G

    Directory of Open Access Journals (Sweden)

    Lindsey M. Skrdlant

    2018-03-01

    Full Text Available Lentiviral vectors are a common tool used to introduce new and corrected genes into cell therapy products for treatment of human diseases. Although lentiviral vectors are ideal for delivery and stable integration of genes of interest into the host cell genome, they potentially pose risks to human health, such as integration-mediated transformation and generation of a replication competent lentivirus (RCL capable of infecting non-target cells. In consideration of the latter risk, all cell-based products modified by lentiviral vectors and intended for patient use must be tested for RCL prior to treatment of the patient. Current Food and Drug Administration (FDA guidelines recommend use of cell-based assays to this end, which can take up to 6 weeks for results. However, qPCR-based assays are a quick alternative for rapid assessment of RCL in products intended for fresh infusion. We describe here the development and qualification of a qPCR assay based on detection of envelope gene sequences (vesicular stomatitis virus G glycoprotein [VSV-G] for RCL in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE guidelines. Our results demonstrate the sensitivity, linearity, specificity, and reproducibility of detection of VSV-G sequences, with a low false-positive rate. These procedures are currently being used in our phase 1 clinical investigations.

  13. Effect of the addition of phytosterols and tocopherols on Streptococcus thermophilus robustness during industrial manufacture and ripening of a functional cheese as evaluated by qPCR and RT-qPCR.

    Science.gov (United States)

    Pega, J; Rizzo, S; Pérez, C D; Rossetti, L; Díaz, G; Ruzal, S M; Nanni, M; Descalzo, A M

    2016-09-02

    The quality of functional food products designed for the prevention of degenerative diseases can be affected by the incorporation of bioactive compounds. In many types of cheese, the performance of starter microorganisms is critical for optimal elaboration and for providing potential probiotic benefits. Phytosterols are plant lipophilic triterpenes that have been used for the design of functional dairy products because of their ability to lower serum cholesterol levels in humans. However, their effect on the starter culture behavior during cheesemaking has not yet been studied. Here, we followed DNA and RNA kinetics of the bacterium Streptococcus thermophilus, an extensively used dairy starter with probiotic potential, during industrial production of a functional, semi-soft, reduced-fat cheese containing phytosterol esters and alpha-tocopherol as bioactive compounds. For this purpose, real-time quantitative PCR (qPCR) and reverse transcription-qPCR (RT-qPCR) assays were optimized and applied to samples obtained during the manufacture and ripening of functional and control cheeses. An experimental set-up was used to evaluate the detection threshold of free nucleic acids for extraction protocols based on pelleted microorganisms. To our knowledge, this straight-forward approach provides the first experimental evidence indicating that DNA is not a reliable marker of cell integrity, whereas RNA may constitute a more accurate molecular signature to estimate both bacterial viability and metabolic activity. Compositional analysis revealed that the bioactive molecules were effectively incorporated into the cheese matrix, at levels considered optimal to exert their biological action. The starter S. thermophilus was detected by qPCR and RT-qPCR during cheese production at the industrial level, from at least 30min after its inoculation until 81days of ripening, supporting the possible role of this species in shaping organoleptic profiles. We also showed for the first time that

  14. Validation of chimerism in pediatric recipients of allogeneic hematopoietic stem cell transplantation (HSCT) a comparison between two methods: real-time PCR (qPCR) vs. variable number tandem repeats PCR (VNTR PCR).

    Science.gov (United States)

    Kletzel, Morris; Huang, Wei; Olszewski, Marie; Khan, Sana

    2013-01-01

    Post-hematopoietic stem cell transplantation (HSCT) chimerism monitoring is important to assess relapse and therapeutic intervention. The purpose of our study is to compare two methods variable number tandem repeats (VNTR) vs. quantitative real- time polymerase chain reaction (qPCR) in terms of determining chimerism. 127 (peripheral blood n=112, bone marrow n=15) samples were simultaneously tested by VNTR using APO-B, D1S80, D1S111, D17S30, gene loci SRY and ZP3 and qPCR using 34 assays (CA001-CA034) that are designed to a bi-allelic insertion/deletion (indel) polymorphism in the human genome. Samples were separated in three subsets: total WBC, T-cell and Myeloid cells. Extraction of DNA was performed then quantified. We analyzed column statistics, paired t-test and regression analysis for both methods. There was complete correlation between the two methods. The simplicity and rapidity of the test results from the qPCR method is more efficient and accurate to assess chimerism.

  15. Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods

    DEFF Research Database (Denmark)

    Olsen, Morten Tange; Bérubé, Martine; Robbins, Jooke

    2012-01-01

    BACKGROUND:Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent...... steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay. RESULTS...... to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays. CONCLUSION:Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control...

  16. Use of next generation sequencing data to develop a qPCR method for specific detection of EU-unauthorized genetically modified Bacillus subtilis overproducing riboflavin.

    Science.gov (United States)

    Barbau-Piednoir, Elodie; De Keersmaecker, Sigrid C J; Delvoye, Maud; Gau, Céline; Philipp, Patrick; Roosens, Nancy H

    2015-11-11

    Recently, the presence of an unauthorized genetically modified (GM) Bacillus subtilis bacterium overproducing vitamin B2 in a feed additive was notified by the Rapid Alert System for Food and Feed (RASFF). This has demonstrated that a contamination by a GM micro-organism (GMM) may occur in feed additives and has confronted for the first time,the enforcement laboratories with this type of RASFF. As no sequence information of this GMM nor any specific detection or identification method was available, Next GenerationSequencing (NGS) was used to generate sequence information. However, NGS data analysis often requires appropriate tools, involving bioinformatics expertise which is not alwayspresent in the average enforcement laboratory. This hampers the use of this technology to rapidly obtain critical sequence information in order to be able to develop a specific qPCRdetection method. Data generated by NGS were exploited using a simple BLAST approach. A TaqMan® qPCR method was developed and tested on isolated bacterial strains and on the feed additive directly. In this study, a very simple strategy based on the common BLAST tools that can be used by any enforcement lab without profound bioinformatics expertise, was successfully used toanalyse the B. subtilis data generated by NGS. The results were used to design and assess a new TaqMan® qPCR method, specifically detecting this GM vitamin B2 overproducing bacterium. The method complies with EU critical performance parameters for specificity, sensitivity, PCR efficiency and repeatability. The VitB2-UGM method also could detect the B. subtilis strain in genomic DNA extracted from the feed additive, without prior culturing step. The proposed method, provides a crucial tool for specifically and rapidly identifying this unauthorized GM bacterium in food and feed additives by enforcement laboratories. Moreover, this work can be seen as a case study to substantiate how the use of NGS data can offer an added value to easily

  17. Characterization of relative abundance of lactic acid bacteria species in French organic sourdough by cultural, qPCR and MiSeq high-throughput sequencing methods.

    Science.gov (United States)

    Michel, Elisa; Monfort, Clarisse; Deffrasnes, Marion; Guezenec, Stéphane; Lhomme, Emilie; Barret, Matthieu; Sicard, Delphine; Dousset, Xavier; Onno, Bernard

    2016-12-19

    In order to contribute to the description of sourdough LAB composition, MiSeq sequencing and qPCR methods were performed in association with cultural methods. A panel of 16 French organic bakers and farmer-bakers were selected for this work. The lactic acid bacteria (LAB) diversity of their organic sourdoughs was investigated quantitatively and qualitatively combining (i) Lactobacillus sanfranciscensis-specific qPCR, (ii) global sequencing with MiSeq Illumina technology and (iii) molecular isolates identification. In addition, LAB and yeast enumeration, pH, Total Titratable Acidity, organic acids and bread specific volume were analyzed. Microbial and physico-chemical data were statistically treated by Principal Component Analysis (PCA) and Hierarchical Ascendant Classification (HAC). Total yeast counts were 6 log 10 to 7.6 log 10 CFU/g while LAB counts varied from 7.2 log 10 to 9.6 log 10 CFU/g. Values obtained by L. sanfranciscensis-specific qPCR were estimated between 7.2 and 10.3 log 10 CFU/g, except for one sample at 4.4 log 10 CFU/g. HAC and PCA clustered the sixteen sourdoughs into three classes described by their variables but without links to bakers' practices. L. sanfranciscensis was the dominant species in 13 of the 16 sourdoughs analyzed by Next Generation Sequencing (NGS), by the culture dependent method this species was dominant only in only 10 samples. Based on isolates identification, LAB diversity was higher for 7 sourdoughs with the recovery of L. curvatus, L. brevis, L. heilongjiangensis, L. xiangfangensis, L. koreensis, L. pontis, Weissella sp. and Pediococcus pentosaceus, as the most representative species. L. koreensis, L. heilongjiangensis and L. xiangfangensis were identified in traditional Asian food and here for the first time as dominant in organic sourdough. This study highlighted that L. sanfranciscensis was not the major species in 6/16 sourdough samples and that a relatively high LAB diversity can be observed in French organic

  18. Systematic analysis of DEMETER-like DNA glycosylase genes shows lineage-specific Smi-miR7972 involved in SmDML1 regulation in Salvia miltiorrhiza.

    Science.gov (United States)

    Li, Jiang; Li, Caili; Lu, Shanfa

    2018-05-08

    DEMETER-like DNA glycosylases (DMLs) initiate the base excision repair-dependent DNA demethylation to regulate a wide range of biological processes in plants. Six putative SmDML genes, termed SmDML1-SmDML6, were identified from the genome of S. miltiorrhiza, an emerging model plant for Traditional Chinese Medicine (TCM) studies. Integrated analysis of gene structures, sequence features, conserved domains and motifs, phylogenetic analysis and differential expression showed the conservation and divergence of SmDMLs. SmDML1, SmDML2 and SmDML4 were significantly down-regulated by the treatment of 5Aza-dC, a general DNA methylation inhibitor, suggesting involvement of SmDMLs in genome DNA methylation change. SmDML1 was predicted and experimentally validated to be target of Smi-miR7972. Computational analysis of forty whole genome sequences and almost all of RNA-seq data from Lamiids revealed that MIR7972s were only distributed in some plants of the three orders, including Lamiales, Solanales and Boraginales, and the number of MIR7972 genes varied among species. It suggests that MIR7972 genes underwent expansion and loss during the evolution of some Lamiids species. Phylogenetic analysis of MIR7972s showed closer evolutionary relationships between MIR7972s in Boraginales and Solanales in comparison with Lamiales. These results provide a valuable resource for elucidating DNA demethylation mechanism in S. miltiorrhiza.

  19. Evaluation of the efficiency of nested q-PCR in the detection of Mycobacterium tuberculosis complex directly from tuberculosis-suspected lesions in post-mortem macroscopic inspections of bovine carcasses slaughtered in the state of Mato Grosso, Brazil.

    Science.gov (United States)

    Carvalho, Ricardo César Tavares; Furlanetto, Leone Vinícius; Maruyama, Fernanda Harumy; Araújo, Cristina Pires de; Barros, Sílvia Letícia Bomfim; Ramos, Carlos Alberto do Nascimento; Dutra, Valéria; Araújo, Flábio Ribeiro de; Paschoalin, Vânia Margaret Flosi; Nakazato, Luciano; Figueiredo, Eduardo Eustáquio de Souza

    2015-08-01

    Bovine tuberculosis (BTB) is a zoonotic disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTC). The quick and specific detection of this species is of extreme importance, since BTB may cause economic impacts, in addition to presenting imminent risks to human health. In the present study a nested real-time PCR test (nested q-PCR) was used in post-mortem evaluations to assess cattle carcasses with BTB-suspected lesions. A total of 41,193 cattle slaughtered in slaughterhouses located in the state of Mato Grosso, were examined. Of the examined animals, 198 (0.48%) showed BTB-suspected lesions. M. bovis was isolated in 1.5% (3/198) of the samples. Multiplex-PCR detected MTC in 7% (14/198) of the samples. The nested q-PCR test detected MTC in 28% (56/198) of the BTB-suspected lesions, demonstrating higher efficiency when compared to the multiplex-PCR and conventional microbiology. Nested q-PCR can therefore be used as a complementary test in the national program for control and eradication of bovine tuberculosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Reference gene validation for qPCR in rat carotid body during postnatal development

    Directory of Open Access Journals (Sweden)

    Carroll John L

    2011-10-01

    Full Text Available Abstract Background The carotid bodies are the main arterial oxygen chemoreceptors in mammals. Afferent neural output from the carotid bodies to brainstem respiratory and cardiovascular nuclei provides tonic input and mediates important protective responses to acute and chronic hypoxia. It is widely accepted that the selection of reference genes for mRNA normalization in quantitative real-time PCR must be validated for a given tissue and set of conditions. This is particularly important for studies in carotid body during early postnatal maturation as the arterial oxygen tension undergoes major changes from fetal to postnatal life, which may affect reference gene expression. In order to determine the most stable and suitable reference genes for the study of rat carotid body during development, six commonly used reference genes, β-actin, RPII (RNA polymerase II, PPIA (peptidyl-proyl-isomerase A, TBP (TATA-box binding protein, GAPDH, and 18s rRNA, were evaluated in two age groups (P0-1 and P14-16 under three environmental oxygen conditions (normoxia, chronic hypoxia and chronic hyperoxia using the three most commonly used software programs, geNorm, NormFinder and BestKeeper. Findings The three programs produced similar results but the reference gene rankings were not identical between programs or experimental conditions. Overall, 18s rRNA was the least stable reference gene for carotid body and, when hyperoxia and/or hypoxia conditions were included, actin was similarly unstable. Conclusions Reference or housekeeping gene expression for qPCR studies of carotid body during postnatal development may vary with developmental stage and environmental conditions. Selection of the best reference gene or combination of reference genes for carotid body development studies should take environmental conditions into account. Two commonly used reference genes, 18s rRNA and actin, may be unsuitable for studies of carotid body maturation, especially if the study

  1. A randomized and blinded comparison of qPCR and NGS-based detection of aneuploidy in a cell line mixture model of blastocyst biopsy mosaicism.

    Science.gov (United States)

    Goodrich, David; Tao, Xin; Bohrer, Chelsea; Lonczak, Agnieszka; Xing, Tongji; Zimmerman, Rebekah; Zhan, Yiping; Scott, Richard T; Treff, Nathan R

    2016-11-01

    A subset of preimplantation stage embryos may possess mosaicism of chromosomal constitution, representing a possible limitation to the clinical predictive value of comprehensive chromosome screening (CCS) from a single biopsy. However, contemporary methods of CCS may be capable of predicting mosaicism in the blastocyst by detecting intermediate levels of aneuploidy within a trophectoderm biopsy. This study evaluates the sensitivity and specificity of aneuploidy detection by two CCS platforms using a cell line mixture model of a mosaic trophectoderm biopsy. Four cell lines with known karyotypes were obtained and mixed together at specific ratios of six total cells (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0). A female euploid and a male trisomy 18 cell line were used for one set, and a male trisomy 13 and a male trisomy 15 cell line were used for another. Replicates of each mixture were prepared, randomized, and blinded for analysis by one of two CCS platforms (quantitative polymerase chain reaction (qPCR) or VeriSeq next-generation sequencing (NGS)). Sensitivity and specificity of aneuploidy detection at each level of mosaicism was determined and compared between platforms. With the default settings for each platform, the sensitivity of qPCR and NGS were not statistically different, and 100 % specificity was observed (no false positives) at all levels of mosaicism. However, the use of previously published custom criteria for NGS increased sensitivity but also significantly decreased specificity (33 % false-positive prediction of aneuploidy). By demonstrating increased false-positive diagnoses when reducing the stringency of predicting an abnormality, these data illustrate the importance of preclinical evaluation of new testing paradigms before clinical implementation.

  2. Whole Genome Sequencing and Multiplex qPCR Methods to Identify Campylobacter jejuni Encoding cst-II or cst-III Sialyltransferase

    Directory of Open Access Journals (Sweden)

    Jason M. Neal-McKinney

    2018-03-01

    Full Text Available Campylobacter jejuni causes more than 2 million cases of gastroenteritis annually in the United States, and is also linked to the autoimmune sequelae Guillan–Barre syndrome (GBS. GBS often results in flaccid paralysis, as the myelin sheaths of nerve cells are degraded by the adaptive immune response. Certain strains of C. jejuni modify their lipooligosaccharide (LOS with the addition of neuraminic acid, resulting in LOS moieties that are structurally similar to gangliosides present on nerve cells. This can trigger GBS in a susceptible host, as antibodies generated against C. jejuni can cross-react with gangliosides, leading to demyelination of nerves and a loss of signal transduction. The goal of this study was to develop a quantitative PCR (qPCR method and use whole genome sequencing data to detect the Campylobactersialyltransferase (cst genes responsible for the addition of neuraminic acid to LOS. The qPCR method was used to screen a library of 89 C. jejuni field samples collected by the Food and Drug Administration Pacific Northwest Lab (PNL as well as clinical isolates transferred to PNL. In silico analysis was used to screen 827 C. jejuni genomes in the FDA GenomeTrakr SRA database. The results indicate that a majority of C. jejuni strains could produce LOS with ganglioside mimicry, as 43.8% of PNL isolates and 46.9% of the GenomeTrakr isolates lacked the cst genes. The methods described in this study can be used by public health laboratories to rapidly determine whether a C. jejuni isolate has the potential to induce GBS. Based on these results, a majority of C. jejuni in the PNL collection and submitted to GenomeTrakr have the potential to produce LOS that mimics human gangliosides.

  3. Microarray detection and qPCR screening of potential biomarkers of Folsomia candida (Collembola: Isotomidae) exposed to Bt proteins (Cry1Ab and Cry1Ac)

    International Nuclear Information System (INIS)

    Yuan, Yiyang; Krogh, Paul Henning; Bai, Xue; Roelofs, Dick; Chen, Fajun; Zhu-Salzman, Keyan; Liang, Yuyong; Sun, Yucheng; Ge, Feng

    2014-01-01

    The impact of Bt proteins on non-target arthropods is less understood than their effects on target organisms where the mechanism of toxic action is known. Here, we report the effects of two Bt proteins, Cry1Ab and Cry1Ac, on gene expression in the non-target collembolan, Folsomia candida. A customized microarray was used to study gene expression in F. candida specimens that were exposed to Cry1Ab and Cry1Ac. All selected transcripts were subsequently confirmed by qPCR. Eleven transcripts were finally verified, and three of them were annotated. The responses of all eleven transcripts were tested in specimens for both Cry1Ab and Cry1Ac at a series of concentrations. These transcripts were separated into two and three groups for Cry1Ab and Cry1Ac, respectively, depend on their expression levels. However, those eleven transcripts did not respond to the Bt proteins in Bt-rice residues. -- Highlights: • We examined the effects of Bt proteins on gene expression of Folsomia candida. • Eleven transcripts were up-regulated by Bt proteins (Cry1Ab and Cry1Ac). • Only three of the eleven transcripts were annotated. • The responses of 11 transcripts were tested on both Cry1Ab and Cry1Ac. • These transcripts did not respond to the Bt proteins in Bt-rice residues. -- Eleven potential molecular biomarkers of Folsomia candida to Cry1Ab and Cry1Ac were screened by microarray and qPCR analysis

  4. Meta-analysis of diffusion tensor imaging (DTI) studies shows altered fractional anisotropy occurring in distinct brain areas in association with depression

    LENUS (Irish Health Repository)

    Murphy, Melissa L

    2011-09-27

    Abstract Fractional anisotropy anomalies occurring in the white matter tracts in the brains of depressed patients may reflect microstructural changes underlying the pathophysiology of this disorder. We conducted a meta-analysis of fractional anisotropy abnormalities occurring in major depressive disorder using voxel-based diffusion tensor imaging studies. Using the Embase, PubMed and Google Scholar databases, 89 relevant data sets were identified, of which 7 (including 188 patients with major depressive disorder and 221 healthy controls) met our inclusion criteria. Authors were contacted to retrieve any additional data required. Coordinates were extracted from clusters of significant white matter fractional anisotropy differences between patients and controls. Relevant demographic, clinical and methodological variables were extracted from each study or obtained directly from authors. The meta-analysis was carried out using Signed Differential Mapping. Patients with depression showed decreased white matter fractional anisotropy values in the superior longitudinal fasciculus and increased fractional anisotropy values in the fronto-occipital fasciculus compared to controls. Using quartile and jackknife sensitivity analysis, we found that reduced fractional anisotropy in the left superior longitudinal fasciculus was very stable, with increases in the right fronto-occipital fasciculus driven by just one study. In conclusion, our meta-analysis revealed a significant reduction in fractional anisotropy values in the left superior longitudinal fasciculus, which may ultimately play an important role in the pathology of depression.

  5. Meta-analysis of diffusion tensor imaging studies shows altered fractional anisotropy occurring in distinct brain areas in association with depression.

    LENUS (Irish Health Repository)

    Murphy, Melissa L

    2011-09-01

    Fractional anisotropy anomalies occurring in the white matter tracts in the brains of depressed patients may reflect microstructural changes underlying the pathophysiology of this disorder. We conducted a meta-analysis of fractional anisotropy abnormalities occurring in major depressive disorder using voxel-based diffusion tensor imaging studies. Using the Embase, PubMed and Google Scholar databases, 89 relevant data sets were identified, of which 7 (including 188 patients with major depressive disorder and 221 healthy controls) met our inclusion criteria. Authors were contacted to retrieve any additional data required. Coordinates were extracted from clusters of significant white matter fractional anisotropy differences between patients and controls. Relevant demographic, clinical and methodological variables were extracted from each study or obtained directly from authors. The meta-analysis was carried out using Signed Differential Mapping. Patients with depression showed decreased white matter fractional anisotropy values in the superior longitudinal fasciculus and increased fractional anisotropy values in the fronto-occipital fasciculus compared to controls. Using quartile and jackknife sensitivity analysis, we found that reduced fractional anisotropy in the left superior longitudinal fasciculus was very stable, with increases in the right fronto-occipital fasciculus driven by just one study. In conclusion, our meta-analysis revealed a significant reduction in fractional anisotropy values in the left superior longitudinal fasciculus, which may ultimately play an important role in the pathology of depression.

  6. Use of high throughput qPCR screening to rapidly clone low frequency tumour specific T-cells from peripheral blood for adoptive immunotherapy

    Directory of Open Access Journals (Sweden)

    Serrano Oscar K

    2008-10-01

    Full Text Available Abstract Background The adoptive transfer of autologous tumor reactive lymphocytes can mediate significant tumor regression in some patients with refractory metastatic cancer. However, a significant obstacle for this promising therapy has been the availability of highly efficient methods to rapidly isolate and expand a variety of potentially rare tumor reactive lymphocytes from the natural repertoire of cancer patients. Methods We developed a novel in vitro T cell cloning methodology using high throughput quantitative RT-PCR (qPCR assay as a rapid functional screen to detect and facilitate the limiting dilution cloning of a variety of low frequency T cells from bulk PBMC. In preclinical studies, this strategy was applied to the isolation and expansion of gp100 specific CD8+ T cell clones from the peripheral blood of melanoma patients. Results In optimization studies, the qPCR assay could detect the reactivity of 1 antigen specific T cell in 100,000 background cells. When applied to short term sensitized PBMC microcultures, this assay could detect T cell reactivity against a variety of known melanoma tumor epitopes. This screening was combined with early limiting dilution cloning to rapidly isolate gp100154–162 reactive CD8+ T cell clones. These clones were highly avid against peptide pulsed targets and melanoma tumor lines. They had an effector memory phenotype and showed significant proliferative capacity to reach cell numbers appropriate for adoptive transfer trials (~1010 cells. Conclusion This report describes a novel high efficiency strategy to clone tumor reactive T cells from peripheral blood for use in adoptive immunotherapy.

  7. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

    Directory of Open Access Journals (Sweden)

    Susan D'Costa

    2016-01-01

    Full Text Available Clinical trials using recombinant adeno-associated virus (rAAV vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR is now the most common method to titer vector genomes (vg; however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new “Free-ITR” qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.

  8. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR.

    Science.gov (United States)

    D'Costa, Susan; Blouin, Veronique; Broucque, Frederic; Penaud-Budloo, Magalie; François, Achille; Perez, Irene C; Le Bec, Christine; Moullier, Philippe; Snyder, Richard O; Ayuso, Eduard

    2016-01-01

    Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new "Free-ITR" qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.

  9. Gene network analysis shows immune-signaling and ERK1/2 as novel genetic markers for multiple addiction phenotypes: alcohol, smoking and opioid addiction.

    Science.gov (United States)

    Reyes-Gibby, Cielito C; Yuan, Christine; Wang, Jian; Yeung, Sai-Ching J; Shete, Sanjay

    2015-06-05

    Addictions to alcohol and tobacco, known risk factors for cancer, are complex heritable disorders. Addictive behaviors have a bidirectional relationship with pain. We hypothesize that the associations between alcohol, smoking, and opioid addiction observed in cancer patients have a genetic basis. Therefore, using bioinformatics tools, we explored the underlying genetic basis and identified new candidate genes and common biological pathways for smoking, alcohol, and opioid addiction. Literature search showed 56 genes associated with alcohol, smoking and opioid addiction. Using Core Analysis function in Ingenuity Pathway Analysis software, we found that ERK1/2 was strongly interconnected across all three addiction networks. Genes involved in immune signaling pathways were shown across all three networks. Connect function from IPA My Pathway toolbox showed that DRD2 is the gene common to both the list of genetic variations associated with all three addiction phenotypes and the components of the brain neuronal signaling network involved in substance addiction. The top canonical pathways associated with the 56 genes were: 1) calcium signaling, 2) GPCR signaling, 3) cAMP-mediated signaling, 4) GABA receptor signaling, and 5) G-alpha i signaling. Cancer patients are often prescribed opioids for cancer pain thus increasing their risk for opioid abuse and addiction. Our findings provide candidate genes and biological pathways underlying addiction phenotypes, which may be future targets for treatment of addiction. Further study of the variations of the candidate genes could allow physicians to make more informed decisions when treating cancer pain with opioid analgesics.

  10. The fading affect bias shows positive outcomes at the general but not the individual level of analysis in the context of social media.

    Science.gov (United States)

    Gibbons, Jeffrey A; Horowitz, Kyle A; Dunlap, Spencer M

    2017-08-01

    Unpleasant affect fades faster than pleasant affect (e.g., Walker, Vogl, & Thompson, 1997); this effect is referred to as the Fading Affect Bias (FAB; Walker, Skowronski, Gibbons, Vogl, & Thompson, 2003a). Research shows that the FAB is consistently related to positive/healthy outcomes at a general but not at a specific level of analysis based on event types and individual differences (e.g., Gibbons et al., 2013). Based on the positive outcomes for FAB and negative outcomes for social media (Bolton et al., 2013; Huang, 2010), the current study examined FAB in the context of social media events along with related individual differences. General positive outcomes were shown in the form of robust FAB effects across social media and non-social media events, a larger FAB for non-social media events than for social media events, negative correlations of FAB with depression, anxiety, and stress as well as a positive correlation of FAB with self-esteem. However, the lack of a negative correlation between FAB and anxiety for social media events in a 3-way interaction did not show positive outcomes at a specific level of analysis. Rehearsal ratings mediated the 3-way interaction. Implications are discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. A comparison of genotyping-by-sequencing analysis methods on low-coverage crop datasets shows advantages of a new workflow, GB-eaSy.

    Science.gov (United States)

    Wickland, Daniel P; Battu, Gopal; Hudson, Karen A; Diers, Brian W; Hudson, Matthew E

    2017-12-28

    Genotyping-by-sequencing (GBS), a method to identify genetic variants and quickly genotype samples, reduces genome complexity by using restriction enzymes to divide the genome into fragments whose ends are sequenced on short-read sequencing platforms. While cost-effective, this method produces extensive missing data and requires complex bioinformatics analysis. GBS is most commonly used on crop plant genomes, and because crop plants have highly variable ploidy and repeat content, the performance of GBS analysis software can vary by target organism. Here we focus our analysis on soybean, a polyploid crop with a highly duplicated genome, relatively little public GBS data and few dedicated tools. We compared the performance of five GBS pipelines using low-coverage Illumina sequence data from three soybean populations. To address issues identified with existing methods, we developed GB-eaSy, a GBS bioinformatics workflow that incorporates widely used genomics tools, parallelization and automation to increase the accuracy and accessibility of GBS data analysis. Compared to other GBS pipelines, GB-eaSy rapidly and accurately identified the greatest number of SNPs, with SNP calls closely concordant with whole-genome sequencing of selected lines. Across all five GBS analysis platforms, SNP calls showed unexpectedly low convergence but generally high accuracy, indicating that the workflows arrived at largely complementary sets of valid SNP calls on the low-coverage data analyzed. We show that GB-eaSy is approximately as good as, or better than, other leading software solutions in the accuracy, yield and missing data fraction of variant calling, as tested on low-coverage genomic data from soybean. It also performs well relative to other solutions in terms of the run time and disk space required. In addition, GB-eaSy is built from existing open-source, modular software packages that are regularly updated and commonly used, making it straightforward to install and maintain

  12. A Content Analysis of How Sexual Behavior and Reproductive Health are Being Portrayed on Primetime Television Shows Being Watched by Teens and Young Adults.

    Science.gov (United States)

    Kinsler, Janni J; Glik, Deborah; de Castro Buffington, Sandra; Malan, Hannah; Nadjat-Haiem, Carsten; Wainwright, Nicole; Papp-Green, Melissa

    2018-02-01

    Television is a leading source of sexual education for teens and young adults, thus it is important to understand how sexual behavior and reproductive health are portrayed in popular primetime programming. This study is a media content analysis of the 19 top-rated scripted English-language primetime television shows aired between January 1, 2015 and May 31, 2015, and viewed by American youth audiences 12-24 years of age. The purpose of this study is to assess how sex/sexuality and reproductive health are being portrayed in a popular medium that reaches many adolescent and young adult audiences. Themes used for this analysis include youth pregnancy/parenting, mentoring/guidance of youth regarding sexual behavior, sex/sexuality, body image/identity, sexual violence/abuse/harassment, gender identity/sexual orientation, and reproductive health. Themes have been classified in one of the following six categories: visual cues, brief mentions, dialogue, minor storylines, major storylines, and multi-episode storylines. Our findings indicate that narratives providing educational information regarding the risks and consequences of sexual behavior were missing from the television shows we analyzed and that storylines promoting low risk sexual behavior were rare. Sexual violence and abuse, casual sex among adults, lack of contraception use, or no portrayal of consequences of risky behaviors were common. Compared to prior research, we found an emergent theme normalizing non-heterosexual gender identity and sexual orientation. Our findings have important implications as exposure to popular media shapes the perceptions and behaviors of teens and young adults. This study has the potential to shed light on the need to create stories and narratives in television shows watched by American teens and young adults with educational messages regarding the risks and consequences of sexual behavior.

  13. Viability qPCR, a new tool for Legionella risk management.

    Science.gov (United States)

    Lizana, X; López, A; Benito, S; Agustí, G; Ríos, M; Piqué, N; Marqués, A M; Codony, F

    2017-11-01

    Viability quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach for only detecting live microorganisms by DNA amplification-based methods This approach is based on the use of a reagent that irreversibly fixes dead cells DNA. In this study, we evaluate the utility of v-qPCR versus culture method for Legionellosis risk management. The present study was performed using 116 real samples. Water samples were simultaneously analysed by culture, v-qPCR and qPCR methods. Results were compared by means of a non-parametric test. In 11.6% of samples using both methods (culture method and v-qPCR) results were positive, in 50.0% of samples both methods gave rise to negative results. As expected, equivalence between methods was not observed in all cases, as in 32.1% of samples positive results were obtained by v-qPCR and all of them gave rise to negative results by culture. Only in 6.3% of samples, with very low Legionella levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth of other bacteria did not allow performing the culture. When comparing both methods, significant differences between culture and v-qPCR were in the samples belonging to the cooling towers-evaporative condensers group. The v-qPCR method detected greater presence and obtained higher concentrations of Legionella spp. (p<0.001). Otherwise, no significant differences between methods were found in the rest of the groups. The v-qPCR method can be used as a quick tool to evaluate Legionellosis risk, especially in cooling towers-evaporative condensers, where this technique can detect higher levels than culture. The combined interpretation of PCR results along with the ratio of live cells is proposed as a tool for understanding the sample context and estimating the Legionellosis risk potential according to 4 levels of hierarchy. Copyright © 2017 Elsevier GmbH. All rights reserved.

  14. Effect of apple pectin on gut microbiota - qPCR in applied microbiology

    DEFF Research Database (Denmark)

    Bergström, Anders; Wilcks, Andrea; Poulsen, Morten

    This study was part of the large European project ISAFRUIT aiming to reveal the biological explanations for the epidemiologically well-established health effects of fruits. The objective was to identify effects of apple and apple product consumption on the composition of the cecal microbial...... community in rats, as well as on a number of cecal parameters, which could be influenced by a changed microbiota. Principal Component Analysis (PCA) of cecal microbiota profiles obtained by PCR-DGGE targeting bacterial 16S rRNA genes showed an effect of whole apples in a long-term feeding study (14 weeks...

  15. Analysis of genetic distance between Peruvian Alpaca (Vicugna Pacos showing two distinct fleece phenotypes, Suri and Huacaya, by means of microsatellite markers

    Directory of Open Access Journals (Sweden)

    Carlo Renieri

    2011-10-01

    Full Text Available Two coat phenotypes exist in Alpaca, Huacaya and Suri. The two coats show different fleece structure, textile characteristics and prices on the market. Although present scientific knowledge suggests a simple genetic model of inheritance, there is a tendency to manage and consider the two phenotypes as two different breeds. A 13 microsatellite panel was used in this study to assess genetic distance between Suri and Huacaya alpacas in a sample of non-related animals from two phenotypically pure flocks at the Illpa-Puno experimental station in Quimsachata, Peru. The animals are part of a germplasm established approximately 20 years ago and have been bred separately according to their coat type since then. Genetic variability parameters were also calculated. The data were statistically analyzed using the software Genalex 6.3, Phylip 3.69 and Fstat 2.9.3.2. The sample was tested for Hardy-Weinberg equilibrium (HWE and after strict Bonferroni correction only one locus (LCA37 showed deviation from equilibrium (Ploci associations showed significant disequilibrium. Observed heterozygosis (Ho= 0.766; SE=0.044, expected heterozygosis (He=0.769; SE=0.033, number of alleles (Na=9.667, SE=0.772 and Fixation index (F=0.004; SE=0.036 are comparable to data from previous studies. Measures of genetic distance were 0.06 for Nei’s and 0.03 for Cavalli-Sforza’s. The analysis of molecular variance reported no existing variance between populations. Considering the origin of the animals, their post domestication evolution and the reproductive practices in place, the results do not show genetic differentiation between the two populations for the studied loci.

  16. Gc, gc-ms analysis of lipophilic fractions of aerial parts of fagonia indica burm.f. showing growth inhibitory effect on ht 29 colorectal cancer cells

    International Nuclear Information System (INIS)

    Farheen, R.; Mahmood, I.

    2016-01-01

    Fagonia indica Burm.f. is a small genus of herbs and under shrubs. The plant contains potentially active substances and has been used traditionally for the treatment of many illnesses including cancer. Many polar compounds have been reported from this plant but its non-polar constituents have only been rarely studied. In the present studies these constituents of aerial parts of Fagonia indica Burm.f. and its sub fractions showing growth inhibitory effect on HT 29 colorectal cancer cells were analyzed using flame ionization detector (GC-FID) and GC-EIMS analysis. The present studies exhibited the presence of free fatty acids and their esters along with structurally diverse constituents including triterpene, heterocyclic organic compound, aromatics, hydrocarbons, alcohols, lactone and sterols which may be responsible for this activity. The results suggest that the non-polar constituents of F. indica bear a potential of further studies. (author)

  17. Show-Bix &

    DEFF Research Database (Denmark)

    2014-01-01

    The anti-reenactment 'Show-Bix &' consists of 5 dias projectors, a dial phone, quintophonic sound, and interactive elements. A responsive interface will enable the Dias projectors to show copies of original dias slides from the Show-Bix piece ”March på Stedet”, 265 images in total. The copies are...

  18. Different methods to quantify Listeria monocytogenesbiofilms cells showed different profile in their viability

    Directory of Open Access Journals (Sweden)

    Lizziane Kretli Winkelströter

    2015-03-01

    Full Text Available Listeria monocytogenes is a foodborne pathogen able to adhere and to form biofilms in several materials commonly present in food processing plants. The aim of this study was to evaluate the resistance of Listeria monocytogenes attached to abiotic surface, after treatment with sanitizers, by culture method, microscopy and Quantitative Real Time Polymerase Chain Reaction (qPCR. Biofilms of L. monocytogenes were obtained in stainless steel coupons immersed in Brain Heart Infusion Broth, under agitation at 37 °C for 24 h. The methods selected for this study were based on plate count, microscopic count with the aid of viability dyes (CTC-DAPI, and qPCR. Results of culture method showed that peroxyacetic acid was efficient to kill sessile L. monocytogenes populations, while sodium hypochlorite was only partially effective to kill attached L. monocytogenes (p < 0.05. When, viability dyes (CTC/DAPI combined with fluorescence microscopy and qPCR were used and lower counts were found after treatments (p < 0.05. Selective quantification of viable cells of L. monocytogenes by qPCR using EMA revelead that the pre-treatment with EMA was not appropriate since it also inhibited amplification of DNA from live cells by ca. 2 log. Thus, the use of CTC counts was the best method to count viable cells in biofilms.

  19. Large Scale Genome Analysis Shows that the Epitopes for Broadly Cross-Reactive Antibodies Are Predominant in the Pandemic 2009 Influenza Virus A H1N1 Strain

    Directory of Open Access Journals (Sweden)

    Edgar E. Lara-Ramírez

    2013-11-01

    Full Text Available The past pandemic strain H1N1 (A (H1N1pdm09 has now become a common component of current seasonal influenza viruses. It has changed the pre-existing immunity of the human population to succeeding infections. In the present study, a total of 14,210 distinct sequences downloaded from National Center for Biotechnology Information (NCBI database were used for the analysis. The epitope compositions in A (H1N1pdm09, classic seasonal strains, swine strains as well as highly virulent avian strain H5N1, identified with the aid of the Immune Epitope DataBase (IEDB, were compared at genomic level. The result showed that A (H1N1 pdm09 contains the 90% of B-cell epitopes for broadly cross-reactive antibodies (EBCA, which is in consonance with the recent reports on the experimental identification of new epitopes or antibodies for this virus and the binding tests with influenza virus protein HA of different subtypes. Our analysis supports that high proportional EBCA depends on the epitope pattern of A (H1N1pdm09 virus. This study may be helpful for better understanding of A (H1N1pdm09 and the production of new influenza vaccines.

  20. Robotic Assisted Radical Cystectomy with Extracorporeal Urinary Diversion Does Not Show a Benefit over Open Radical Cystectomy: A Systematic Review and Meta-Analysis of Randomised Controlled Trials.

    Directory of Open Access Journals (Sweden)

    Wei Shen Tan

    Full Text Available The number of robotic assisted radical cystectomy (RARC procedures is increasing despite the lack of Level I evidence showing any advantages over open radical cystectomy (ORC. However, several systematic reviews with meta-analyses including non-randomised studies, suggest an overall benefit for RARC compared to ORC. We performed a systematic review with meta-analysis of randomised controlled trials (RCTs to evaluate the perioperative morbidity and efficacy of RARC compared to ORC in patients with bladder cancer.Literature searches of Medline/Pubmed, Embase, Web of Science and clinicaltrials.gov databases up to 10th March 2016 were performed. The inclusion criteria for eligible studies were RCTs which compared perioperative outcomes of ORC and RARC for bladder cancer. Primary objective was perioperative and histopathological outcomes of RARC versus ORC while the secondary objective was quality of life assessment (QoL, oncological outcomes and cost analysis.Four RCTs (from 5 articles met the inclusion criteria, with a total of 239 patients all with extracorporeal urinary diversion. Patient demographics and clinical characteristics of RARC and ORC patients were evenly matched. There was no significant difference between groups in perioperative morbidity, length of stay, positive surgical margin, lymph node yield and positive lymph node status. RARC group had significantly lower estimated blood loss (p<0.001 and wound complications (p = 0.03 but required significantly longer operating time (p<0.001. QoL was not measured uniformly across trials and cost analysis was reported in one RCTs. A test for heterogeneity did highlight differences across operating time of trials suggesting that surgeon experience may influence outcomes.This study does not provide evidence to support a benefit for RARC compared to ORC. These results may not have inference for RARC with intracorporeal urinary diversion. Well-designed trials with appropriate endpoints conducted

  1. Gene expression analysis of two extensively drug-resistant tuberculosis isolates show that two-component response systems enhance drug resistance.

    Science.gov (United States)

    Yu, Guohua; Cui, Zhenling; Sun, Xian; Peng, Jinfu; Jiang, Jun; Wu, Wei; Huang, Wenhua; Chu, Kaili; Zhang, Lu; Ge, Baoxue; Li, Yao

    2015-05-01

    Global analysis of expression profiles using DNA microarrays was performed between a reference strain H37Rv and two clinical extensively drug-resistant isolates in response to three anti-tuberculosis drug exposures (isoniazid, capreomycin, and rifampicin). A deep analysis was then conducted using a combination of genome sequences of the resistant isolates, resistance information, and related public microarray data. Certain known resistance-associated gene sets were significantly overrepresented in upregulated genes in the resistant isolates relative to that observed in H37Rv, which suggested a link between resistance and expression levels of particular genes. In addition, isoniazid and capreomycin response genes, but not rifampicin, either obtained from published works or our data, were highly consistent with the differentially expressed genes of resistant isolates compared to those of H37Rv, indicating a strong association between drug resistance of the isolates and genes differentially regulated by isoniazid and capreomycin exposures. Based on these results, 92 genes of the studied isolates were identified as candidate resistance genes, 10 of which are known resistance-related genes. Regulatory network analysis of candidate resistance genes using published networks and literature mining showed that three two-component regulatory systems and regulator CRP play significant roles in the resistance of the isolates by mediating the production of essential envelope components. Finally, drug sensitivity testing indicated strong correlations between expression levels of these regulatory genes and sensitivity to multiple anti-tuberculosis drugs in Mycobacterium tuberculosis. These findings may provide novel insights into the mechanism underlying the emergence and development of drug resistance in resistant tuberculosis isolates and useful clues for further studies on this issue. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. An Analysis of Trafficking Receptors Shows that CD44 and P-Selectin Glycoprotein Ligand-1 Collectively Control the Migration of Activated Human T-Cells

    KAUST Repository

    Ali, Amal J.

    2017-05-03

    Selectins guide the traffic of activated T-cells through the blood stream by mediating their tethering and rolling onto inflamed endothelium, in this way acting as beacons to help navigate them to sites of inflammation. Here, we present a comprehensive analysis of E-selectin ligands expressed on activated human T-cells. We identified several novel glycoproteins that function as E-selectin ligands. Specifically, we compared the role of P-selectin glycoprotein ligand-1 (PSGL-1) and CD43, known E-selectin ligands, to CD44, a ligand that has not previously been characterized as an E-selectin ligand on activated human T-cells. We showed that CD44 acts as a functional E-selectin ligand when expressed on both CD4+ and CD8+ T-cells. Moreover, the CD44 protein carries a binding epitope identifying it as hematopoietic cell E- and/or L-selectin ligand (HCELL). Furthermore, by knocking down these ligands individually or together in primary activated human T-cells, we demonstrated that CD44/HCELL, and not CD43, cooperates with PSGL-1 as a major E-selectin ligand. Additionally, we demonstrated the relevance of our findings to chronic autoimmune disease, by showing that CD44/HCELL and PSGL-1, but not CD43, from T-cells isolated from psoriasis patients, bind E-selectin.

  3. Limitations of Using Propidium Monoazide with qPCR to Discriminate between Live and Dead in Biofilm Samples

    Directory of Open Access Journals (Sweden)

    Michael J. Taylor

    2014-01-01

    Full Text Available Accurately quantifying Legionella for regulatory purposes to protect public health is essential. Real-time PCR (qPCR has been proposed as a better method for detecting and enumerating Legionella in samples than conventional culture method. However, since qPCR amplifies any target DNA in the sample, the technique's inability to discriminate between live and dead cells means that counts are generally significantly overestimated. Propidium monoazide (PMA has been used successfully in qPCR to aid live/dead discrimination. We tested PMA use as a method to count only live Legionella cells in samples collected from a modified chemostat that generates environmentally comparable samples. Counts from PMA-treated samples that were pretreated with either heat or three types of disinfectants (to kill the cells were highly variable, with the only consistent trend being the relationship between biofilm mass and numbers of Legionella cells. Two possibilities explain this result: 1. PMA treatment worked and the subsequent muted response of Legionella to disinfection treatment is a factor of biofilm/microbiological effects; although this does not account for the relationship between the amount of biofilm sampled and the viable Legionella count as determined by PMA-qPCR; or 2. PMA treatment did not work, and any measured decrease or increase in detectable Legionella is because of other factors affecting the method. This is the most likely explanation for our results, suggesting that higher concentrations of PMA might be needed to compensate for the presence of other compounds in an environmental sample or that lower amounts of biofilm need to be sampled. As PMA becomes increasingly toxic at higher concentrations and is very expensive, augmenting the method to include higher PMA concentrations is both counterproductive and cost prohibitive. Conversely, if smaller volumes of biofilm are used, the reproducibility of the method is reduced. Our results suggest that

  4. Development of a multiplex Q-PCR to detect Trichoderma harzianum Rifai strain T22 in plant roots.

    Science.gov (United States)

    Horn, Ivo R; van Rijn, Menno; Zwetsloot, Tom J J; Basmagi, Said; Dirks-Mulder, Anita; van Leeuwen, Willem B; Ravensberg, Willem J; Gravendeel, Barbara

    2016-02-01

    The fungal species Trichoderma harzianum is widely used as a biological agent in crop protection. To verify the continued presence of this fungus on plant roots manually inoculated with T. harzianum strain T22, a Q-PCR was designed using specific probes for this particular strain. To develop these molecular diagnostic tools, genome mining was first carried out to retrieve putative new regions by which different strains of T. harzianum could be distinguished. Subsequently, Sanger sequencing of the L-aminoacid oxidase gene (aox1) in T. harzianum was applied to determine the mutations differing between various strains isolated from the Trichoderma collection of Koppert Biological Systems. Based on the sequence information obtained, a set of hydrolysis probes was subsequently developed which discriminated T. harzianum T22 strains varying in only a single nucleotide. Probes designed for two strains uniquely recognized the respective strains in Q-PCR with a detection limit of 12,5ng DNA. Titration assays in which T. harzianum DNA from distinct strains was varied further underscored the specificity of the probes. Lastly, fungal DNA extracted from roots of greenhouse cultured tomato plants was analyzed using the probe-based assay. DNA from T. harzianum strain T22 could readily be identified on roots of greenhouse reared tomato plants inoculated with varying concentrations up to one week after treatment with a detection limit of 3e6 colony forming units of T. harzianum T22. We conclude that the Q-PCR method is a reliable and robust method for assessing the presence and quantity of T. harzianum strain T22 in manually inoculated plant material. Our method provides scope for the development of DNA based strain specific identification of additional strains of Trichoderma and other fungal biological control agents. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. A pipeline to determine RT-QPCR control genes for evolutionary studies: application to primate gene expression across multiple tissues.

    Directory of Open Access Journals (Sweden)

    Olivier Fedrigo

    Full Text Available Because many species-specific phenotypic differences are assumed to be caused by differential regulation of gene expression, many recent investigations have focused on measuring transcript abundance. Despite the availability of high-throughput platforms, quantitative real-time polymerase chain reaction (RT-QPCR is often the method of choice because of its low cost and wider dynamic range. However, the accuracy of this technique heavily relies on the use of multiple valid control genes for normalization. We created a pipeline for choosing genes potentially useful as RT-QPCR control genes for measuring expression between human and chimpanzee samples across multiple tissues, using published microarrays and a measure of tissue-specificity. We identified 13 genes from the pipeline and from commonly used control genes: ACTB, USP49, ARGHGEF2, GSK3A, TBP, SDHA, EIF2B2, GPDH, YWHAZ, HPTR1, RPL13A, HMBS, and EEF2. We then tested these candidate genes and validated their expression stability across species. We established the rank order of the most preferable set of genes for single and combined tissues. Our results suggest that for at least three tissues (cerebral cortex, liver, and skeletal muscle, EIF2B2, EEF2, HMBS, and SDHA are useful genes for normalizing human and chimpanzee expression using RT-QPCR. Interestingly, other commonly used control genes, including TBP, GAPDH, and, especially ACTB do not perform as well. This pipeline could be easily adapted to other species for which expression data exist, providing taxonomically appropriate control genes for comparisons of gene expression among species.

  6. Talking with TV shows

    DEFF Research Database (Denmark)

    Sandvik, Kjetil; Laursen, Ditte

    2014-01-01

    User interaction with radio and television programmes is not a new thing. However, with new cross-media production concepts such as X Factor and Voice, this is changing dramatically. The second-screen logic of these productions encourages viewers, along with TV’s traditional one-way communication...... mode, to communicate on interactive (dialogue-enabling) devices such as laptops, smartphones and tablets. Using the TV show Voice as our example, this article shows how the technological and situational set-up of the production invites viewers to engage in new ways of interaction and communication...

  7. Talk Show Science.

    Science.gov (United States)

    Moore, Mitzi Ruth

    1992-01-01

    Proposes having students perform skits in which they play the roles of the science concepts they are trying to understand. Provides the dialog for a skit in which hot and cold gas molecules are interviewed on a talk show to study how these properties affect wind, rain, and other weather phenomena. (MDH)

  8. Obesity in show cats.

    Science.gov (United States)

    Corbee, R J

    2014-12-01

    Obesity is an important disease with a high prevalence in cats. Because obesity is related to several other diseases, it is important to identify the population at risk. Several risk factors for obesity have been described in the literature. A higher incidence of obesity in certain cat breeds has been suggested. The aim of this study was to determine whether obesity occurs more often in certain breeds. The second aim was to relate the increased prevalence of obesity in certain breeds to the official standards of that breed. To this end, 268 cats of 22 different breeds investigated by determining their body condition score (BCS) on a nine-point scale by inspection and palpation, at two different cat shows. Overall, 45.5% of the show cats had a BCS > 5, and 4.5% of the show cats had a BCS > 7. There were significant differences between breeds, which could be related to the breed standards. Most overweight and obese cats were in the neutered group. It warrants firm discussions with breeders and cat show judges to come to different interpretations of the standards in order to prevent overweight conditions in certain breeds from being the standard of beauty. Neutering predisposes for obesity and requires early nutritional intervention to prevent obese conditions. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.

  9. Honored Teacher Shows Commitment.

    Science.gov (United States)

    Ratte, Kathy

    1987-01-01

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

  10. The energy show

    International Nuclear Information System (INIS)

    1988-01-01

    The Energy Show is a new look at the problems of world energy, where our supplies come from, now and in the future. The programme looks at how we need energy to maintain our standards of living. Energy supply is shown as the complicated set of problems it is - that Fossil Fuels are both raw materials and energy sources, that some 'alternatives' so readily suggested as practical options are in reality a long way from being effective. (author)

  11. Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR.

    Science.gov (United States)

    Dobnik, David; Demšar, Tina; Huber, Ingrid; Gerdes, Lars; Broeders, Sylvia; Roosens, Nancy; Debode, Frederic; Berben, Gilbert; Žel, Jana

    2018-01-01

    Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection. Graphical abstract The output of three different PCR-based platforms was assessed in an inter-laboratory comparison.

  12. A high-throughput qPCR system for simultaneous quantitative detection of dairy Lactococcus lactis and Leuconostoc bacteriophages

    DEFF Research Database (Denmark)

    Muhammed, Musemma Kedir; Krych, Lukasz; Nielsen, Dennis Sandris

    2017-01-01

    simultaneous quantitative detection of Lc. lactis 936 (now SK1virus), P335, c2 (now C2virus) and Leuconostoc phage groups. Component assays are designed to have high efficiencies and nearly the same dynamic detection ranges, i.e., from 1.1 x 105 to 1.1 x 101 phage genomes per reaction, which corresponds to 9 x......Simultaneous quantitative detection of Lactococcus (Lc.) lactis and Leuconostoc species bacteriophages (phages) has not been reported in dairies using undefined mixed-strain DL-starters, probably due to the lack of applicable methods. We optimized a high-throughput qPCR system that allows...... 107 to 9 x 103 phage particles mL-1 without any additional up-concentrating steps. The amplification efficiencies of the corresponding assays were 100.1±2.6, 98.7±2.3, 101.0±2.3 and 96.2±6.2. The qPCR system was tested on samples obtained from a dairy plant that employed traditional mother...

  13. Seasonal Dynamics of Microcystis spp. and Their Toxigenicity as Assessed by qPCR in a Temperate Reservoir

    Directory of Open Access Journals (Sweden)

    Agostinho Antunes

    2011-09-01

    Full Text Available Blooms of toxic cyanobacteria are becoming increasingly frequent, mainly due to water quality degradation. This work applied qPCR as a tool for early warning of microcystin(MC-producer cyanobacteria and risk assessment of water supplies. Specific marker genes for cyanobacteria, Microcystis and MC-producing Microcystis, were quantified to determine the genotypic composition of the natural Microcystis population. Correlations between limnological parameters, pH, water temperature, dissolved oxygen and conductivity and MC concentrations as well as Microcystis abundance were assessed. A negative significant correlation was observed between toxic (with mcy genes to non-toxic (without mcy genes genotypes ratio and the overall Microcystis density. The highest proportions of toxic Microcystis genotypes were found 4–6 weeks before and 8–10 weeks after the peak of the bloom, with the lowest being observed at its peak. These results suggest positive selection of non-toxic genotypes under favorable environmental growth conditions. Significant positive correlations could be found between quantity of toxic genotypes and MC concentration, suggesting that the method applied can be useful to predict potential MC toxicity risk. No significant correlation was found between the limnological parameters measured and MC concentrations or toxic genotypes proportions indicating that other abiotic and biotic factors should be governing MC production and toxic genotypes dynamics. The qPCR method here applied is useful to rapidly estimate the potential toxicity of environmental samples and so, it may contribute to the more efficient management of water use in eutrophic systems.

  14. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients.

    Directory of Open Access Journals (Sweden)

    Julia Stadler

    Full Text Available Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent "gold standard". Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution, at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients.

  15. Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR).

    Science.gov (United States)

    Hornyák, Akos; Bálint, Adám; Farsang, Attila; Balka, Gyula; Hakhverdyan, Mikhayil; Rasmussen, Thomas Bruun; Blomberg, Jonas; Belák, Sándor

    2012-05-01

    Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCoV) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection and the quantitation of the genome copies of FCoV. In order to detect the broadest spectrum of potential FCoV variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (PriProET) principle. This technology was chosen since PriProET is very robust to tolerate the nucleotide substitutions in the target area. Therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. The detection specificity of the new assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. The sensitivity of the P-sg-QPCR method was high, since as few as 10 genome copies of FCoV were detected. The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of

  16. Showing Value (Editorial

    Directory of Open Access Journals (Sweden)

    Denise Koufogiannakis

    2009-06-01

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

  17. Individual patient data meta-analysis shows a significant association between the ATM rs1801516 SNP and toxicity after radiotherapy in 5456 breast and prostate cancer patients

    DEFF Research Database (Denmark)

    Andreassen, Christian Nicolaj; Rosenstein, Barry S; Kerns, Sarah L

    2016-01-01

    PURPOSE: Several small studies have indicated that the ATM rs1801516 SNP is associated with risk of normal tissue toxicity after radiotherapy. However, the findings have not been consistent. In order to test this SNP in a well-powered study, an individual patient data meta-analysis was carried ou...

  18. An Analysis of Trafficking Receptors Shows that CD44 and P-Selectin Glycoprotein Ligand-1 Collectively Control the Migration of Activated Human T-Cells

    KAUST Repository

    Ali, Amal J.; AbuElela, Ayman; Merzaban, Jasmeen

    2017-01-01

    -selectin ligands, to CD44, a ligand that has not previously been characterized as an E-selectin ligand on activated human T-cells. We showed that CD44 acts as a functional E-selectin ligand when expressed on both CD4+ and CD8+ T-cells. Moreover, the CD44 protein

  19. Spatial Heterodyne Observations of Water (SHOW) vapour in the upper troposphere and lower stratosphere from a high altitude aircraft: Modelling and sensitivity analysis

    Science.gov (United States)

    Langille, J. A.; Letros, D.; Zawada, D.; Bourassa, A.; Degenstein, D.; Solheim, B.

    2018-04-01

    A spatial heterodyne spectrometer (SHS) has been developed to measure the vertical distribution of water vapour in the upper troposphere and the lower stratosphere with a high vertical resolution (∼500 m). The Spatial Heterodyne Observations of Water (SHOW) instrument combines an imaging system with a monolithic field-widened SHS to observe limb scattered sunlight in a vibrational band of water (1363 nm-1366 nm). The instrument has been optimized for observations from NASA's ER-2 aircraft as a proof-of-concept for a future low earth orbit satellite deployment. A robust model has been developed to simulate SHOW ER-2 limb measurements and retrievals. This paper presents the simulation of the SHOW ER-2 limb measurements along a hypothetical flight track and examines the sensitivity of the measurement and retrieval approach. Water vapour fields from an Environment and Climate Change Canada forecast model are used to represent realistic spatial variability along the flight path. High spectral resolution limb scattered radiances are simulated using the SASKTRAN radiative transfer model. It is shown that the SHOW instrument onboard the ER-2 is capable of resolving the water vapour variability in the UTLS from approximately 12 km - 18 km with ±1 ppm accuracy. Vertical resolutions between 500 m and 1 km are feasible. The along track sampling capability of the instrument is also discussed.

  20. Early detection of sugar beet pathogen Ramularia beticola in leaf and air samples using qPCR

    DEFF Research Database (Denmark)

    Wieczorek, Thies Marten; Jørgensen, Lise Nistrup; Hansen, Anne Lisbet

    2014-01-01

    A quantitative PCR method (qPCR) was developed for the detection and quantification of Ramularia beticola causing Ramularia leaf spot in sugar beet. R. beticola specific primers were designed based on the internal transcribed spacer region 2 (ITS2). The assay was applied on DNA extracted from...... spores trapped on tape from Burkard spore traps placed in an artificially inoculated sugar beet field trial and in two sugar beet fields with natural infections. R. beticola DNA was detected at variable amounts in the air samples 14 to 16 days prior to first visible symptoms. R. beticola DNA was detected...... in air samples from fields with natural infection at significant and increasing levels from development of the first symptoms, indicating that spore production within the crop plays a major role in the epidemic development of the disease. Sugar beet leaves sampled from the inoculated field trial were...

  1. Seawater detection and biological assessments regarding transmission of the oyster parasite Mikrocytos mackini using qPCR.

    Science.gov (United States)

    Polinski, Mark P; Meyer, Gary R; Lowe, Geoffrey J; Abbott, Cathryn L

    2017-10-18

    Mikrocytos mackini is an intracellular parasite of oysters and causative agent of Denman Island disease in Pacific oysters Crassostrea gigas. Although M. mackini has been investigated for decades, its natural mode of transmission, mechanism for host entry, and environmental stability are largely unknown. We explored these biological characteristics of M. mackini using a recently described quantitative PCR (qPCR) assay. We detected M. mackini in the flow-through tank water of experimentally infected oysters and during disease remission in host tissues following 6 wk of elevated water temperature. Waterborne exposure of oysters to M. mackini further confirmed the potential for extracellular seawater transmission of this parasite and also identified host gill to have the highest early and continued prevalence for M. mackini DNA compared to stomach, mantle, labial palps, or adductor muscle samples. However, infections following waterborne challenge were slow to develop despite a substantial exposure (>106 M. mackini l-1 for 24 h), and further investigation demonstrated that M. mackini occurrence and infectivity severely declined following extracellular seawater incubation of more than 24 h. This study demonstrates a potential for using qPCR to monitor M. mackini in wild or farmed oyster populations during periods of disease remission or from environmental seawater samples. This work also suggests that gill tissues may provide a primary site for waterborne entry and possibly shedding of M. mackini in oysters. Further, although extracellular seawater transmission of M. mackini was possible, poor environmental stability and infection efficiency likely restricts the geographic transmission of M. mackini between oysters in natural environs and may help to explain localized areas of infection.

  2. Diffusion tensor MRI shows progressive changes in the hippocampus and dentate gyrus after status epilepticus in rat - histological validation with Fourier-based analysis.

    Science.gov (United States)

    Salo, Raimo A; Miettinen, Tuukka; Laitinen, Teemu; Gröhn, Olli; Sierra, Alejandra

    2017-05-15

    Imaging markers for monitoring disease progression, recovery, and treatment efficacy are a major unmet need for many neurological diseases, including epilepsy. Recent evidence suggests that diffusion tensor imaging (DTI) provides high microstructural contrast even outside major white matter tracts. We hypothesized that in vivo DTI could detect progressive microstructural changes in the dentate gyrus and the hippocampal CA3bc in the rat brain after status epilepticus (SE). To test this hypothesis, we induced SE with systemic kainic acid or pilocarpine in adult male Wistar rats and subsequently scanned them using in vivo DTI at five time-points: prior to SE, and 10, 20, 34, and 79 days post SE. In order to tie the DTI findings to changes in the tissue microstructure, myelin- and glial fibrillary acidic protein (GFAP)-stained sections from the same animals underwent Fourier analysis. We compared the Fourier analysis parameters, anisotropy index and angle of myelinated axons or astrocyte processes, to corresponding DTI parameters, fractional anisotropy (FA) and the orientation angle of the principal eigenvector. We found progressive detectable changes in DTI parameters in both the dentate gyrus (FA, axial diffusivity [D || ], linear anisotropy [CL] and spherical anisotropy [CS], pFourier analysis revealed that both myelinated axons and astrocyte processes played a role in the water diffusion anisotropy changes detected by DTI in individual portions of the dentate gyrus (suprapyramidal blade, mid-portion, and infrapyramidal blade). In the whole dentate gyrus, myelinated axons markedly contributed to the water diffusion changes. In CA3bc as well as in CA3b and CA3c, both myelinated axons and astrocyte processes contributed to water diffusion anisotropy and orientation. Our study revealed that DTI is a promising method for noninvasive detection of microstructural alterations in the hippocampus proper. These alterations may be potential imaging markers for epileptogenesis

  3. 'You want to show you're a valuable employee': A critical discourse analysis of multi-perspective portrayals of employed women with fibromyalgia.

    Science.gov (United States)

    Oldfield, Margaret; MacEachen, Ellen; MacNeill, Margaret; Kirsh, Bonnie

    2018-06-01

    Background Advice on fibromyalgia, a chronic illness primarily affecting women, often presents it as incompatible with work and rarely covers how to remain employed. Yet many women do. Objectives We aimed to understand how these women, their family members, and workmates portrayed employees with fibromyalgia, and how these portrayals helped women retain employment. Methods We interviewed 22 participants, comprising five triads and three dyads of people who knew each other. Using the methodology of critical discourse analysis, we analysed the interview data within and across the triads/dyads through coding, narrative summaries, and relational mapping. Results Participants reported stereotypes that employees with fibromyalgia are lazy, malingering, and less productive than healthy workers. Countering these assumptions, participants portrayed the women as normal, valuable employees who did not 'give in' to their illness. The portrayals drew on two discourses, normalcy and mind-controlling-the-body, and a related narrative, overcoming disability. We propose that participants' portrayals helped women manage their identities in competitive workplaces and thereby remain employed. Discussion Our findings augment the very sparse literature on employment with fibromyalgia. Using a new approach, critical discourse analysis, we expand on known job-retention strategies and add the perspectives of two key stakeholders: family members and workmates.

  4. Analysis of S gene mutation of the hepatitis B virus in adult liver transplant recipients showing resistance to hepatitis B immunoglobulin therapy.

    Science.gov (United States)

    Park, G-C; Hwang, S; Ahn, C-S; Kim, K-H; Moon, D-B; Ha, T-Y; Song, G-W; Jung, D-H; Shin, Y W; Kim, S-H; Chang, K-H; Namgoong, J-M; Park, C-S; Park, H-W; Park, Y-H; Kang, S-H; Jung, B-H; Lee, S-G

    2013-10-01

    A considerable proportion of recipients of liver transplantations who are presented hepatitis B immunoglobulin (HBIG) monotherapy for hepatitis B virus (HBV) prophylaxis develop HBIG resistance. In this study, we investigated the mutation patterns in the major hydrophilic region (MHR) of amino acid sequences 100 to 160. Using the gene sequence analyzer for amino acid sequences 0 to 226 in the S/pre-S region we analyzed blood samples of 15 patients showing HBIG resistance after high-dose HBIG prophylaxis. Various mutations in the MHR were observed in 14/15 samples: Gly145Arg mutation in 8/13 Adr subtype and 1/2 Ayw subtype samples (60%). The next most common mutation was Gly165Trp in 8/13 Adr subtype but neither of 2 Ayw subtype samples (53.3%). Concurrent antiviral resistance was noted in 5 patients: lamivudine (n = 5), or entecavir (n = 3), but not adefovir, suggesting the occurrence of simultaneous, antiviral cross-resistances. Two patients underwent retransplantation due to the progression of HBV infection despite vigorous antiviral therapy. At diagnosis of HBV recurrence, the mean HBV DNA load was 6.5 × 10(6) copies/mL; 4 patients showed paradoxical coexistence of anti-HBs and HBsAg. Currently, 2 subjects show low-level HBV DNA replication in peripheral blood, although the other 12 had no DNA replication after prolonged antiviral therapy. This study suggested that various mutations in the "a" determinant were associated with HBIG resistance. Since treatment failure to rescue antiviral therapy was often associated with delayed detection of HBV recurrence rather than concurrent antiviral resistance, frequent HBV surveillance using more sensitive screening tests, such as HBeAg and HBV DNA polymerase chain reaction assay, seems to be mandatory. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Combined analysis of 19 common validated type 2 diabetes susceptibility gene variants shows moderate discriminative value and no evidence of gene-gene interaction

    DEFF Research Database (Denmark)

    Sparsø, T; Grarup, N; Andreasen, C.

    2009-01-01

    study; and additional type 2 diabetic patients and glucose-tolerant individuals. The case-control studies involved 4,093 type 2 diabetic patients and 5,302 glucose-tolerant individuals. RESULTS: Single-variant analyses demonstrated allelic odds ratios ranging from 1.04 (95% CI 0.98-1.11) to 1.33 (95% CI...... analysis of the 19 validated variants enables detection of subgroups at substantially increased risk of type 2 diabetes; however, the discrimination between glucose-tolerant and type 2 diabetes individuals is still too inaccurate to achieve clinical value.......AIMS/HYPOTHESIS: The list of validated type 2 diabetes susceptibility variants has recently been expanded from three to 19. The variants identified are common and have low penetrance in the general population. The aim of the study is to investigate the combined effect of the 19 variants by applying...

  6. Analysis of Static and Dynamic E-Reference Content at a Multi-Campus University Shows, that Updated Content is Associated with Greater Annual Usage

    Directory of Open Access Journals (Sweden)

    Laura Costello

    2016-03-01

    Full Text Available Objective – To discover whether there is a difference in use over time between dynamically updated and changing subscription e-reference titles and collections, and static purchased e-reference titles and collections. Design – Case study. Setting – A multi-campus Canadian university with 9,200 students enrolled in both graduate and undergraduate programs. Subjects – E-reference book packages and individual e-reference titles. Methods – The author compared data from individual e-reference books and packages. First, individual subscription e-reference books that periodically added updated content were compared to individually purchased e-reference books that remained static after purchase. The author then compared two e-reference book packages that provided new and updated content to two static e-reference book packages. The author compared data from patron usage to new content added over time using regression analysis. Main Results – As the library acquired e-reference titles, dynamic title subscriptions added to the collection were associated with 2,246 to 4,635 views per subscription while static title additions were associated with 8 to 123 views per purchase. The author also found that there was a strong linear relationship between views and dynamic titles added to the collection (R2=0.79 and a very weak linear relationship (R2=0.18 with views when static titles are added to the collection. Regression analysis of dynamic e-reference collections revealed that the number of titles added to each collection was strongly associated with views of the material (R2=0.99, while static e-reference collections were less strongly linked (R2=0.43. Conclusion – Dynamic e-reference titles and collections experienced increases in usage each year while static titles and collections experienced decreases in usage. This indicates that collections and titles that offer new content to users each year will continue to see growth in usage while static

  7. Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR)

    NARCIS (Netherlands)

    Ruiz-Villalba, Adrián; van Pelt-Verkuil, Elizabeth; Gunst, Quinn D.; Ruijter, Jan M.; van den Hoff, Maurice J. B.

    2017-01-01

    Quantitative PCR allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, a survey with 93 validated assays for genes in the Wnt-pathway showed that the amplification of nonspecific products occurs frequently and is unrelated

  8. Genotypic and phenotypic analysis of familial male breast cancer shows under representation of the HER2 and basal subtypes in BRCA-associated carcinomas

    International Nuclear Information System (INIS)

    Deb, Siddhartha; Jene, Nicholas; Fox, Stephen B

    2012-01-01

    Male breast cancer (MBC) is an uncommon and relatively uncharacterised disease accounting for <1% of all breast cancers. A significant proportion occurs in families with a history of breast cancer and in particular those carrying BRCA2 mutations. Here we describe clinicopathological features and genomic BRCA1 and BRCA2 mutation status in a large cohort of familial MBCs. Cases (n=60) included 3 BRCA1 and 25 BRCA2 mutation carries, and 32 non-BRCA1/2 (BRCAX) carriers with strong family histories of breast cancer. The cohort was examined with respect to mutation status, clinicopathological parameters including TNM staging, grade, histological subtype and intrinsic phenotype. Compared to the general population, MBC incidence was higher in all subgroups. In contrast to female breast cancer (FBC) there was greater representation of BRCA2 tumours (41.7% vs 8.3%, p=0.0008) and underrepresentation of BRCA1 tumours (5.0% vs 14.4%, p=0.0001). There was no correlation between mutation status and age of onset, disease specific survival (DSS) or other clincopathological factors. Comparison with sporadic MBC studies showed similar clinicopathological features. Prognostic variables affecting DSS included primary tumour size (p=0.003, HR:4.26 95%CI 1.63-11.11), age (p=0.002, HR:4.09 95%CI 1.65-10.12), lymphovascular (p=0.019, HR:3.25 95%CI 1.21-8.74) and perineural invasion (p=0.027, HR:2.82 95%CI 1.13-7.06). Unlike familial FBC, the histological subtypes seen in familial MBC were more similar to those seen in sporadic MBC with 46 (76.7%) pure invasive ductal carcinoma of no special type (IDC-NST), 2 (3.3%) invasive lobular carcinomas and 4 (6.7%) invasive papillary carcinoma. A further 8 (13.3%) IDC-NST had foci of micropapillary differentiation, with a strong trend for co-occurrence in BRCA2 carriers (p=0.058). Most tumours were of the luminal phenotype (89.7%), with infrequent HER2 (8.6%) and basal (1.7%) phenotype tumours seen. MBC in BRCA1/2 carriers and BRCAX families is

  9. Surface plasmon resonance analysis shows an IgG-isotype-specific defect in ABO blood group antibody formation in patients with common variable immunodeficiency

    Directory of Open Access Journals (Sweden)

    Michael Bernhard Fischer

    2015-05-01

    Full Text Available Background: Common variable immunodeficiency (CVID is the most common clinically severe primary immunodeficiency and comprises a heterogeneous group of patients with recurrent severe bacterial infections due to the failure to produce IgG antibodies after exposure to infectious agents and immunization. Diagnostic recommendations for antibody failure include assessment of isoagglutinins. We have readdressed this four decades old but still accepted recommendation with up to date methodology.Methods: Anti-A/B IgM- and IgG-antibodies were measured by Diamed-ID Micro Typing, surface plasmon resonance (SPR using the Biacore® device and flow cytometry.Results: When Diamed-ID Micro Typing was used, CVID patients (n=34 showed IgG- and IgM-isoagglutinins that were comparable to healthy volunteers (n=28, while all XLA patients (n=8 had none. Anti-A/B IgM-antibodies were present in more than 2/3 of the CVID patients and showed binding kinetics comparable to anti-A/B IgM-antibodies from healthy individuals. A correlation could be found in CVID patients between levels of anti-A/B IgM-antibodies and levels of serum IgM and PnP-IgM-antibodies. In contrast in CVID patients as a group ABO antibodies were significantly decreased when assessed by SPR, which correlated with levels of switched memory, non-switched memory and naïve B cells, but all CVID patients had low/undetectable anti-A/B IgG-antibodies.Conclusion: These results indicate that conventional isoagglutinin assessment and assessment of anti-A/B IgM antibodies are not suited for the diagnosis of impaired antibody production in CVID. Examination of anti-A/B IgG antibodies by SPR provides a useful method for the diagnosis of IgG antibody failure in all CVID patients studied, thus indicating an important additional rationale to start immunoglobulin replacement therapy early in these patients, before post-infectious sequelae develop.

  10. Program impact pathway analysis of a social franchise model shows potential to improve infant and young child feeding practices in Vietnam.

    Science.gov (United States)

    Nguyen, Phuong H; Menon, Purnima; Keithly, Sarah C; Kim, Sunny S; Hajeebhoy, Nemat; Tran, Lan M; Ruel, Marie T; Rawat, Rahul

    2014-10-01

    By mapping the mechanisms through which interventions are expected to achieve impact, program impact pathway (PIP) analysis lays out the theoretical causal links between program activities, outcomes, and impacts. This study examines the pathways through which the Alive & Thrive (A&T) social franchise model is intended to improve infant and young child feeding (IYCF) practices in Vietnam. Mixed methods were used, including qualitative interviews with franchise management board members (n = 12), surveys with health providers (n = 120), counseling observations (n = 160), and household surveys (n = 2045). Six PIP components were assessed: 1) franchise management, 2) training and IYCF knowledge of health providers, 3) service delivery, 4) program exposure and utilization, 5) maternal behavioral determinants (knowledge, beliefs, and intentions) toward optimal IYCF practices, and 6) IYCF practices. Data were collected from A&T-intensive areas (A&T-I; mass media + social franchise) and A&T-nonintensive areas (A&T-NI; mass media only) by using a cluster-randomized controlled trial design. Data from 2013 were compared with baseline where similar measures were available. Results indicate that mechanisms are in place for effective management of the franchise system, despite challenges to routine monitoring. A&T training was associated with increased capacity of providers, resulting in higher-quality IYCF counseling (greater technical knowledge and communication skills during counseling) in A&T-I areas. Franchise utilization increased from 10% in 2012 to 45% in 2013 but fell below the expected frequency of 9-15 contacts per mother-child dyad. Improvements in breastfeeding knowledge, beliefs, intentions, and practices were greater among mothers in A&T-I areas than among those in A&T-NI areas. In conclusion, there are many positive changes along the impact pathway of the franchise services, but challenges in utilization and demand creation should be addressed to achieve the full

  11. Seasonality shows evidence for polygenic architecture and genetic correlation with schizophrenia and bipolar disorder – a meta-analysis of genetic studies

    Science.gov (United States)

    Byrne, Enda M; Raheja, Uttam; Stephens, Sarah H.; Heath, Andrew C; Madden, Pamela AF; Vaswani, Dipika; Nijjar, Gagan V.; Ryan, Kathleen A.; Youssufi, Hassaan; Gehrman, Philip R; Shuldiner, Alan R; Martin, Nicholas G; Montgomery, Grant W; Wray, Naomi R; Nelson, Elliot C; Mitchell, Braxton D; Postolache, Teodor T

    2015-01-01

    Objective To test common genetic variants for association with seasonality (seasonal changes in mood and behavior) and to investigate whether there are shared genetic risk factors between psychiatric disorders and seasonality. Methods A meta-analysis of genome-wide association studies (GWAS) conducted in Australian and Amish populations in whom the Seasonal Pattern Assessment Questionnaire (SPAQ) had been administered. The total sample size was 4,156 individuals. Genetic risk scores based on results from prior large GWAS studies of bipolar disorder (BD), major depressive disorder (MDD), and schizophrenia (SCZ) were calculated to test for overlap in risk between psychiatric disorders and seasonality. Results The most significant association was with rs11825064 (p = 1.7 × 10−6, β = 0.64, S.E = 0.13), an intergenic SNP found on chromosome 11. The evidence for overlap in risk factors was strongest for SCZ and seasonality, with the SCZ genetic profile scores explaining 3% of the variance in log-transformed GSS. BD genetic profile scores were also significantly associated with seasonality, although at much weaker levels, and no evidence for overlap in risk was detected between MDD and seasonality. Conclusions Common SNPs of very large effect likely do not exist for seasonality in the populations examined. As expected, there was overlapping genetic risk factors for BD (but not MDD) with seasonality. Unexpectedly, the risk for SCZ and seasonality had the largest overlap, an unprecedented finding that requires replication in other populations, and has potential clinical implications considering overlapping cognitive deficits in seasonal affective disorders and SCZ PMID:25562672

  12. PIXE analysis showed that the preirradiation enhanced recovery of bone marrow elements after challenging irradiation in C57BL/6N Mice

    International Nuclear Information System (INIS)

    Matsuda, Y.; Yonezawa, M.; Nishiyama, F.

    2000-01-01

    Priming X-irradiation with 0.3-0.5 Gy induces radio-resistance in C57BL/6 strain of mice 2 weeks afterward. Elements in the bone marrow, sampled 11 days after challenging exposure to 5.0 Gy, were determined by PIXE. The challenging irradiation decreased Mg, P, S, K, Ca and Zn as well as dried bone marrow weight. The pre-irradiation enhanced recovery of these levels, indicating stimulated recovery of the metabolism int he tissue. Fe in both control (without pre-irradiation) and experimental groups increased to about twice the original value, showing elevated hemoglobin synthesis after challenging exposure. In previous studies we have reported that recovery of peripheral blood cell counts after sub-lethal irradiation was enhanced by the pre-irradiation. Further, study on accumulation of p53 and Bax proteins, which lead to apoptotic cell death, revealed that the pre-irradiation significantly suppressed accumulation of these proteins in the spleen after challenging irradiation with 3 Gy. These results and our present study suggest that the pre-irradiation decreased the spleen cell death, and favored re-growth of the spleen cells, resulting in stimulated recovery of metabolism for hematopoiesis in the bone marrow as well as in the spleen after challenging high dose irradiation. Stimulated recovery of Mg, P, S, K, Ca and Zn levels might indicate the importance of these elements in hematopoiesis. (author)

  13. Comprehensive Behavioral Analysis of Male Ox1r−/− Mice Showed Implication of Orexin Receptor-1 in Mood, Anxiety, and Social Behavior

    Science.gov (United States)

    Abbas, Md. G.; Shoji, Hirotaka; Soya, Shingo; Hondo, Mari; Miyakawa, Tsuyoshi; Sakurai, Takeshi

    2015-01-01

    Neuropeptides orexin A and orexin B, which are exclusively produced by neurons in the lateral hypothalamic area, play an important role in the regulation of a wide range of behaviors and homeostatic processes, including regulation of sleep/wakefulness states and energy homeostasis. The orexin system has close anatomical and functional relationships with systems that regulate the autonomic nervous system, emotion, mood, the reward system, and sleep/wakefulness states. Recent pharmacological studies using selective antagonists have suggested that orexin receptor-1 (OX1R) is involved in physiological processes that regulate emotion, the reward system, and autonomic nervous system. Here, we examined Ox1r−/− mice with a comprehensive behavioral test battery to screen additional OX1R functions. Ox1r−/− mice showed increased anxiety-like behavior, altered depression-like behavior, slightly decreased spontaneous locomotor activity, reduced social interaction, increased startle response, and decreased prepulse inhibition. These results suggest that OX1R plays roles in social behavior and sensory motor gating in addition to roles in mood and anxiety. PMID:26696848

  14. High Content Analysis of Hippocampal Neuron-Astrocyte Co-cultures Shows a Positive Effect of Fortasyn Connect on Neuronal Survival and Postsynaptic Maturation.

    Science.gov (United States)

    van Deijk, Anne-Lieke F; Broersen, Laus M; Verkuyl, J Martin; Smit, August B; Verheijen, Mark H G

    2017-01-01

    Neuronal and synaptic membranes are composed of a phospholipid bilayer. Supplementation with dietary precursors for phospholipid synthesis -docosahexaenoic acid (DHA), uridine and choline- has been shown to increase neurite outgrowth and synaptogenesis both in vivo and in vitro . A role for multi-nutrient intervention with specific precursors and cofactors has recently emerged in early Alzheimer's disease, which is characterized by decreased synapse numbers in the hippocampus. Moreover, the medical food Souvenaid, containing the specific nutrient combination Fortasyn Connect (FC), improves memory performance in early Alzheimer's disease patients, possibly via maintaining brain connectivity. This suggests an effect of FC on synapses, but the underlying cellular mechanism is not fully understood. Therefore, we investigated the effect of FC (consisting of DHA, eicosapentaenoic acid (EPA), uridine, choline, phospholipids, folic acid, vitamins B12, B6, C and E, and selenium), on synaptogenesis by supplementing it to primary neuron-astrocyte co-cultures, a cellular model that mimics metabolic dependencies in the brain. We measured neuronal developmental processes using high content screening in an automated manner, including neuronal survival, neurite morphology, as well as the formation and maturation of synapses. Here, we show that FC supplementation resulted in increased numbers of neurons without affecting astrocyte number. Furthermore, FC increased postsynaptic PSD95 levels in both immature and mature synapses. These findings suggest that supplementation with FC to neuron-astrocyte co-cultures increased both neuronal survival and the maturation of postsynaptic terminals, which might aid the functional interpretation of FC-based intervention strategies in neurological diseases characterized by neuronal loss and impaired synaptic functioning.

  15. High Content Analysis of Hippocampal Neuron-Astrocyte Co-cultures Shows a Positive Effect of Fortasyn Connect on Neuronal Survival and Postsynaptic Maturation

    Directory of Open Access Journals (Sweden)

    Anne-Lieke F. van Deijk

    2017-08-01

    Full Text Available Neuronal and synaptic membranes are composed of a phospholipid bilayer. Supplementation with dietary precursors for phospholipid synthesis –docosahexaenoic acid (DHA, uridine and choline– has been shown to increase neurite outgrowth and synaptogenesis both in vivo and in vitro. A role for multi-nutrient intervention with specific precursors and cofactors has recently emerged in early Alzheimer's disease, which is characterized by decreased synapse numbers in the hippocampus. Moreover, the medical food Souvenaid, containing the specific nutrient combination Fortasyn Connect (FC, improves memory performance in early Alzheimer's disease patients, possibly via maintaining brain connectivity. This suggests an effect of FC on synapses, but the underlying cellular mechanism is not fully understood. Therefore, we investigated the effect of FC (consisting of DHA, eicosapentaenoic acid (EPA, uridine, choline, phospholipids, folic acid, vitamins B12, B6, C and E, and selenium, on synaptogenesis by supplementing it to primary neuron-astrocyte co-cultures, a cellular model that mimics metabolic dependencies in the brain. We measured neuronal developmental processes using high content screening in an automated manner, including neuronal survival, neurite morphology, as well as the formation and maturation of synapses. Here, we show that FC supplementation resulted in increased numbers of neurons without affecting astrocyte number. Furthermore, FC increased postsynaptic PSD95 levels in both immature and mature synapses. These findings suggest that supplementation with FC to neuron-astrocyte co-cultures increased both neuronal survival and the maturation of postsynaptic terminals, which might aid the functional interpretation of FC-based intervention strategies in neurological diseases characterized by neuronal loss and impaired synaptic functioning.

  16. Tyrosine kinase inhibitors show different anti-brain metastases efficacy in NSCLC: A direct comparative analysis of icotinib, gefitinib, and erlotinib in a nude mouse model.

    Science.gov (United States)

    Tan, Jianlong; Li, Min; Zhong, Wen; Hu, Chengping; Gu, Qihua; Xie, Yali

    2017-11-17

    Brain metastasis is an increasing problem in non-small cell lung cancer (NSCLC) patients. Tyrosine kinase inhibitors (TKIs), including gefitinib, erlotinib, and icotinib, are reported to be effective in patients with brain metastases. However, direct comparative studies of the pharmacokinetics and efficacy of these three drugs in treating brain metastases are lacking. In the present investigation, we found that gefitinib penetrated the blood-tumor barrier and was distributed to brain metastases more effectively than erlotinib or icotinib in a nude mouse model. The 1-h ratio of brain metastases to plasma concentration for gefitinib, erlotinib, and icotinib was 9.82±1.03%, 4.83±0.25%, and 2.62±0.21%, respectively. The 2-h ratio of brain metastases to plasma concentration for gefitinib, erlotinib, and icotinib was 15.11±2.00%, 5.73±1.31%, and 2.69±0.31%, respectively. Gefitinib exhibited the strongest antitumor activity ( p gefitinib vs. erlotinib =0.005; p gefitinib vs. icotinib =0.002). Notably, erlotinib exhibited a better treatment efficacy than icotinib ( p =0.037). Consistently, immunohistochemical data showed that TKIs differentially inhibit the proliferation of metastatical tumor cells. Gefitinib and erlotinib markedly inhibited the proliferation of tumor cells, while there were more ki-67-positive tumor cells in the icotinib group. Additionally, gefitinib inhibited the phosphorylation of EGFR better than the other drugs, whereas pEGFR expression levels in erlotinib groups were lower than levels in the icotinib group ( p gefitinib vs. erlotinib =0.995; p gefitinib vs. icotinib =0.028; p erlotinib vs. icotinib =0.042).Altogether, our findings suggest that gefitinib and erlotinib can inhibit the growth of PC-9-luc brain tumors. Gefitinib demonstrated better antitumor activity and penetration rate in brain metastases than erlotinib or icotinib.

  17. Retrotransposon-centered analysis of piRNA targeting shows a shift from active to passive retrotransposon transcription in developing mouse testes

    Directory of Open Access Journals (Sweden)

    Mourier Tobias

    2011-09-01

    Full Text Available Abstract Background Piwi-associated RNAs (piRNAs bind transcripts from retrotransposable elements (RTE in mouse germline cells and seemingly act as guides for genomic methylation, thereby repressing the activity of RTEs. It is currently unknown if and how Piwi proteins distinguish RTE transcripts from other cellular RNAs. During germline development, the main target of piRNAs switch between different types of RTEs. Using the piRNA targeting of RTEs as an indicator of RTE activity, and considering the entire population of genomic RTE loci along with their age and location, this study aims at further elucidating the dynamics of RTE activity during mouse germline development. Results Due to the inherent sequence redundancy between RTE loci, assigning piRNA targeting to specific loci is problematic. This limits the analysis, although certain features of piRNA targeting of RTE loci are apparent. As expected, young RTEs display a much higher level of piRNA targeting than old RTEs. Further, irrespective of age, RTE loci near protein-coding coding genes are targeted to a greater extent than RTE loci far from genes. During development, a shift in piRNA targeting is observed, with a clear increase in the relative piRNA targeting of RTEs residing within boundaries of protein-coding gene transcripts. Conclusions Reanalyzing published piRNA sequences and taking into account the features of individual RTE loci provide novel insight into the activity of RTEs during development. The obtained results are consistent with some degree of proportionality between what transcripts become substrates for Piwi protein complexes and the level by which the transcripts are present in the cell. A transition from active transcription of RTEs to passive co-transcription of RTE sequences residing within protein-coding transcripts appears to take place in postnatal development. Hence, the previously reported increase in piRNA targeting of SINEs in postnatal testis development

  18. Post-mortem analysis of suicide victims shows ABCB1 haplotype 1236T-2677T-3435T as a candidate predisposing factor behind adverse drug reactions in females.

    Science.gov (United States)

    Rahikainen, Anna-Liina; Palo, Jukka U; Haukka, Jari; Sajantila, Antti

    2018-04-01

    Genetic variation in efflux transporter, permeability glycoprotein (P-gp), has recently been associated with completed violent suicides and also violent suicide attempts. As depression is known to be a risk factor for suicide and many antidepressants are P-gp substrates, it has been speculated that inadequate antidepressant treatment response or adverse side effects could be involved. The aim of this study was to investigate whether there is an association between the P-gp coding ABCB1 gene and completed suicides in citalopram users. Also, the effect of sex and suicide method used (violent vs. non-violent) was evaluated. All cases included in the study population, 349 completed suicide victims and 284 controls, were shown to be positive for antidepressant citalopram in a post-mortem toxicological drug screen. ABCB1 1236C>T, 2677G>T/A and 3435C>T polymorphisms were determined by TaqMan genotyping assays. Haplotypes were constructed from genotype data using the PHASE software. The association between the manner of death and the ABCB1 haplotype was tested with logistic regression analysis. No statistically significant differences were observed in the ABCB1 allele or genotype frequencies between the suicide and control groups. However, the ABCB1 1236T-2677T-3435T haplotype was associated with completed suicides of female citalopram users (odds ratio: 2.23; 95% confidence interval: 1.22-4.07; P=0.009). After stratification by the method used for suicide, the association emerged in fatal intoxications (odds ratio: 2.51; 95% confidence interval: 1.29-4.87; P=0.007). In other groups, no statistically significant associations were observed. Our results suggest that female citalopram users with ABCB1 1236T-2677T-3435T are more vulnerable to adverse effects of the drugs as this haplotype was enriched in non-violent suicides of female citalopram users. Even though the biological mechanism behind this observation is unknown, the results provide another example of the importance

  19. Standardization of enterococci density estimates by EPA qPCR methods and comparison of beach action value exceedances in river waters with culture methods

    Science.gov (United States)

    The U.S.EPA has published recommendations for calibrator cell equivalent (CCE) densities of enterococci in recreational waters determined by a qPCR method in its 2012 Recreational Water Quality Criteria (RWQC). The CCE quantification unit stems from the calibration model used to ...

  20. Human-associated fecal qPCR measurements and predicted risk of gastrointestinal illness in recreational waters contaminated with raw sewage

    Science.gov (United States)

    We used quantitative microbial risk assessment (QMRA) to estimate the risk of gastrointestinal (GI) illness associated with swimming in recreational waters containing different concentrations of human-associated fecal qPCR markers from raw sewage– HF183 and HumM2. The volume/volu...

  1. Blood and dried blood spot telomere length measurement by qPCR: assay considerations.

    Directory of Open Access Journals (Sweden)

    DeAnna L Zanet

    Full Text Available Measurement of telomere length is crucial for the study of telomere maintenance and its role in molecular pathophysiology of diseases and in aging. Several methods are used to measure telomere length, the choice of which usually depends on the type and size of sample to be assayed, as well as cost and throughput considerations. The goal of this study was to investigate the factors that may influence the reliability of qPCR-based relative telomere length measurements in whole blood. Day to day intra-individual variability, types of blood anticoagulant, sample storage conditions, processing and site of blood draw were investigated. Two qPCR-based methods to measure telomere length (monoplex vs. multiplex were also investigated and showed a strong correlation between them. Freezing and thawing of the blood and storage of the blood at 4°C for up to 4 days did not affect telomere length values. Telomere lengths in dried blood spots were significantly higher than both whole blood and peripheral mononuclear blood cells, and were highly correlated with both. We found that telomere length measurements were significantly higher in dried blood spots collected directly from fingertip prick compared to dried blood spots prepared with anticoagulated whole blood collected from the finger, and non-blotted whole blood taken from both finger and arm venipuncture. This suggests that DNA from cells blotted on paper is not equivalent to that collected from venipuncture whole blood, and caution should be taken when comparing between blood sample types.

  2. Accuracy of qPCR for quantifying Leishmania kDNA in different skin layers of patients with American tegumentary leishmaniasis.

    Science.gov (United States)

    Sevilha-Santos, L; Dos Santos Júnior, A C M; Medeiros-Silva, V; Bergmann, J O; da Silva, E F; Segato, L F; Arabi, A Y M; de Paula, N A; Sampaio, R N R; Lima, B D; Gomes, C M

    2018-05-03

    Superficial swab sampling of American tegumentary leishmaniasis (ATL) lesions shows higher amounts of Leishmania than those from biopsy. Subcutaneous involvement is also important in ATL, but parasite quantification according to lesion depth has not been evaluated. We aim to present the best depth at which sampling should be performed for molecular exams of ATL. Patients with a clinical presentation compatible with ATL were allocated to ATL and control groups. Qualitative and quantitative qPCR assays were performed using SYBR Green and primers amplifying the kDNA minicircle of Leishmania spp. in different skin layers, including the epidermis, the superior dermis, the inferior dermis, and the hypodermis. Fifty-nine patients were included in this study, including 40 who had been diagnosed with ATL and 19 controls. The number of parasites was greater in samples of the epidermis and superior dermis (159.1 × 10 6 , range 4.0-781.7, and 75.4 × 10 6 , range 8.0-244.5, mean Leishmania parasite equivalents per μg of tissue DNA, respectively) than those in samples of the inferior dermis and hypodermis (54.6, range 8.0-256.6, and 16.8 × 10 6 , range 8.0-24.1, mean Leishmania parasite equivalents per μg of tissue DNA, respectively). The best diagnostic accuracy was achieved in the superior dermis (77.9%) and was significantly greater than that in the hypodermis (63.3%; p 0.039). We conclude that superficial sampling can retrieve a greater quantity of parasites. Future studies of the role of transepidermal elimination as a mechanism of host defence in ATL must be performed as there is a considerable quantity of Leishmania kDNA in the epidermis. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  3. A rapid Q-PCR titration protocol for adenovirus and helper-dependent adenovirus vectors that produces biologically relevant results

    Science.gov (United States)

    Gallaher, Sean D.; Berk, Arnold J.

    2013-01-01

    Adenoviruses are employed in the study of cellular processes and as expression vectors used in gene therapy. The success and reproducibility of these studies is dependent in part on having accurate and meaningful titers of replication competent and helper-dependent adenovirus stocks, which is problematic due to the use of varied and divergent titration protocols. Physical titration methods, which quantify the total number of viral particles, are used by many, but are poor at estimating activity. Biological titration methods, such as plaque assays, are more biologically relevant, but are time consuming and not applicable to helper-dependent gene therapy vectors. To address this, a protocol was developed called “infectious genome titration” in which viral DNA is isolated from the nuclei of cells ~3 h post-infection, and then quantified by Q-PCR. This approach ensures that only biologically active virions are counted as part of the titer determination. This approach is rapid, robust, sensitive, reproducible, and applicable to all forms of adenovirus. Unlike other Q-PCR-based methods, titers determined by this protocol are well correlated with biological activity. PMID:23624118

  4. In silico prediction and qPCR validation of novel sRNAs in Propionibacterium acnes KPA171202

    Directory of Open Access Journals (Sweden)

    Praveen P. Balgir

    2016-06-01

    Full Text Available Propionibacterium acnes is an anaerobic, Gram-positive, opportunistic pathogen known to be involved in a wide variety of diseases ranging from mild acne to prostate cancer. Bacterial small non-coding RNAs are novel regulators of gene expression and are known to be involved in, virulence, pathogenesis, stress tolerance and adaptation to environmental changes in bacteria. The present study was undertaken keeping in view the lack of predicted sRNAs of P. acnes KPA171202 in databases. This report represents the first attempt to identify sRNAs in P. acnes KPA171202. A total of eight potential candidate sRNAs were predicted using SIPHT, one was found to have a Rfam homolog and seven were novel. Out of these seven predicted sRNAs, five were validated by reverse transcriptase-polymerase chain reaction (RT-PCR and sequencing. The expression of these sRNAs was quantified in different growth phases by qPCR (quantitative PCR. They were found to be expressed in both exponential and stationary stages of growth but with maximum expression in stationary phase which points to a regulatory role for them. Further investigation of their targets and regulatory functions is in progress.

  5. Detection and quantification of Spongospora subterranea f. sp. subterranea in bait plants and potato fields in Colombia using QPCR

    International Nuclear Information System (INIS)

    Garcia Bastidas, Nevar; Morales, Juan Gonzalo; Gonzalez Jaimes, Paola; Gutierrez, Pablo Andres; Marin Montoya, Mauricio

    2013-01-01

    In recent years, potato crops (Solanum tuberosum, S. phureja) have been seriously affected by powdery scab; a disease caused by Spongospora subterranea f.sp. subterranea (Sss). In Colombia, asymptomatic detection of Sss has been achieved with bait plants, PCR of its regions and ELISA tests. Unfortunately, these techniques have low sensitivity and may require long processing times. In this work, quantitative real time PCR (qPCR) was tested for detection of Sss using different sets of primers. Primers SsTQF1-SsTQR1, Spon421F-Spon494R and SscolF-SscolR (designed in this study), were tested using SYBR green, while primers sponfsponr were tested using the Taqman probe sponp. Primers Spon421F-Spon494R was discarded due to lack of specificity. Standard curves were obtained from serial dilutions of Cystosori. the 20 N. benthamiana and potato bait plants evaluated tested positive for Sss using primers SsTQF1-SsTQR1 (Ct: 10.57-29.34) and Sscolf-SscolR (Ct: 14.39-34.08) and 19 samples were positive with primers SponF-SponR-SponP, with Ct values ranging between 15,63 and 38,93. Sss was detected in 17 out of 20 root samples from potato crops in la Union (Antioquia) using primers SscolF-SscolRt with an estimated concentration of 6470 to 1,39x10 1 0 cystosori/ mL. these results suggest high levels of sss in the potato fields from this region and recall the importance of strengthening seed-certification programs in Colombia.

  6. gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples.

    Directory of Open Access Journals (Sweden)

    Luigi Vezzulli

    Full Text Available The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA is a chitin-binding protein involved in V. cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V. cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V. cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR samples. Overall, the method is sensitive (50 gene copies, highly specific for V. cholerae and failed to amplify strains of the closely-related species Vibrio mimicus. The sensitivity of the assay applied to environmental and stool samples spiked with V. cholerae ATCC 39315 was comparable to that of pure cultures and was of 102 genomic units/l for drinking and seawater samples, 101 genomic units/g for sediment and 102 genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V. cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V. cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies.

  7. Quantitative analysis of food and feed samples with droplet digital PCR.

    Directory of Open Access Journals (Sweden)

    Dany Morisset

    Full Text Available In this study, the applicability of droplet digital PCR (ddPCR for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs. Real-time quantitative polymerase chain reaction (qPCR is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.

  8. Development of a rapid HRM qPCR for the diagnosis of the four most prevalent Plasmodium lineages in New Zealand.

    Science.gov (United States)

    Schoener, E R; Hunter, S; Howe, L

    2017-07-01

    Although wildlife rehabilitation and translocations are important tools in wildlife conservation in New Zealand, disease screening of birds has not been standardized. Additionally, the results of the screening programmes are often difficult to interpret due to missing disease data in resident or translocating avian populations. Molecular methods have become the most widespread method for diagnosing avian malaria (Plasmodium spp.) infections. However, these methods can be time-consuming, expensive and are less specific in diagnosing mixed infections. Thus, this study developed a new real-time PCR (qPCR) method that was able to detect and specifically identify infections of the three most common lineages of avian malaria in New Zealand (Plasmodium (Novyella) sp. SYAT05, Plasmodium elongatum GRW6 and Plasmodium spp. LINN1) as well as a less common, pathogenic Plasmodium relictum GRW4 lineage. The assay was also able to discern combinations of these parasites in the same sample and had a detection limit of five parasites per microlitre. Due to concerns relating to the presence of the potentially highly pathogenic P. relictum GRW4 lineage in avian populations, an additional confirmatory high resolution (HRM) qPCR was developed to distinguish between commonly identified P. elongatum GRW6 from P. relictum GRW4. The new qPCR assays were tested using tissue samples containing Plasmodium schizonts from three naturally infected dead birds resulting in the identified infection of P. elongatum GRW6. Thus, these rapid qPCR assays have shown to be cost-effective and rapid screening tools for the detection of Plasmodium infection in New Zealand native birds.

  9. Quantification by qPCR of Pathobionts in Chronic Periodontitis: Development of Predictive Models of Disease Severity at Site-Specific Level

    OpenAIRE

    Tomás, Inmaculada; Regueira-Iglesias, Alba; López, Maria; Arias-Bujanda, Nora; Novoa, Lourdes; Balsa-Castro, Carlos; Tomás, Maria

    2017-01-01

    Currently, there is little evidence available on the development of predictive models for the diagnosis or prognosis of chronic periodontitis based on the qPCR quantification of subgingival pathobionts. Our objectives were to: (1) analyze and internally validate pathobiont-based models that could be used to distinguish different periodontal conditions at site-specific level within the same patient with chronic periodontitis; (2) develop nomograms derived from predictive models. Subgingival pl...

  10. Analytical Performance of Four Polymerase Chain Reaction (PCR and Real Time PCR (qPCR Assays for the Detection of Six Leishmania Species DNA in Colombia

    Directory of Open Access Journals (Sweden)

    Cielo M. León

    2017-10-01

    Full Text Available Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR, limit of detection (LoD and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia. Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America.

  11. Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

    Science.gov (United States)

    León, Cielo M.; Muñoz, Marina; Hernández, Carolina; Ayala, Martha S.; Flórez, Carolina; Teherán, Aníbal; Cubides, Juan R.; Ramírez, Juan D.

    2017-01-01

    Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America. PMID:29046670

  12. Polymorphism in promoter of SIX4 gene shows association with its transcription and body measurement traits in Qinchuan cattle.

    Science.gov (United States)

    Wei, Dawei; Raza, Sayed Haidar Abbas; Zhang, Jiupan; Gui, Linsheng; Rahman, Siddiq Ur; Khan, Rajwali; Hosseini, Seyed Mahdi; Kaleri, Hubdar Ali; Zan, Linsen

    2018-05-20

    The sine oculis homeobox homolog 4 (SIX4) gene belongs to the SIX gene family, which plays a critical role in muscle regeneration and early stages of ontogeny. This study aimed to detect promoter variations of bovine SIX4 genes in Qinchuan cattle, and to evaluate the effect of transcription regulations and body measurement traits. Quantitative real-time PCR (qPCR) results showed that the mRNA expression levels of SIX4 gene were found significantly highest in longissimus thoracis tissue and individual before attaining the stage of physiological maturity. Using sequencing technology on a total of 428 Qinchuan cattle, seven single nucleotide polymorphisms (SNPs) were identified in the promoter region of SIX4, and seven haplotypes representing 18 potential transcription factor compositions of polymorphic potential cis-acting elements. Association analysis indicated that the H 3 -H 3 diplotype performed greater withers height, chest depth, chest circumference, back fat thickness and ultrasound loin muscle area (P cattle. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. The polymorphic insertion of the luteinizing hormone receptor "insLQ" show a negative association to LHR gene expression and to the follicular fluid hormonal profile in human small antral follicles

    DEFF Research Database (Denmark)

    Borgbo, T; Chrudimska, J; Macek, M

    2018-01-01

    (AMHR2) and LHCGR, respectively, were observed for insLQ/insLQ compared to -/insLQ and the -/- genotypes. Moreover, LHCGR and CYP19a1 together with oestradiol and inhibin-B were significantly increased in -/insLQ compared to the -/- genotype. The homozygous insLQ genotype showed strong significant......The luteinizing hormone receptor (LHCGR) has a little studied polymorphic 6 bp insertion (rs4539842/insLQ). This study has evaluated the insLQ polymorphism in relation to potential associations with hormonal characteristics of human small antral follicles (hSAFs). In total, 310 hSAFs were collected...... from 86 women undergoing fertility preservation. Analysis included hormonal profile of 297 follicular fluid (FF) samples and 148 corresponding granulosa cells samples were evaluated by qPCR for selected genes. Significantly reduced and non-detectable mRNA levels of anti-Müllerian hormone receptor II...

  14. Detection and characterization of Leishmania (Leishmania and Leishmania (Viannia by SYBR green-based real-time PCR and high resolution melt analysis targeting kinetoplast minicircle DNA.

    Directory of Open Access Journals (Sweden)

    Marcello Ceccarelli

    Full Text Available Leishmaniasis is a neglected disease with a broad clinical spectrum which includes asymptomatic infection. A thorough diagnosis, able to distinguish and quantify Leishmania parasites in a clinical sample, constitutes a key step in choosing an appropriate therapy, making an accurate prognosis and performing epidemiological studies. Several molecular techniques have been shown to be effective in the diagnosis of leishmaniasis. In particular, a number of PCR methods have been developed on various target DNA sequences including kinetoplast minicircle constant regions. The first aim of this study was to develop a SYBR green-based qPCR assay for Leishmania (Leishmania infantum detection and quantification, using kinetoplast minicircle constant region as target. To this end, two assays were compared: the first used previously published primer pairs (qPCR1, whereas the second used a nested primer pairs generating a shorter PCR product (qPCR2. The second aim of this study was to evaluate the possibility to discriminate among subgenera Leishmania (Leishmania and Leishmania (Viannia using the qPCR2 assay followed by melting or High Resolution Melt (HRM analysis. Both assays used in this study showed good sensitivity and specificity, and a good correlation with standard IFAT methods in 62 canine clinical samples. However, the qPCR2 assay allowed to discriminate between Leishmania (Leishmania and Leishmania (Viannia subgenera through melting or HRM analysis. In addition to developing assays, we investigated the number and genetic variability of kinetoplast minicircles in the Leishmania (L. infantum WHO international reference strain (MHOM/TN/80/IPT1, highlighting the presence of minicircle subclasses and sequence heterogeneity. Specifically, the kinetoplast minicircle number per cell was estimated to be 26,566±1,192, while the subclass of minicircles amplifiable by qPCR2 was estimated to be 1,263±115. This heterogeneity, also observed in canine clinical

  15. Interlaboratory comparison of three microbial source tracking quantitative polymerase chain reaction (qPCR) assays from fecal-source and environmental samples

    Science.gov (United States)

    Stelzer, Erin A.; Strickler, Kriston M.; Schill, William B.

    2012-01-01

    During summer and early fall 2010, 15 river samples and 6 fecal-source samples were collected in West Virginia. These samples were analyzed by three laboratories for three microbial source tracking (MST) markers: AllBac, a general fecal indicator; BacHum, a human-associated fecal indicator; and BoBac, a ruminant-associated fecal indicator. MST markers were analyzed by means of the quantitative polymerase chain reaction (qPCR) method. The aim was to assess interlaboratory precision when the three laboratories used the same MST marker and shared deoxyribonucleic acid (DNA) extracts of the samples, but different equipment, reagents, and analyst experience levels. The term assay refers to both the markers and the procedure differences listed above. Interlaboratory precision was best for all three MST assays when using the geometric mean absolute relative percent difference (ARPD) and Friedman's statistical test as a measure of interlaboratory precision. Adjustment factors (one for each MST assay) were calculated using results from fecal-source samples analyzed by all three laboratories and applied retrospectively to sample concentrations to account for differences in qPCR results among labs using different standards and procedures. Following the application of adjustment factors to qPCR results, ARPDs were lower; however, statistically significant differences between labs were still observed for the BacHum and BoBac assays. This was a small study and two of the MST assays had 52 percent of samples with concentrations at or below the limit of accurate quantification; hence, more testing could be done to determine if the adjustment factors would work better if the majority of sample concentrations were above the quantification limit.

  16. Effectiveness of onsite wastewater reuse system in reducing bacterial contaminants measured with human-specific IMS/ATP and qPCR.

    Science.gov (United States)

    Agidi, Senyo; Vedachalam, Sridhar; Mancl, Karen; Lee, Jiyoung

    2013-01-30

    Water shortages and the drive to recycle is increasing interest in reuse of reclaimed wastewater. Timely and cost-effective ways to detect fecal pollutants prior to reuse increases confidence of residents and neighbors concerned about reuse of reclaimed wastewater. The on-site wastewater treatment and reuse systems (OWTRS) used in this study include a septic tank, peat bioreactor, ClO(2) disinfection and land spray irrigation system. Bacteroides fragilis, Escherichia coli and Enterococcus spp., were tested with immunomagnetic separation/ATP bioluminescence (IMS/ATP), qPCR and culture-based methods. The results displayed a 2-log reduction in fecal bacteria in the peat bioreactor and a 5-log reduction following chloride dioxide disinfection. The fecal bacteria levels measured by IMS/ATP correlated with qPCR results: HuBac 16S (R(2) = 0.903), Bf-group 16S (R(2) = 0.956), gyrB (R(2) = 0.673), and Ent 23S (R(2) = 0.724). This is the first study in which the newly developed human-specific IMS/ATP and previously developed IMS/ATP were applied for determining OWTRS efficiency. Results of the study revealed that IMS/ATP is a timely and cost-effective way to detect fecal contaminants, and results were validated with qPCR and culture based methods. The new IMS/ATP can also be applied broadly in the detection of human-originated fecal contamination. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

    Science.gov (United States)

    Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

    2016-01-01

    Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

  18. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

    Directory of Open Access Journals (Sweden)

    Cengiz Ikten

    Full Text Available Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.. Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

  19. Fluorescence in situ hybridization and qPCR to detect Merkel cell polyomavirus physical status and load in Merkel cell carcinomas.

    Science.gov (United States)

    Haugg, Anke M; Rennspiess, Dorit; zur Hausen, Axel; Speel, Ernst-Jan M; Cathomas, Gieri; Becker, Jürgen C; Schrama, David

    2014-12-15

    The Merkel cell polyomavirus (MCPyV) is detected in 80% of Merkel cell carcinomas (MCC). Clonal integration and tumor-specific mutations in the large T antigen are strong arguments that MCPyV is a human tumor virus. However, the relationship between viral presence and cancer induction remains discussed controversially. Since almost all studies on virus prevalence are based on PCR techniques, we performed MCPyV fluorescence in situ hybridization (FISH) on MCC to gain information about the quality of the viral presence on the single cell level. MCPyV-FISH was performed on tissue microarrays containing 62 formalin-fixed and paraffin-embedded tissue samples including all tumor grades of 42 patients. The hybridization patterns were correlated to the qPCR data determined on corresponding whole tissue sections. Indeed, MCPyV-FISH and qPCR data were highly correlated, i.e. 83% for FISH-positive and 93% for FISH-negative cores. Accordingly, the mean of the qPCR values of all MCPyV-positive cores differed significantly from the mean of the negative cores (p = 0.0076). Importantly, two hybridization patterns were definable in the MCPyV-FISH: a punctate pattern (85%) indicating viral integration, which correlated with a moderate viral abundance and a combination of the punctate with a diffuse pattern (15%), suggesting a possible coexistence of integrated and episomal virus which was associated with very high viral load and VP1 expression. Thus, MCPyV-FISH adds important information on the single cell level within the histomorphological context and could therefore be an important tool to further elucidate MCPyV related carcinogenesis. © 2014 UICC.

  20. Evaluation of a low-cost procedure for sampling, long-term storage, and extraction of RNA from blood for qPCR analyses

    DEFF Research Database (Denmark)

    Mærkedahl, Rasmus Baadsgaard; Frøkiær, Hanne; Lauritzen, Lotte

    2015-01-01

    Abstract Background: In large clinical trials, where RNA cannot be extracted immediately after sampling, preserving RNA in whole blood is a crucial initial step in obtaining robust qPCR data. The current golden standard for RNA preservation is costly and designed for time-consuming column-based RNA....../binding solution over time and between samples stored and extracted by the two systems. Conclusions: The MagMAX system can be used for storage of human blood for up to 4 months and is equivalent to the PAXgene system for RNA extraction. It furthermore, provides a means for significant cost reduction in large...

  1. Risk Aversion in Game Shows

    DEFF Research Database (Denmark)

    Andersen, Steffen; Harrison, Glenn W.; Lau, Morten I.

    2008-01-01

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

  2. Measuring performance at trade shows

    DEFF Research Database (Denmark)

    Hansen, Kåre

    2004-01-01

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

  3. Regional assignment of seven loci to 12p 13. 2-pter by PCR analysis of somatic cell hybrids containing the der(12) or the der(X) chromosome from a mesothelioma showing t(X; 12)(q22; p13)

    Energy Technology Data Exchange (ETDEWEB)

    Aerssens, J.; Chaffanet, M.; Baens, M.; Matthijs, G.; Van Den Berche, H.; Cassiman, J.J.; Marynen, P. (Arthritis and Metabolic Bone Disease Research Unit, Leuven (Belgium))

    1994-03-01

    Two somatic cell hybrids containing the der(12) or the der(X) from a mesothelioma with a translocation t(X;12) (q22;p13) as the only chromosomal change were generated to characterize the region of 12p12 containing the translocation breakpoint. Fluorescence in situ hybridization analysis showed the breakpoint on chromosome 12 to occur between VWF and D12S158. On the linkage map developed by J. Weissenbach et al., the breakpoints were located between DXS1106 and DCS1001 on chromosome X. PCR analysis based on genomic sequences, with DNA from both somatic cell hybrids, enabled mapping of CACNL1A1, FGF6, D12S370, D12S38OE, D12S381E, and D12S382E distally to the 12p13 breakpoint and to VWF. 11 refs., 2 figs., 1 tab.

  4. Tokyo Motor Show 2003; Tokyo Motor Show 2003

    Energy Technology Data Exchange (ETDEWEB)

    Joly, E.

    2004-01-01

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

  5. Powerful qPCR assays for the early detection of latent invaders: interdisciplinary approaches in clinical cancer research and plant pathology.

    Science.gov (United States)

    Luchi, Nicola; Capretti, Paolo; Pazzagli, Mario; Pinzani, Pamela

    2016-06-01

    Latent invaders represent the first step of disease before symptoms occur in the host. Based on recent findings, tumors are considered to be ecosystems in which cancer cells act as invasive species that interact with the native host cell species. Analogously, in plants latent fungal pathogens coevolve within symptomless host tissues. For these reasons, similar detection approaches can be used for an early diagnosis of the invasion process in both plants and humans to prevent or reduce the spread of the disease. Molecular tools based on the evaluation of nucleic acids have been developed for the specific, rapid, and early detection of human diseases. During the last decades, these techniques to assess and quantify the proliferation of latent invaders in host cells have been transferred from the medical field to different areas of scientific research, such as plant pathology. An improvement in molecular biology protocols (especially referring to qPCR assays) specifically designed and optimized for detection in host plants is therefore advisable. This work is a cross-disciplinary review discussing the use of a methodological approach that is employed within both medical and plant sciences. It provides an overview of the principal qPCR tools for the detection of latent invaders, focusing on comparisons between clinical cancer research and plant pathology, and recent advances in the early detection of latent invaders to improve prevention and control strategies.

  6. Correlation of crAssphage-based qPCR markers with culturable and molecular indicators of human fecal pollution in an impacted urban watershed.

    Science.gov (United States)

    Stachler, Elyse; Akyon, Benay; Aquino de Carvalho, Nathalia; Ference, Christian; Bibby, Kyle

    2018-06-06

    Environmental waters are monitored for fecal pollution to protect public health. Many previously developed human-specific fecal pollution indicators lack adequate sensitivity to be reliably detected in environmental waters or do not correlate well with viral pathogens. Recently, two novel human sewage-associated source tracking qPCR markers were developed based on the bacteriophage crAssphage, CPQ_056 and CPQ_064. These assays are highly human specific, abundant in sewage, and are viral-based, suggesting great promise for environmental application as human fecal pollution indicators. A 30-day sampling study was conducted in an urban stream impacted by combined sewer overflows to evaluate the crAssphage markers' performance in an environmental system. The crAssphage markers were present at concentrations of 4.02-6.04 log10 copies/100 mL throughout the study period, indicating their high abundance and ease of detection in polluted environmental waters. In addition, the crAssphage assays were correlated with rain events, molecular markers for human polyomavirus and HF183, as well as culturable E. coli, enterococci, and somatic coliphage. The CPQ_064 assay correlated strongly to a greater number of biological indicators than the CPQ_056 assay. This study is the first to evaluate both crAssphage qPCR assays in an extended environmental application of crAssphage markers for monitoring of environmental waters. It is also the first study to compare crAssphage marker concentration with other viral-based indicators.

  7. Comparative validation using quantitative real-time PCR (qPCR and conventional PCR of bovine semen centrifuged in continuous density gradient

    Directory of Open Access Journals (Sweden)

    M.V. Resende

    2011-06-01

    Full Text Available The objective of the present study was to determine the sperm enrichment with X-bearing spermatozoa, after one centrifugation in a Percoll or OptiPrep continuous density gradient, using quantitative real-time polymerase chain reaction (qPCR of sperm DNA and resultant in vitro-produced bovine embryos by PCR. Frozen/thawed sperm was layered on density gradients and the tubes were centrifuged. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Cleavage and blastocyst rates were determined through in vitro production of embryos and PCR was performed to identify the embryos' genetic sex. A difference in blastocyst rate was found in the Percoll treatment compared to OptiPrep (P<0.05. The percentage of female embryos in the Percoll and OptiPrep groups was 62.0% and 47.1%, respectively. These results were confirmed by qPCR of spermatozoa DNA and underestimation was seen only in the Percoll group. It was possible to sexing sperm using simple approach.

  8. Reality show: um paradoxo nietzschiano

    Directory of Open Access Journals (Sweden)

    Ilana Feldman

    2011-01-01

    Full Text Available

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

  9. Dietary fiber showed no preventive effect against colon and rectal cancers in Japanese with low fat intake: an analysis from the results of nutrition surveys from 23 Japanese prefectures

    Directory of Open Access Journals (Sweden)

    Sugawara Kazuo

    2001-10-01

    Full Text Available Abstract Background Since Fuchs' report in 1999, the reported protective effect of dietary fiber from colorectal carcinogenesis has led many researchers to question its real benefit. The aim of this study is to evaluate the association between diet, especially dietary fiber and fat and colorectal cancer in Japan. Methods A multiple regression analysis (using the stepwise variable selection method was performed using the standardized mortality ratios (SMRs of colon and rectal cancer in 23 Japanese prefectures as objective variables and dietary fiber, nutrients and food groups as explanatory variables. Results As for colon cancer, the standardized partial correlation coefficients were positively significant for fat (1,13, P = 0.000, seaweeds (0.41, P = 0.026 and beans (0.45, P = 0.017 and were negatively significant for vitamin A (-0.63, P = 0.003, vitamin C (-0.42, P = 0.019 and yellow-green vegetables (-0.37, P = 0.046. For rectal cancer, the standardized partial correlation coefficient in fat (0.60, P = 0.002 was positively significant. Dietary fiber was not found to have a significant relationship with either colon or rectal cancers. Conclusions This study failed to show any protective effect of dietary fiber in subjects with a low fat intake (Japanese in this analysis, which supports Fuchs' findings in subjects with a high fat intake (US Americans.

  10. Major depressive disorder and generalized anxiety disorder show different autonomic dysregulations revealed by heart-rate variability analysis in first-onset drug-naïve patients without comorbidity.

    Science.gov (United States)

    Shinba, Toshikazu

    2017-02-01

    The aim of the present study was to examine whether depression and anxiety disorder manifest different autonomic dysregulations using heart-rate variability (HRV) and heart rate (HR) measurements. HRV and HR were recorded both at rest and during task execution (random-number generation) in first-onset drug-naïve patients with major depressive disorder (MDD, n = 14) and generalized anxiety disorder (GAD, n = 11) as well as in healthy controls (n = 41). The patients showed no comorbidity of depression and anxiety disorder. GAD patients did not exhibit panic or phobic symptoms at the time of measurement. Following power spectrum analysis of HR trend, the high- (HF) and low-frequency (LF) components, the sum (LF + HF), and the LF/HF ratio were compared among the groups. In the MDD patients, as previously reported, HF was low and the LF/HF ratio was high during the initial-rest condition, and HF was less reactive to the task. In contrast, GAD patients showed significantly high HF, although autonomic reactivity was not impaired. The results indicate that baseline autonomic activity and its reactivity to behavioral changes are different between MDD and GAD in the early stage of illness. High parasympathetic tone in GAD may reflect responses of the parasympathetic system to anxiety. MDD is accompanied by an autonomic shift toward sympathetic activation and a reduced reactivity to task. © 2016 The Authors. Psychiatry and Clinical Neurosciences © 2016 Japanese Society of Psychiatry and Neurology.

  11. Comparison of bacterial culture and qPCR testing of rectal and pen floor samples as diagnostic approaches to detect enterotoxic Escherichia coli in nursery pigs

    DEFF Research Database (Denmark)

    Weber, N. R.; Nielsen, J. P.; Hjulsager, Charlotte Kristiane

    2017-01-01

    Enterotoxigenic E. coli (ETEC) are a major cause of diarrhoea in weaned pigs. The objective of this study was to evaluate the agreement at pen level among three different diagnostic approaches for the detection of ETEC in groups of nursery pigs with diarrhoea. The diagnostic approaches used were......: bacterial culturing of faecal samples from three pigs (per pen) with clinical diarrhoea and subsequent testing for virulence genes in E. coli isolates; bacterial culturing of pen floor samples and subsequent testing for virulence genes in E. coli isolates; qPCR testing of pen floor samples in order...... to determine the quantity of F18 and F4 genes. The study was carried out in three Danish pig herds and included 31 pens with a pen-level diarrhoea prevalence of > 25%, as well as samples from 93 diarrhoeic nursery pigs from these pens. All E. coli isolates were analysed by PCR and classified as ETEC when genes...

  12. Longitudinal data analysis of polymorphisms in the κ-casein and β-lactoglobulin genes shows differential effects along the trajectory of the lactation curve in tropical dairy goats.

    Science.gov (United States)

    Cardona, Samir Julián Calvo; Cadavid, Henry Cardona; Corrales, Juan David; Munilla, Sebastián; Cantet, Rodolfo J C; Rogberg-Muñoz, Andrés

    2016-09-01

    The κ-casein (CSN-3) and β-lactoglobulin (BLG) genes are extensively polymorphic in ruminants. Several association studies have estimated the effects of polymorphisms in these genes on milk yield, milk composition, and cheese-manufacturing properties. Usually, these results are based on production integrated over the lactation curve or on cross-sectional studies at specific days in milk (DIM). However, as differential expression of milk protein genes occurs over lactation, the effect of the polymorphisms may change over time. In this study, we fitted a mixed-effects regression model to test-day records of milk yield and milk quality traits (fat, protein, and total solids yields) from Colombian tropical dairy goats. We used the well-characterized A/B polymorphisms in the CSN-3 and BLG genes. We argued that this approach provided more efficient estimators than cross-sectional designs, given the same number and pattern of observations, and allowed exclusion of between-subject variation from model error. The BLG genotype AA showed a greater performance than the BB genotype for all traits along the whole lactation curve, whereas the heterozygote showed an intermediate performance. We observed no such constant pattern for the CSN-3 gene between the AA homozygote and the heterozygote (the BB genotype was absent from the sample). The differences among the genotypic effects of the BLG and the CSN-3 polymorphisms were statistically significant during peak and mid lactation (around 40-160 DIM) for the BLG gene and only for mid lactation (80-145 DIM) for the CSN-3 gene. We also estimated the additive and dominant effects of the BLG locus. The locus showed a statistically significant additive behavior along the whole lactation trajectory for all quality traits, whereas for milk yield the effect was not significant at later stages. In turn, we detected a statistically significant dominance effect only for fat yield in the early and peak stages of lactation (at about 1-45 DIM

  13. Synaptonemal complex analysis of interracial hybrids between the Moscow and Neroosa chromosomal races of the common shrew Sorex araneus showing regular formation of a complex meiotic configuration (ring-of-four).

    Science.gov (United States)

    Matveevsky, Sergey N; Pavlova, Svetlana V; Maret M Acaeva; Oxana L Kolomiets

    2012-01-01

    Immunocytochemical and electron microscopic analysis of synaptonemal complexes (SCs) was carried out for the first time in homozygotes and complex Robertsonian heterozygotes (hybrids) of the common shrew, Sorex araneus Linnaeus, 1758, from a newly discovered hybrid zone between the Moscow and the Neroosa chromosomal races. These races differ in four monobrachial homologous metacentrics, and closed SC tetravalent is expected to be formed in meiosis of a hybrid. Indeed, such a multivalent was found at meiotic prophase I in hybrids. Interactions between multivalent and both autosomes and/or the sex chromosomes were observed. For the first time we have used immunocytochemical techniques to analyse asynapsis in Sorex araneus and show that the multivalent pairs in an orderly fashion with complete synapsis. Despite some signs of spermatocytes arrested in the meiotic prophase I, hybrids had large number of active sperm. Thus, Moscow - Neroosa hybrid males that form a ring-of-four meiotic configuration are most likely not sterile. Our results support previous demonstrations that monobrachial homology of metacentrics of the common shrew does not lead to complete reproductive isolation between parapatric chromosomal races of the species.

  14. Synaptonemal complex analysis of interracial hybrids between the Moscow and Neroosa chromosomal races of the common shrew Sorex araneus showing regular formation of a complex meiotic configuration (ring-of-four

    Directory of Open Access Journals (Sweden)

    Sergey Matveevsky

    2012-09-01

    Full Text Available Immunocytochemical and electron microscopic analysis of synaptonemal complexes (SCs was carried out for the first time in homozygotes and complex Robertsonian heterozygotes (hybrids of the common shrew, Sorex araneus Linnaeus, 1758, from a newly discovered hybrid zone between the Moscow and the Neroosa chromosomal races. These races differ in four monobrachial homologous metacentrics, and closed SC tetravalent is expected to be formed in meiosis of a hybrid. Indeed, such a multivalent was found at meiotic prophase I in hybrids. Interactions between multivalent and both autosomes and/or the sex chromosomes were observed. For the first time we have used immunocytochemical techniques to analyse asynapsis in S. araneus and show that the multivalent pairs in an orderly fashion with complete synapsis. Despite some signs of spermatocytes arrested in the meiotic prophase I, hybrids had large number of active sperm. Thus, Moscow – Neroosa hybrid males that form a ring-of-four meiotic configuration are most likely not sterile. Our results support previous demonstrations that monobrachial homology of metacentrics of the common shrew does not lead to complete reproductive isolation between parapatric chromosomal races of the species.

  15. Systematic review and meta-analysis shows a specific micronutrient profile in people with Down Syndrome: Lower blood calcium, selenium and zinc, higher red blood cell copper and zinc, and higher salivary calcium and sodium.

    Directory of Open Access Journals (Sweden)

    Amene Saghazadeh

    Full Text Available Different metabolic profiles as well as comorbidities are common in people with Down Syndrome (DS. Therefore it is relevant to know whether micronutrient levels in people with DS are also different. This systematic review was designed to review the literature on micronutrient levels in people with DS compared to age and sex-matched controls without DS. We identified sixty nine studies from January 1967 to April 2016 through main electronic medical databases PubMed, Scopus, and Web of knowledge. We carried out meta-analysis of the data on four essential trace elements (Cu, Fe, Se, and Zn, six minerals (Ca, Cl, K, Mg, Na, and P, and five vitamins (vitamin A, B9, B12, D, and E. People with DS showed lower blood levels of Ca (standard mean difference (SMD = -0.63; 95% confidence interval (CI: -1.16 to -0.09, Se (SMD = -0.99; 95% CI: -1.55 to -0.43, and Zn (SMD = -1.30; 95% CI: -1.75 to -0.84, while red cell levels of Zn (SMD = 1.88; 95% CI: 0.48 to 3.28 and Cu (SMD = 2.77; 95% CI: 1.96 to 3.57 were higher. They had also higher salivary levels of Ca (SMD = 0.85; 95% CI: 0.38 to 1.33 and Na (SMD = 1.04; 95% CI: 0.39 to 1.69. Our findings that micronutrient levels are different in people with DS raise the question whether these differences are related to the different metabolic profiles, the common comorbidities or merely reflect DS.

  16. Systems-wide RNAi analysis of CASP8AP2/FLASH shows transcriptional deregulation of the replication-dependent histone genes and extensive effects on the transcriptome of colorectal cancer cells.

    Science.gov (United States)

    Hummon, Amanda B; Pitt, Jason J; Camps, Jordi; Emons, Georg; Skube, Susan B; Huppi, Konrad; Jones, Tamara L; Beissbarth, Tim; Kramer, Frank; Grade, Marian; Difilippantonio, Michael J; Ried, Thomas; Caplen, Natasha J

    2012-01-04

    Colorectal carcinomas (CRC) carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF) alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival. A small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently CASP8AP2/FLASH. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of CASP8AP2/FLASH resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of CASP8AP2/FLASH also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of CASP8AP2/FLASH silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH), a protein recently linked to regulation of the AKT1/ß-catenin pathway. We have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of expression of the recently described tumor suppressor gene NEFH.

  17. Energy flow analysis of amputee walking shows a proximally-directed transfer of energy in intact limbs, compared to a distally-directed transfer in prosthetic limbs at push-off.

    Science.gov (United States)

    Weinert-Aplin, R A; Howard, D; Twiste, M; Jarvis, H L; Bennett, A N; Baker, R J

    2017-01-01

    Reduced capacity and increased metabolic cost of walking occurs in amputees, despite advances in prosthetic componentry. Joint powers can quantify deficiencies in prosthetic gait, but do not reveal how energy is exchanged between limb segments. This study aimed to quantify these energy exchanges during amputee walking. Optical motion and forceplate data collected during walking at a self-selected speed for cohorts of 10 controls, 10 unilateral trans-tibial, 10 unilateral trans-femoral and 10 bilateral trans-femoral amputees were used to determine the energy exchanges between lower limb segments. At push-off, consistent thigh and shank segment powers were observed between amputee groups (1.12W/kg vs. 1.05W/kg for intact limbs and 0.97W/kg vs. 0.99W/kg for prosthetic limbs), and reduced prosthetic ankle power, particularly in trans-femoral amputees (3.12W/kg vs. 0.87W/kg). Proximally-directed energy exchange was observed in the intact limbs of amputees and controls, while prosthetic limbs displayed distally-directed energy exchanges at the knee and hip. This study used energy flow analysis to show a reversal in the direction in which energy is exchanged between prosthetic limb segments at push-off. This reversal was required to provide sufficient energy to propel the limb segments and is likely a direct result of the lack of push-off power at the prosthetic ankle, particularly in trans-femoral amputees, and leads to their increased metabolic cost of walking. Copyright © 2016 IPEM. Published by Elsevier Ltd. All rights reserved.

  18. Determining the optimal number of individual samples to pool for quantification of average herd levels of antimicrobial resistance genes in Danish pig herds using high-throughput qPCR

    DEFF Research Database (Denmark)

    Clasen, Julie; Mellerup, Anders; Olsen, John Elmerdahl

    2016-01-01

    The primary objective of this study was to determine the minimum number of individual fecal samples to pool together in order to obtain a representative sample for herd level quantification of antimicrobial resistance (AMR) genes in a Danish pig herd, using a novel high-throughput qPCR assay...

  19. This research is to study the factors which influence the business success of small business ‘processed rotan’. The data employed in the study are primary data within the period of July to August 2013, 30 research observations through census method. Method of analysis used in the study is multiple linear regressions. The results of analysis showed that the factors of labor, innovation and promotion have positive and significant influence on the business success of small business ‘processed rotan’ simultaneously. The analysis also showed that partially labor has positive and significant influence on the business success, yet innovation and promotion have insignificant and positive influence on the business success.

    OpenAIRE

    Nasution, Inggrita Gusti Sari; Muchtar, Yasmin Chairunnisa

    2013-01-01

    This research is to study the factors which influence the business success of small business ‘processed rotan’. The data employed in the study are primary data within the period of July to August 2013, 30 research observations through census method. Method of analysis used in the study is multiple linear regressions. The results of analysis showed that the factors of labor, innovation and promotion have positive and significant influence on the business success of small busine...

  20. Systems-wide RNAi analysis of CASP8AP2/FLASH shows transcriptional deregulation of the replication-dependent histone genes and extensive effects on the transcriptome of colorectal cancer cells

    Directory of Open Access Journals (Sweden)

    Hummon Amanda B

    2012-01-01

    Full Text Available Abstract Background Colorectal carcinomas (CRC carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival. Results A small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently CASP8AP2/FLASH. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of CASP8AP2/FLASH resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of CASP8AP2/FLASH also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of CASP8AP2/FLASH silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH, a protein recently linked to regulation of the AKT1/ß-catenin pathway. Conclusions We have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of

  1. Online Italian fandoms of American TV shows

    Directory of Open Access Journals (Sweden)

    Eleonora Benecchi

    2015-06-01

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

  2. DETECCIÓN Y CUANTIFICACIÓN DE Spongospora subterranea f. sp. subterranea EN PLANTAS SEÑUELO Y CULTIVOS DE PAPA EN COLOMBIA MEDIANTE qPCR

    Directory of Open Access Journals (Sweden)

    Nevar Alirio García Bastidas

    2013-01-01

    Full Text Available La sarna polvosa de la papa (Solanum tuberosum , S. phureja causada por Spongospora subterranea f. sp. subterranea (Sss, es una de las enfermedades más limitantes de este cultivo. En Colombia, se han empleado diferentes métodos de detección asintomática de Sss, incluyendo bioensayos con plantas señuelo, PCR de ITS y pruebas de ELISA. Sin embargo, sus niveles de sensibilidad son bajos o requieren tiempos extensos. Una alternativa para complementar dichas herramientas es la PCR cuantitativa en tiempo real (qPCR. En este trabajo se evaluó dicha técnica utilizando los juegos de cebadores SsTQF1-SsTQR1; Spon421F-Spon494R y SscolF-SscolR (diseñados en este estudio, bajo la metodología de SYBR Green®; mientras que con Taqman® se evaluaron los cebadores SponF-SponR y la sonda SponP. Una vez determinada la funcionalidad de los cebadores, se descartó por inespecificidad, el par Spon421F-Spon494R; para los restantes se realizaron curvas estándar basadas en diluciones seriadas de quistosoros. Las pruebas de qPCR detectaron a Sss en las 20 muestras evaluadas de plantas señuelo de Nicotiana benthamiana y papa, utilizando los cebadores SsTQF1-SsTQR1 (Ct: 10,57- 29,34 y SscolF-SscolR (Ct: 14,39-34,08; mientras que 19 de las muestras fueron positivas con SponF-SponR-SponP (Ct: 15,63-38,93. A partir de 20 muestras de raíces de papa de cultivos de La Unión (Antioquia, Colombia, fue posible detectar el patógeno en 17 de ellas con SscolF-SscolR, estimándose una concentración de 6470 a 1,39 x 1010 quistorosos/mL. Estos resultados indican la ocurrencia de altos niveles de inóculo de Sss en esta región y enfatizan en la necesidad de fortalecer los programas de certificación de tubérculo-semilla en Colombia.

  3. Deciphering the Anti-Aflatoxinogenic Properties of Eugenol Using a Large-Scale q-PCR Approach

    Directory of Open Access Journals (Sweden)

    Isaura Caceres

    2016-04-01

    Full Text Available Produced by several species of Aspergillus, Aflatoxin B1 (AFB1 is a carcinogenic mycotoxin contaminating many crops worldwide. The utilization of fungicides is currently one of the most common methods; nevertheless, their use is not environmentally or economically sound. Thus, the use of natural compounds able to block aflatoxinogenesis could represent an alternative strategy to limit food and feed contamination. For instance, eugenol, a 4-allyl-2-methoxyphenol present in many essential oils, has been identified as an anti-aflatoxin molecule. However, its precise mechanism of action has yet to be clarified. The production of AFB1 is associated with the expression of a 70 kB cluster, and not less than 21 enzymatic reactions are necessary for its production. Based on former empirical data, a molecular tool composed of 60 genes targeting 27 genes of aflatoxin B1 cluster and 33 genes encoding the main regulatory factors potentially involved in its production, was developed. We showed that AFB1 inhibition in Aspergillus flavus following eugenol addition at 0.5 mM in a Malt Extract Agar (MEA medium resulted in a complete inhibition of the expression of all but one gene of the AFB1 biosynthesis cluster. This transcriptomic effect followed a down-regulation of the complex composed by the two internal regulatory factors, AflR and AflS. This phenomenon was also influenced by an over-expression of veA and mtfA, two genes that are directly linked to AFB1 cluster regulation.

  4. Deciphering the Anti-Aflatoxinogenic Properties of Eugenol Using a Large-Scale q-PCR Approach

    International Nuclear Information System (INIS)

    Atoui, A.; Caceres, I.; El Khoury, R.; Lippi, Y.; Naylies, Cl.; Oswald, I.; Bailly, J.; Puel, O.; El Khoury, A.; Medina, A.

    2016-01-01

    Produced by several species of Aspergillus, Aflatoxin B1 (AFB1) is a carcinogenic mycotoxin contaminating many crops worldwide. The utilization of fungicides is currently one of the most common methods; nevertheless, their use is not environmentally or economically sound. Thus, the use of natural compounds able to block aflatoxinogenesis could represent an alternative strategy to limit food and feed contamination. For instance, eugenol, a 4-allyl-2-methoxyphenol present in many essential oils, has been identified as an anti-aflatoxin molecule. However, its precise mechanism of action has yet to be clarified. The production of AFB1 is associated with the expression of a 70 kB cluster, and not less than 21 enzymatic reactions are necessary for its production. Based on former empirical data, a molecular tool composed of 60 genes targeting 27 genes of aflatoxin B1 cluster and 33 genes encoding the main regulatory factors potentially involved in its production, was developed. We showed that AFB1 inhibition in Aspergillus flavus following eugenol addition at 0.5 mM in a Malt Extract Agar (MEA) medium resulted in a complete inhibition of the expression of all but one gene of the AFB1 biosynthesis cluster. This transcriptomic effect followed a down-regulation of the complex composed by the two internal regulatory factors, AflR and AflS. This phenomenon was also influenced by an over-expression of veA and mtfA, two genes that are directly linked to AFB1 cluster regulation.(author)

  5. Microarray detection and qPCR screening of potential biomarkers of Folsomia candida (Collembola: Isotomidae) exposed to Bt proteins (Cry1Ab and Cry1Ac)

    DEFF Research Database (Denmark)

    Yuan, Yiyang; Krogh, Paul Henning; Bai, Xue

    2014-01-01

    The impact of Bt proteins on non-target arthropods is less understood than their effects on target organisms where the mechanism of toxic action is known. Here, we report the effects of two Bt proteins, Cry1Ab and Cry1Ac, on gene expression in the non-target collembolan, Folsomia candida....... A customized microarray was used to study gene expression in F. candida specimens that were exposed to Cry1Ab and Cry1Ac. All selected transcripts were subsequently confirmed by qPCR. Eleven transcripts were finally verified, and three of them were annotated. The responses of all eleven transcripts were...... tested in specimens for both Cry1Ab and Cry1Ac at a series of concentrations. These transcripts were separated into two and three groups for Cry1Ab and Cry1Ac, respectively, depend on their expression levels. However, those eleven transcripts did not respond to the Bt proteins in Bt-rice residues....

  6. Restriction Cascade Exponential Amplification (RCEA) assay with an attomolar detection limit: a novel, highly specific, isothermal alternative to qPCR.

    Science.gov (United States)

    Ghindilis, Andrey L; Smith, Maria W; Simon, Holly M; Seoudi, Ihab A; Yazvenko, Nina S; Murray, Iain A; Fu, Xiaoqing; Smith, Kenneth; Jen-Jacobson, Linda; Xu, Shuang-Yong

    2015-01-13

    An alternative to qPCR was developed for nucleic acid assays, involving signal rather than target amplification. The new technology, Restriction Cascade Exponential Amplification (RCEA), relies on specific cleavage of probe-target hybrids by restriction endonucleases (REase). Two mutant REases for amplification (Ramp), S17C BamHI and K249C EcoRI, were conjugated to oligonucleotides, and immobilized on a solid surface. The signal generation was based on: (i) hybridization of a target DNA to a Ramp-oligonucleotide probe conjugate, followed by (ii) specific cleavage of the probe-target hybrid using a non-immobilized recognition REase. The amount of Ramp released into solution upon cleavage was proportionate to the DNA target amount. Signal amplification was achieved through catalysis, by the free Ramp, of a restriction cascade containing additional oligonucleotide-conjugated Ramp and horseradish peroxidase (HRP). Colorimetric quantification of free HRP indicated that the RCEA achieved a detection limit of 10 aM (10(-17) M) target concentration, or approximately 200 molecules, comparable to the sensitivity of qPCR-based assays. The RCEA assay had high specificity, it was insensitive to non-specific binding, and detected target sequences in the presence of foreign DNA. RCEA is an inexpensive isothermal assay that allows coupling of the restriction cascade signal amplification with any DNA target of interest.

  7. Comparison of bacterial culture and qPCR testing of rectal and pen floor samples as diagnostic approaches to detect enterotoxic Escherichia coli in nursery pigs

    DEFF Research Database (Denmark)

    Weber, N. R.; Nielsen, J. P.; Hjulsager, Charlotte Kristiane

    2017-01-01

    Enterotoxigenic E. coli (ETEC) are a major cause of diarrhoea in weaned pigs. The objective of this study was to evaluate the agreement at pen level among three different diagnostic approaches for the detection of ETEC in groups of nursery pigs with diarrhoea. The diagnostic approaches used were...... to determine the quantity of F18 and F4 genes. The study was carried out in three Danish pig herds and included 31 pens with a pen-level diarrhoea prevalence of > 25%, as well as samples from 93 diarrhoeic nursery pigs from these pens. All E. coli isolates were analysed by PCR and classified as ETEC when genes...... was observed between the detection of ETEC by bacterial culture and qPCR in the same pen floor sample in 26 (83.9%, Kappa = 0.679) pens. Conclusion: We observed an acceptable agreement for the detection of ETEC-positive diarrhoeic nursery pigs in pen samples for both bacterial culture of pen floor samples...

  8. Citrus huanglongbing: validation of Real-Time PCR (qPCR for the detection of Candidatus Liberibacter asiaticus and Candidatus Liberibacter americanus in Colombia

    Directory of Open Access Journals (Sweden)

    Jorge Evelio Ángel

    2014-12-01

    Full Text Available Citrus huanglongbing (HLB is the most destructive citrus disease. Two of the three known HLB-associated Candidatus Liberibacter species were recently found to be present in the Americas. In this study, eggs, nymphs and adults of Diaphorina citri Kuwayama (Hemiptera: Liviidae and suspect citrus plant materials were collected in 25 municipalities in the departments of Cundinamarca, Santander, Valle del Cauca, Meta and Quindio (Colombia. The detection sensitivity, specificity and assay performance of the 16S rDNA-based real-time PCR (qPCR were validated for the field survey of the disease in Colombia. The validation confirmed the reliability and robustness of the real-time PCR method for the detection of HLB bacteria in host citrus plant tissues and the vector D. citri. The diagnosis was performed for Candidatus Liberibacter asiaticus (Ca. L. asiaticus and for Candidatus Liberibacter americanus (Ca. L. americanus on 168 citrus plant material samples and 239 insect samples. Neither Ca. L. asiaticus nor Ca. L. americanus were detected in the host plants or insects vector, confirming the absence of the disease in the citrus-producing areas of Colombia.

  9. Review: Martin Spetsmann-Kunkel (2004. Die Moral der Daytime Talkshow. Eine soziologische Analyse eines umstrittenen Fernsehformats [The Morality of the Daytime Talk Show. A Sociological Analysis of a Controversial Television Format

    Directory of Open Access Journals (Sweden)

    Nicola Döring

    2006-05-01

    Full Text Available This book deals with the phenomenon of the daytime talk show from a sociological perspective. The author questions the common cultural pessimism of this TV format ("exhibitionist guests," "voyeuristic spectators". He first describes the characteristics of the daytime talk show and summarizes the results of previous surveys that reveal a broad variety of talk show guests' and recipients' motives—beyond pathology. Drawing on concepts like civilization and individualisation, the book outlines the societal functions of the daytime talk show. A participatory observation study in the editorial office of "Hans Meiser" and free interpretations of three series from "Vera am Mittag" are presented as "empirical evidence." Unfortunately the book lacks theoretical and methodological rigor and a sound empirical basis. The bibliography could have been more comprehensive. The work is useful, though, as an inspired, readable introduction into the topic. URN: urn:nbn:de:0114-fqs0603119

  10. A map of the cosmic microwave background radiation from the Wilkinson Microwave Anisotropy Probe (WMAP), showing the large-scale fluctuations (the quadrupole and octopole) isolated by an analysis done partly by theorists at CERN.

    CERN Multimedia

    2004-01-01

    A recent analysis, in part by theorists working at CERN, suggests a new view of the cosmic microwave background radiation. It seems the solar system, rather than the universe, causes the radiation's large-scale fluctuations, similar to the bass in a song.

  11. Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.

    Science.gov (United States)

    Fox, Bridget C; Devonshire, Alison S; Baradez, Marc-Olivier; Marshall, Damian; Foy, Carole A

    2012-08-15

    Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Detection of Angiostrongylus cantonensis in the Blood and Peripheral Tissues of Wild Hawaiian Rats (Rattus rattus by a Quantitative PCR (qPCR Assay.

    Directory of Open Access Journals (Sweden)

    Susan I Jarvi

    Full Text Available The nematode Angiostrongylus cantonensis is a rat lungworm, a zoonotic pathogen that causes human eosinophilic meningitis and ocular angiostrongyliasis characteristic of rat lungworm (RLW disease. Definitive diagnosis is made by finding and identifying A. cantonensis larvae in the cerebral spinal fluid or by using a custom immunological or molecular test. This study was conducted to determine if genomic DNA from A. cantonensis is detectable by qPCR in the blood or tissues of experimentally infected rats. F1 offspring from wild rats were subjected to experimental infection with RLW larvae isolated from slugs, then blood or tissue samples were collected over multiple time points. Blood samples were collected from 21 rats throughout the course of two trials (15 rats in Trial I, and 6 rats in Trial II. In addition to a control group, each trial had two treatment groups: the rats in the low dose (LD group were infected by approximately 10 larvae and the rats in the high dose (HD group were infected with approximately 50 larvae. In Trial I, parasite DNA was detected in cardiac bleed samples from five of five LD rats and five of five HD rats at six weeks post-infection (PI, and three of five LD rats and five of five HD rats from tail tissue. In Trial II, parasite DNA was detected in peripheral blood samples from one of two HD rats at 53 minutes PI, one of two LD rats at 1.5 hours PI, one of two HD rats at 18 hours PI, one of two LD rats at five weeks PI and two of two at six weeks PI, and two of two HD rats at weeks five and six PI. These data demonstrate that parasite DNA can be detected in peripheral blood at various time points throughout RLW infection in rats.

  13. Detection of Angiostrongylus cantonensis in the Blood and Peripheral Tissues of Wild Hawaiian Rats (Rattus rattus) by a Quantitative PCR (qPCR) Assay.

    Science.gov (United States)

    Jarvi, Susan I; Pitt, William C; Farias, Margaret E; Shiels, Laura; Severino, Michael G; Howe, Kathleen M; Jacquier, Steven H; Shiels, Aaron B; Amano, Karis K; Luiz, Blaine C; Maher, Daisy E; Allison, Maureen L; Holtquist, Zachariah C; Scheibelhut, Neil T

    2015-01-01

    The nematode Angiostrongylus cantonensis is a rat lungworm, a zoonotic pathogen that causes human eosinophilic meningitis and ocular angiostrongyliasis characteristic of rat lungworm (RLW) disease. Definitive diagnosis is made by finding and identifying A. cantonensis larvae in the cerebral spinal fluid or by using a custom immunological or molecular test. This study was conducted to determine if genomic DNA from A. cantonensis is detectable by qPCR in the blood or tissues of experimentally infected rats. F1 offspring from wild rats were subjected to experimental infection with RLW larvae isolated from slugs, then blood or tissue samples were collected over multiple time points. Blood samples were collected from 21 rats throughout the course of two trials (15 rats in Trial I, and 6 rats in Trial II). In addition to a control group, each trial had two treatment groups: the rats in the low dose (LD) group were infected by approximately 10 larvae and the rats in the high dose (HD) group were infected with approximately 50 larvae. In Trial I, parasite DNA was detected in cardiac bleed samples from five of five LD rats and five of five HD rats at six weeks post-infection (PI), and three of five LD rats and five of five HD rats from tail tissue. In Trial II, parasite DNA was detected in peripheral blood samples from one of two HD rats at 53 minutes PI, one of two LD rats at 1.5 hours PI, one of two HD rats at 18 hours PI, one of two LD rats at five weeks PI and two of two at six weeks PI, and two of two HD rats at weeks five and six PI. These data demonstrate that parasite DNA can be detected in peripheral blood at various time points throughout RLW infection in rats.

  14. De Novo Transcriptome Analysis Shows That SAV-3 Infection Upregulates Pattern Recognition Receptors of the Endosomal Toll-Like and RIG-I-Like Receptor Signaling Pathways in Macrophage/Dendritic Like TO-Cells

    Directory of Open Access Journals (Sweden)

    Cheng Xu

    2016-04-01

    Full Text Available A fundamental step in cellular defense mechanisms is the recognition of “danger signals” made of conserved pathogen associated molecular patterns (PAMPs expressed by invading pathogens, by host cell germ line coded pattern recognition receptors (PRRs. In this study, we used RNA-seq and the Kyoto encyclopedia of genes and genomes (KEGG to identify PRRs together with the network pathway of differentially expressed genes (DEGs that recognize salmonid alphavirus subtype 3 (SAV-3 infection in macrophage/dendritic like TO-cells derived from Atlantic salmon (Salmo salar L headkidney leukocytes. Our findings show that recognition of SAV-3 in TO-cells was restricted to endosomal Toll-like receptors (TLRs 3 and 8 together with RIG-I-like receptors (RLRs and not the nucleotide-binding oligomerization domain-like receptors NOD-like receptor (NLRs genes. Among the RLRs, upregulated genes included the retinoic acid inducible gene I (RIG-I, melanoma differentiation association 5 (MDA5 and laboratory of genetics and physiology 2 (LGP2. The study points to possible involvement of the tripartite motif containing 25 (TRIM25 and mitochondrial antiviral signaling protein (MAVS in modulating RIG-I signaling being the first report that links these genes to the RLR pathway in SAV-3 infection in TO-cells. Downstream signaling suggests that both the TLR and RLR pathways use interferon (IFN regulatory factors (IRFs 3 and 7 to produce IFN-a2. The validity of RNA-seq data generated in this study was confirmed by quantitative real time qRT-PCR showing that genes up- or downregulated by RNA-seq were also up- or downregulated by RT-PCR. Overall, this study shows that de novo transcriptome assembly identify key receptors of the TLR and RLR sensors engaged in host pathogen interaction at cellular level. We envisage that data presented here can open a road map for future intervention strategies in SAV infection of salmon.

  15. Hierarchical Cluster Analysis of Three-Dimensional Reconstructions of Unbiased Sampled Microglia Shows not Continuous Morphological Changes from Stage 1 to 2 after Multiple Dengue Infections in Callithrix penicillata

    Science.gov (United States)

    Diniz, Daniel G.; Silva, Geane O.; Naves, Thaís B.; Fernandes, Taiany N.; Araújo, Sanderson C.; Diniz, José A. P.; de Farias, Luis H. S.; Sosthenes, Marcia C. K.; Diniz, Cristovam G.; Anthony, Daniel C.; da Costa Vasconcelos, Pedro F.; Picanço Diniz, Cristovam W.

    2016-01-01

    It is known that microglial morphology and function are related, but few studies have explored the subtleties of microglial morphological changes in response to specific pathogens. In the present report we quantitated microglia morphological changes in a monkey model of dengue disease with virus CNS invasion. To mimic multiple infections that usually occur in endemic areas, where higher dengue infection incidence and abundant mosquito vectors carrying different serotypes coexist, subjects received once a week subcutaneous injections of DENV3 (genotype III)-infected culture supernatant followed 24 h later by an injection of anti-DENV2 antibody. Control animals received either weekly anti-DENV2 antibodies, or no injections. Brain sections were immunolabeled for DENV3 antigens and IBA-1. Random and systematic microglial samples were taken from the polymorphic layer of dentate gyrus for 3-D reconstructions, where we found intense immunostaining for TNFα and DENV3 virus antigens. We submitted all bi- or multimodal morphological parameters of microglia to hierarchical cluster analysis and found two major morphological phenotypes designated types I and II. Compared to type I (stage 1), type II microglia were more complex; displaying higher number of nodes, processes and trees and larger surface area and volumes (stage 2). Type II microglia were found only in infected monkeys, whereas type I microglia was found in both control and infected subjects. Hierarchical cluster analysis of morphological parameters of 3-D reconstructions of random and systematic selected samples in control and ADE dengue infected monkeys suggests that microglia morphological changes from stage 1 to stage 2 may not be continuous. PMID:27047345

  16. Reference Genes for qPCR Analysis in Resin-Tapped Adult Slash Pine As a Tool to Address the Molecular Basis of Commercial Resinosis

    Directory of Open Access Journals (Sweden)

    Júlio C. de Lima

    2016-06-01

    Full Text Available Pine oleoresin is a major source of terpenes, consisting of turpentine (mono- and sesquiterpenes and rosin (diterpenes fractions. Higher oleoresin yields are of economic interest, since oleoresin derivatives make up a valuable source of materials for chemical industries. Oleoresin can be extracted from living trees, often by the bark streak method, in which bark removal is done periodically, followed by application of stimulant paste containing sulfuric acid and other chemicals on the freshly wounded exposed surface. To better understand the molecular basis of chemically-stimulated and wound induced oleoresin production, we evaluated the stability of 11 putative reference genes for the purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis. Samples for RNA extraction were collected from field-grown adult trees under tapping operations using stimulant pastes with different compositions and at various time points after paste application. Statistical methods established by geNorm, NormFinder, and BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3 and UBI. To confirm expression stability of the candidate reference genes, expression profiles of putative P. elliottii orthologs of resin biosynthesis-related genes encoding Pinus contorta β-pinene synthase [PcTPS-(−β-pin1], P. contorta levopimaradiene/abietadiene synthase (PcLAS1, Pinus taeda α-pinene synthase [PtTPS-(+αpin], and P. taeda α-farnesene synthase (PtαFS were examined following stimulant paste application. Increased oleoresin yields observed in stimulated treatments using phytohormone-based pastes were consistent with higher expression of pinene synthases. Overall, the expression of all genes examined matched the expected profiles of oleoresin-related transcript changes reported for previously examined conifers.

  17. The numbers game: quantitative analysis of Neorickettsia sp. propagation through complex life cycle of its digenean host using real-time qPCR.

    Science.gov (United States)

    Greiman, Stephen E; Tkach, Vasyl V

    2016-07-01

    Bacteria of the genus Neorickettsia are obligate intracellular endosymbionts of parasitic flukes (Digenea) and are passed through the entire complex life cycle of the parasite by vertical transmission. Several species of Neorickettsia are known to cause diseases in domestic animals, wildlife, and humans. Quantitative data on the transmission of the bacteria through the digenean life cycle is almost completely lacking. This study quantified for the first time the abundance of Neorickettsia within multiple stages of the life cycle of the digenean Plagiorchis elegans. Snails Lymnaea stagnalis collected from a pond in North Dakota were screened for the presence of digenean cercariae, which were subsequently tested for the presence of Neorickettsia. Three L. stagnalis were found shedding P. elegans cercariae infected with Neorickettsia. These snails were used to initiate three separate laboratory life cycles and obtain all life cycle stages for bacterial quantification. A quantitative real-time PCR assay targeting the GroEL gene was developed to enumerate Neorickettsia sp. within different stages of the digenean life cycle. The number of bacteria significantly increased throughout all stages, from eggs to adults. The two largest increases in number of bacteria occurred during the period from eggs to cercariae and from 6-day metacercariae to 48-h juvenile worms. These two periods seem to be the most important for Neorickettsia propagation through the complex digenean life cycle and maturation in the definitive host.

  18. Genes co-regulated with LBD16 in nematode feeding sites inferred from in silico analysis show similarities to regulatory circuits mediated by the auxin/cytokinin balance in Arabidopsis.

    Science.gov (United States)

    Cabrera, Javier; Fenoll, Carmen; Escobar, Carolina

    2015-01-01

    Plant endoparasitic nematodes, root-knot and cyst nematodes (RKNs and CNs) induce within the root vascular cylinder transfer cells used for nourishing, termed giant cells (GCs) and syncytia. Understanding the molecular mechanisms behind this process is essential to develop tools for nematode control. Based on the crucial role in gall development of LBD16, also a key component of the auxin pathway leading to the divisions in the xylem pole pericycle during lateral root (LR) formation, we investigated genes co-regulated with LBD16 in different transcriptomes and analyzed their similarities and differences with those of RKNs and CNs feeding sites (FS). This analysis confirmed LBD16 and its co-regulated genes, integrated in signaling cascades mediated by auxins during LR and callus formation, as a particular feature of RKN-FS distinct to CNs. However, LBD16, and its positively co-regulated genes, were repressed in syncytia, suggesting a selective down- regulation of the LBD16 auxin mediated pathways in CNs-FS. Interestingly, cytokinin-induced genes are enriched in syncytia and we encountered similarities between the transcriptome of shoot regeneration from callus, modulated by cytokinins, and that of syncytia. These findings establish differences in the regulatory networks leading to both FS formation, probably modulated by the auxin/cytokinin balance.

  19. Evaluation of 16S rRNA qPCR for detection of Mycobacterium leprae DNA in nasal secretion and skin biopsy samples from multibacillary and paucibacillary leprosy cases.

    Science.gov (United States)

    Marques, Lívia Érika Carlos; Frota, Cristiane Cunha; Quetz, Josiane da Silva; Bindá, Alexandre Havt; Mota, Rosa Maria Salane; Pontes, Maria Araci de Andrade; Gonçalves, Heitor de Sá; Kendall, Carl; Kerr, Ligia Regina Franco Sansigolo

    2017-12-26

    Mycobacterium leprae bacilli are mainly transmitted by the dissemination of nasal aerosols from multibacillary (MB) patients to susceptible individuals through inhalation. The upper respiratory tract represents the main entry and exit routes of M. leprae. Therefore, this study aimed to evaluate the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) in detecting M. leprae in nasal secretion (NS) and skin biopsy (SB) samples from MB and paucibacillary (PB) cases. Fifty-four NS samples were obtained from leprosy patients at the Dona Libânia National Reference Centre for Sanitary Dermatology in Ceará, Brazil. Among them, 19 MB cases provided both NS and SB samples. Bacilloscopy index assays were conducted and qPCR amplification was performed using specific primers for M. leprae 16S rRNA gene, generating a 124-bp fragment. Primer specificity was verified by determining the amplicon melting temperature (T m  = 79.5 °C) and detection limit of qPCR was 20 fg of M. leprae DNA. Results were positive for 89.7 and 73.3% of NS samples from MB and PB cases, respectively. SB samples from MB patients were 100% positive. The number of bacilli detected in NS samples were 1.39 × 10 3 -8.02 × 10 5 , and in SB samples from MB patients were 1.87 × 10 3 -1.50 × 10 6 . Therefore, qPCR assays using SYBR Green targeting M. leprae 16S rRNA region can be employed in detecting M. leprae in nasal swabs from leprosy patients, validating this method for epidemiological studies aiming to identify healthy carriers among household contacts or within populations of an endemic area.

  20. Has the sanctity of life law 'gone too far'?: analysis of the sanctity of life doctrine and English case law shows that the sanctity of life law has not 'gone too far'.

    Science.gov (United States)

    Rabiu, Abdul-Rasheed; Sugand, Kapil

    2014-02-22

    The medical profession consistently strives to uphold patient empowerment, equality and safety. It is ironic that now, at a time where advances in technology and knowledge have given us an increased capacity to preserve and prolong life, we find ourselves increasingly asking questions about the value of the lives we are saving. A recent editorial by Professor Raanan Gillon questions the emphasis that English law places on the sanctity of life doctrine. In what was described by Reverend Nick Donnelly as a "manifesto for killing patients", Professor Gillon argues that the sanctity of life law has gone too far because of its disregard for distributive justice and an incompetent person's previously declared autonomy. This review begins by outlining the stance of the sanctity of life doctrine on decisions about administering, withholding and withdrawing life-prolonging treatment. Using this as a foundation for a rebuttal, a proposal is made that Professor Gillon's assertions do not take the following into account:1) A sanctity of life law does not exist since English Common Law infringes the sanctity doctrine by tolerating quality of life judgements and a doctor's intention to hasten death when withdrawing life-prolonging treatment.2) Even if a true sanctity of life law did exist:a) The sanctity of life doctrine allows for resource considerations in the wider analysis of benefits and burdens.b) The sanctity of life doctrine yields to a competent person's autonomous decision.This review attempts to demonstrate that at present, and with the legal precedent that restricts it, a sanctity of life law cannot go too far.

  1. Ultrasonography-guided core needle biopsy for the thyroid nodule: does the procedure hold any benefit for the diagnosis when fine-needle aspiration cytology analysis shows inconclusive results?

    Science.gov (United States)

    Hahn, S Y; Han, B-K; Ko, E Y; Ko, E S

    2013-01-01

    Objective: We evaluated the diagnostic role of ultrasonography-guided core needle biopsy (CNB) according to ultrasonography features of thyroid nodules that had inconclusive ultrasonography-guided fine-needle aspiration (FNA) results. Methods: A total of 88 thyroid nodules in 88 patients who underwent ultrasonography-guided CNB because of previous inconclusive FNA results were evaluated. The patients were classified into three groups based on ultrasonography findings: Group A, which was suspicious for papillary thyroid carcinoma (PTC); Group B, which was suspicious for follicular (Hurthle cell) neoplasm; and Group C, which was suspicious for lymphoma. The final diagnoses of the thyroid nodules were determined by surgical confirmation or follow-up after ultrasonography-guided CNB. Results: Of the 88 nodules, the malignant rate was 49.1% in Group A, 12.0% in Group B and 90.0% in Group C. The rates of conclusive ultrasonography-guided CNB results after previous incomplete ultrasonography-guided FNA results were 96.2% in Group A, 64.0% in Group B and 90.0% in Group C (p=0.001). 12 cases with inconclusive ultrasonography-guided CNB results were finally diagnosed as 8 benign lesions, 3 PTCs and 1 lymphoma. The number of previous ultrasonography-guided FNA biopsies was not significantly different between the conclusive and the inconclusive result groups of ultrasonography-guided CNB (p=0.205). Conclusion: Ultrasonography-guided CNB has benefit for the diagnosis of thyroid nodules with inconclusive ultrasonography-guided FNA results. However, it is still not helpful for the differential diagnosis in 36% of nodules that are suspicious for follicular neoplasm seen on ultrasonography. Advances in knowledge: This study shows the diagnostic contribution of ultrasonography-guided CNB as an alternative to repeat ultrasonography-guided FNA or surgery. PMID:23564885

  2. Novel recurrent chromosomal aberrations detected in clonal plasma cells of light chain amyloidosis patients show potential adverse prognostic effect: first results from a genome-wide copy number array analysis.

    Science.gov (United States)

    Granzow, Martin; Hegenbart, Ute; Hinderhofer, Katrin; Hose, Dirk; Seckinger, Anja; Bochtler, Tilmann; Hemminki, Kari; Goldschmidt, Hartmut; Schönland, Stefan O; Jauch, Anna

    2017-07-01

    Immunoglobulin light chain (AL) amyloidosis is a rare plasma cell dyscrasia characterized by the deposition of abnormal amyloid fibrils in multiple organs, thus impairing their function. In the largest cohort studied up to now of 118 CD138-purified plasma cell samples from previously untreated immunoglobulin light chain amyloidosis patients, we assessed in parallel copy number alterations using high-density copy number arrays and interphase fluorescence in situ hybridization (iFISH). We used fluorescence in situ hybridization probes for the IgH translocations t(11;14), t(4;14), and t(14;16) or any other IgH rearrangement as well as numerical aberrations of the chromosome loci 1q21, 8p21, 5p15/5q35, 11q22.3 or 11q23, 13q14, 15q22, 17p13, and 19q13. Recurrent gains included chromosomes 1q (36%), 9 (24%), 11q (24%), as well as 19 (15%). Recurrent losses affected chromosome 13 (29% monosomy) and partial losses of 14q (19%), 16q (14%) and 13q (12%), respectively. In 88% of patients with translocation t(11;14), the hallmark chromosomal aberration in AL amyloidosis, a concomitant gain of 11q22.3/11q23 detected by iFISH was part of the unbalanced translocation der(14)t(11;14)(q13;q32) with the breakpoint in the CCND1/MYEOV gene region. Partial loss of chromosome regions 14q and 16q were significantly associated to gain 1q. Gain 1q21 detected by iFISH almost always resulted from a gain of the long arm of chromosome 1 and not from trisomy 1, whereas deletions on chromosome 1p were rarely found. Overall and event-free survival analysis found a potential adverse prognostic effect of concomitant gain 1q and deletion 14q as well as of deletion 1p. In conclusion, in the first whole genome report of clonal plasma cells in AL amyloidosis, novel aberrations and hitherto unknown potential adverse prognostic effects were uncovered. Copyright© 2017 Ferrata Storti Foundation.

  3. Comprehensive proteome analysis of nasal lavage samples after controlled exposure to welding nanoparticles shows an induced acute phase and a nuclear receptor, LXR/RXR, activation that influence the status of the extracellular matrix.

    Science.gov (United States)

    Ali, Neserin; Ljunggren, Stefan; Karlsson, Helen M; Wierzbicka, Aneta; Pagels, Joakim; Isaxon, Christina; Gudmundsson, Anders; Rissler, Jenny; Nielsen, Jörn; Lindh, Christian H; Kåredal, Monica

    2018-01-01

    Epidemiological studies have shown that many welders experience respiratory symptoms. During the welding process a large number of airborne nanosized particles are generated, which might be inhaled and deposited in the respiratory tract. Knowledge of the underlying mechanisms behind observed symptoms is still partly lacking, although inflammation is suggested to play a central role. The aim of this study was to investigate the effects of welding fume particle exposure on the proteome expression level in welders suffering from respiratory symptoms, and changes in protein mediators in nasal lavage samples were analyzed. Such mediators will be helpful to clarify the pathomechanisms behind welding fume particle-induced effects. In an exposure chamber, 11 welders with work-related symptoms in the lower airways during the last month were exposed to mild-steel welding fume particles (1 mg/m 3 ) and to filtered air, respectively, in a double-blind manner. Nasal lavage samples were collected before, immediately after, and the day after exposure. The proteins in the nasal lavage were analyzed with two different mass spectrometry approaches, label-free discovery shotgun LC-MS/MS and a targeted selected reaction monitoring LC-MS/MS analyzing 130 proteins and four in vivo peptide degradation products. The analysis revealed 30 significantly changed proteins that were associated with two main pathways; activation of acute phase response signaling and activation of LXR/RXR, which is a nuclear receptor family involved in lipid signaling. Connective tissue proteins and proteins controlling the degradation of such tissues, including two different matrix metalloprotease proteins, MMP8 and MMP9, were among the significantly changed enzymes and were identified as important key players in the pathways. Exposure to mild-steel welding fume particles causes measurable changes on the proteome level in nasal lavage matrix in exposed welders, although no clinical symptoms were manifested. The

  4. Molecular Analysis of Methanogen Richness in Landfill and Marshland Targeting 16S rDNA Sequences.

    Science.gov (United States)

    Yadav, Shailendra; Kundu, Sharbadeb; Ghosh, Sankar K; Maitra, S S

    2015-01-01

    Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic) and Thaumarchaeota (mesophilic), were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about "methanogenic archaea composition" and "abundance" in the contrasting ecosystems like "landfill" and "marshland" may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process.

  5. Molecular Analysis of Methanogen Richness in Landfill and Marshland Targeting 16S rDNA Sequences

    Directory of Open Access Journals (Sweden)

    Shailendra Yadav

    2015-01-01

    Full Text Available Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic and Thaumarchaeota (mesophilic, were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about “methanogenic archaea composition” and “abundance” in the contrasting ecosystems like “landfill” and “marshland” may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process.

  6. Comparative Transcriptome Analysis Reveals Different Silk Yields of Two Silkworm Strains.

    Directory of Open Access Journals (Sweden)

    Juan Li

    Full Text Available Cocoon and silk yields are the most important characteristics of sericulture. However, few studies have examined the genes that modulate these features. Further studies of these genes will be useful for improving the products of sericulture. JingSong (JS and Lan10 (L10 are two strains having significantly different cocoon and silk yields. In the current study, RNA-Seq and quantitative polymerase chain reaction (qPCR were performed on both strains in order to determine divergence of the silk gland, which controls silk biosynthesis in silkworms. Compared with L10, JS had 1375 differentially expressed genes (DEGs; 738 up-regulated genes and 673 down-regulated genes. Nine enriched gene ontology (GO terms were identified by GO enrichment analysis based on these DEGs. KEGG enrichment analysis results showed that the DEGs were enriched in three pathways, which were mainly associated with the processing and biosynthesis of proteins. The representative genes in the enrichment pathways and ten significant DEGs were further verified by qPCR, the results of which were consistent with the RNA-Seq data. Our study has revealed differences in silk glands between the two silkworm strains and provides a perspective for understanding the molecular mechanisms determining silk yield.

  7. Micro-epidemiology and spatial heterogeneity of P. vivax parasitaemia in riverine communities of the Peruvian Amazon: A multilevel analysis.

    Science.gov (United States)

    Carrasco-Escobar, Gabriel; Gamboa, Dionicia; Castro, Marcia C; Bangdiwala, Shrikant I; Rodriguez, Hugo; Contreras-Mancilla, Juan; Alava, Freddy; Speybroeck, Niko; Lescano, Andres G; Vinetz, Joseph M; Rosas-Aguirre, Angel; Llanos-Cuentas, Alejandro

    2017-08-14

    Malaria has steadily increased in the Peruvian Amazon over the last five years. This study aimed to determine the parasite prevalence and micro-geographical heterogeneity of Plasmodium vivax parasitaemia in communities of the Peruvian Amazon. Four cross-sectional active case detection surveys were conducted between May and July 2015 in four riverine communities in Mazan district. Analysis of 2785 samples of 820 individuals nested within 154 households for Plasmodium parasitaemia was carried out using light microscopy and qPCR. The spatio-temporal distribution of Plasmodium parasitaemia, dominated by P. vivax, was shown to cluster at both household and community levels. Of enrolled individuals, 47% had at least one P. vivax parasitaemia and 10% P. falciparum, by qPCR, both of which were predominantly sub-microscopic and asymptomatic. Spatial analysis detected significant clustering in three communities. Our findings showed that communities at small-to-moderate spatial scales differed in P. vivax parasite prevalence, and multilevel Poisson regression models showed that such differences were influenced by factors such as age, education, and location of households within high-risk clusters, as well as factors linked to a local micro-geographic context, such as travel and occupation. Complex transmission patterns were found to be related to human mobility among communities in the same micro-basin.

  8. Evolutionary Analysis of Minor Histocompatibility Genes In Hydra

    KAUST Repository

    Aalismail, Nojood

    2016-01-01

    In the present study we took initiative to study the self/nonself recognition in hydra and its relation to the immune response. Moreover, performing phylogenetic analysis to look for annotated immune genes in hydra gave us a potential to analyze the expression of minor histocompatibility genes that have been shown to play a major role in grafting and transplantation in mammals. Here we obtained the cDNA library that shows expression of minor histocompatibility genes and confirmed that the annotated sequences in databases are actually present. In addition, grafting experiments suggested, although still preliminary, that homograft showed less rejection response than in heterograft. Involvement of possible minor histocompatibility gene orthologous in immune response was examined by qPCR.

  9. High Throughput qPCR Expression Profiling of Circulating MicroRNAs Reveals Minimal Sex- and Sample Timing-Related Variation in Plasma of Healthy Volunteers.

    Directory of Open Access Journals (Sweden)

    Catherine Mooney

    Full Text Available MicroRNAs are a class of small non-coding RNA that regulate gene expression at a post-transcriptional level. MicroRNAs have been identified in various body fluids under normal conditions and their stability as well as their dysregulation in disease opens up a new field for biomarker study. However, diurnal and day-to-day variation in plasma microRNA levels, and differential regulation between males and females, may affect biomarker stability. A QuantStudio 12K Flex Real-Time PCR System was used to profile plasma microRNA levels using OpenArray in male and female healthy volunteers, in the morning and afternoon, and at four time points over a one month period. Using this system we were able to run four OpenArray plates in a single run, the equivalent of 32 traditional 384-well qPCR plates or 12,000 data points. Up to 754 microRNAs can be identified in a single plasma sample in under two hours. 108 individual microRNAs were identified in at least 80% of all our samples which compares favourably with other reports of microRNA profiles in serum or plasma in healthy adults. Many of these microRNAs, including miR-16-5p, miR-17-5p, miR-19a-3p, miR-24-3p, miR-30c-5p, miR-191-5p, miR-223-3p and miR-451a are highly expressed and consistent with previous studies using other platforms. Overall, microRNA levels were very consistent between individuals, males and females, and time points and we did not detect significant differences in levels of microRNAs. These results suggest the suitability of this platform for microRNA profiling and biomarker discovery and suggest minimal confounding influence of sex or sample timing. However, the platform has not been subjected to rigorous validation which must be demonstrated in future biomarker studies where large differences may exist between disease and control samples.

  10. MicroRNAs and cardiac sarcoplasmic reticulum calcium ATPase-2 in human myocardial infarction: expression and bioinformatic analysis.

    Science.gov (United States)

    Boštjančič, Emanuela; Zidar, Nina; Glavač, Damjan

    2012-10-15

    Cardiac sarco(endo)plasmic reticulum calcium ATPase-2 (SERCA2) plays one of the central roles in myocardial contractility. Both, SERCA2 mRNA and protein are reduced in myocardial infarction (MI), but the correlation has not been always observed. MicroRNAs (miRNAs) act by targeting 3'-UTR mRNA, causing translational repression in physiological and pathological conditions, including cardiovascular diseases. One of the aims of our study was to identify miRNAs that could influence SERCA2 expression in human MI. The protein SERCA2 was decreased and 43 miRNAs were deregulated in infarcted myocardium compared to corresponding remote myocardium, analyzed by western blot and microRNA microarrays, respectively. All the samples were stored as FFPE tissue and in RNAlater. miRNAs binding prediction to SERCA2 including four prediction algorithms (TargetScan, PicTar, miRanda and mirTarget2) identified 213 putative miRNAs. TAM and miRNApath annotation of deregulated miRNAs identified 18 functional and 21 diseased states related to heart diseases, and association of the half of the deregulated miRNAs to SERCA2. Free-energy of binding and flanking regions (RNA22, RNAfold) was calculated for 10 up-regulated miRNAs from microarray analysis (miR-122, miR-320a/b/c/d, miR-574-3p/-5p, miR-199a, miR-140, and miR-483), and nine miRNAs deregulated from microarray analysis were used for validation with qPCR (miR-21, miR-122, miR-126, miR-1, miR-133, miR-125a/b, and miR-98). Based on qPCR results, the comparison between FFPE and RNAlater stored tissue samples, between Sybr Green and TaqMan approaches, as well as between different reference genes were also performed. Combing all the results, we identified certain miRNAs as potential regulators of SERCA2; however, further functional studies are needed for verification. Using qPCR, we confirmed deregulation of nine miRNAs in human MI, and show that qPCR normalization strategy is important for the outcome of miRNA expression analysis in human MI.

  11. Analysis of audiometric database shows evidence of employee fraud

    Science.gov (United States)

    Erdreich, John

    2003-10-01

    Following a lengthy strike, several hundred delivery drivers filed workers compensation claims for occupational hearing loss. We were asked to evaluate the noise exposure of the drivers during their in-plant tasks. In-plant exposures were not predictive of any hearing loss. A comparison of audiometric data for the claimants revealed consistent hearing loss independent of duration of employment or age. These discrepancies between observations and common understanding of dose-response relationships between noise exposure and hearing loss led to further investigation, ultimately resulting in the dismissal of all claims against the employer who then filed an action against the claimant's attorneys and physician under the Racketeering in Corrupt Organizations Act (RICO). The details of the legal complaint, which reads like a detective novel, can be found at the United States District Court for the Southern District of New York [93 Civ. 7222 (LAP)].

  12. Using real-time PCR and Bayesian analysis to distinguish susceptible tubificid taxa important in the transmission of Myxobolus cerebralis, the cause of salmonid whirling disease.

    Science.gov (United States)

    Fytilis, Nikolaos; Rizzo, Donna M; Lamb, Ryan D; Kerans, Billie L; Stevens, Lori

    2013-05-01

    Aquatic oligochaetes have long been appreciated for their value in assessing habitat quality because they are ubiquitous sediment-dwelling filter feeders. Many oligochaete taxa are also important in the transmission of fish diseases. Distinguishing resistant and susceptible taxa is important for managing fish disease, yet challenging in practice. Tubifex tubifex (Oligochaeta: Tubificidae) is the definitive host for the complex life-cycle parasite, Myxobolus cerebralis, the causative agent of salmonid whirling disease. We developed two hydrolysis probe-based qualitative real-time PCR (qPCR) multiplex assays that distinguish among tubificid taxa collected from the Madison River, Montana, USA. The first assay distinguishes T. tubifex from Rhyacodrilus spp.; while the second classifies T. tubifex identified by the first assay into two genetic lineages (I and III). Specificity and sensitivity were optimized for each assay; the two assays showed specificity of 94.3% and 98.6% for the target oligochaetes, respectively. DNA sequencing verified the results. The development of these assays allowed us to more fully describe tubificid community composition (the taxa and their abundance at a site) and estimate the relative abundances of host taxa. To relate tubificid relative abundance to fish disease risk, we determined M. cerebralis infection prevalence in samples identified as T. tubifex using similar molecular techniques. Given prior information (i.e., morphological identification of sexually mature worms), Bayesian analysis inferred that the first qPCR assay improved taxonomic identification. Bayesian inference of the relative abundance of T. tubifex, combined with infection assay results, identified sites with a high prevalence of infected T. tubifex. To our knowledge, this study represents both the first assessment of oligochaete community composition using a qPCR assay based on fluorescent probes and the first use of Bayesian analysis to fully characterize the dominant

  13. Tomato Fruits Show Wide Phenomic Diversity but Fruit Developmental Genes Show Low Genomic Diversity.

    Directory of Open Access Journals (Sweden)

    Vijee Mohan

    Full Text Available Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS, carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1 involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima'D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future.

  14. UV Photography Shows Hidden Sun Damage

    Science.gov (United States)

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

  15. Development of a qPCR strategy to select bean genes involved in plant defence response and regulated by the Trichoderma velutinum - Rhizoctonia solani interaction

    Directory of Open Access Journals (Sweden)

    Sara Mayo

    2016-08-01

    Full Text Available Bean production is affected by a wide diversity of fungal pathogens, among them Rhizoctonia solani is one of the most important. A strategy to control bean infectious diseases, mainly those caused by fungi, is based on the use of biocontrol agents that can reduce the negative effects of plant pathogens and also can promote positive responses in the plant. Trichoderma is a fungal genus that is able to induce the expression of genes involved in plant defence response and also to promote plant growth, root development and nutrient uptake. In this article, a strategy that combines in silico analysis and real time PCR to detect additional bean defence-related genes, regulated by the presence of Trichoderma velutinum and/or R. solani has been applied. Based in this strategy, from From the 48 bean genes initially analysed, 14 were selected, and only WRKY33, CH5b and hGS showed an up-regulatory response in the presence of T. velutinum. The other genes were or not affected (OSM34 or down-regulated by the presence of this fungus. R. solani infection resulted in a down-regulation of most of the genes analyzed, except PR1, OSM34 and CNGC2 that were not affected, and the presence of both, T. velutinum and R. solani, up-regulates hGS and down-regulates all the other genes analyzed, except CH5b which was not significantly affected.As conclusion, the strategy described in the present work has been shown to be effective to detect genes involved in plant defence, which respond to the presence of a biocontrol agent or to a pathogen and also to the presence of both. The selected genes show significant homology with previously described plant defence genes and they are expressed in bean leaves of plants treated with T. velutinum and/or infected with R. solani.

  16. Educational Outreach: The Space Science Road Show

    Science.gov (United States)

    Cox, N. L. J.

    2002-01-01

    The poster presented will give an overview of a study towards a "Space Road Show". The topic of this show is space science. The target group is adolescents, aged 12 to 15, at Dutch high schools. The show and its accompanying experiments would be supported with suitable educational material. Science teachers at schools can decide for themselves if they want to use this material in advance, afterwards or not at all. The aims of this outreach effort are: to motivate students for space science and engineering, to help them understand the importance of (space) research, to give them a positive feeling about the possibilities offered by space and in the process give them useful knowledge on space basics. The show revolves around three main themes: applications, science and society. First the students will get some historical background on the importance of space/astronomy to civilization. Secondly they will learn more about novel uses of space. On the one hand they will learn of "Views on Earth" involving technologies like Remote Sensing (or Spying), Communication, Broadcasting, GPS and Telemedicine. On the other hand they will experience "Views on Space" illustrated by past, present and future space research missions, like the space exploration missions (Cassini/Huygens, Mars Express and Rosetta) and the astronomy missions (Soho and XMM). Meanwhile, the students will learn more about the technology of launchers and satellites needed to accomplish these space missions. Throughout the show and especially towards the end attention will be paid to the third theme "Why go to space"? Other reasons for people to get into space will be explored. An important question in this is the commercial (manned) exploration of space. Thus, the questions of benefit of space to society are integrated in the entire show. It raises some fundamental questions about the effects of space travel on our environment, poverty and other moral issues. The show attempts to connect scientific with

  17. Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation.

    Science.gov (United States)

    Sihto, Henna-Maria; Tasara, Taurai; Stephan, Roger; Johler, Sophia

    2014-07-01

    Staphylococcus aureus represents the most prevalent cause of food-borne intoxications worldwide. While being repressed by competing bacteria in most matrices, this pathogen exhibits crucial competitive advantages during growth at high salt concentrations or low pH, conditions frequently encountered in food production and preservation. We aimed to identify reference genes that could be used to normalize qPCR mRNA expression levels during growth of S. aureus in food-related osmotic (NaCl) and acidic (lactic acid) stress adaptation models. Expression stability of nine housekeeping genes was evaluated in full (LB) and nutrient-deficient (CYGP w/o glucose) medium under conditions of osmotic (4.5% NaCl) and acidic stress (lactic acid, pH 6.0) after 2-h exposure. Among the set of candidate reference genes investigated, rplD, rpoB,gyrB, and rho were most stably expressed in LB and thus represent the most suitable reference genes for normalization of qPCR data in osmotic or lactic acid stress models in a rich medium. Under nutrient-deficient conditions, expression of rho and rpoB was highly stable across all tested conditions. The presented comprehensive data on changes in expression of various S. aureus housekeeping genes under conditions of osmotic and lactic acid stress facilitate selection of reference genes for qPCR-based stress response models. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  18. 2008 LHC Open Days Physics: the show

    CERN Multimedia

    2008-01-01

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

  19. Duchenne muscular dystrophy models show their age

    OpenAIRE

    Chamberlain, Jeffrey S.

    2010-01-01

    The lack of appropriate animal models has hampered efforts to develop therapies for Duchenne muscular dystrophy (DMD). A new mouse model lacking both dystrophin and telomerase (Sacco et al., 2010) closely mimics the pathological progression of human DMD and shows that muscle stem cell activity is a key determinant of disease severity.

  20. Show Them You Really Want the Job

    Science.gov (United States)

    Perlmutter, David D.

    2012-01-01

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

  1. A Talk Show from the Past.

    Science.gov (United States)

    Gallagher, Arlene F.

    1991-01-01

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

  2. Laser entertainment and light shows in education

    Science.gov (United States)

    Sabaratnam, Andrew T.; Symons, Charles

    2002-05-01

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

  3. The Last Great American Picture Show

    NARCIS (Netherlands)

    Elsaesser, Thomas; King, Noel; Horwath, Alexander

    2004-01-01

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

  4. Reality, ficción o show

    Directory of Open Access Journals (Sweden)

    Sandra Ruíz Moreno

    2002-01-01

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

  5. Molecular cloning and expression analysis of a zeta-class ...

    African Journals Online (AJOL)

    Jane

    2011-07-27

    Jul 27, 2011 ... sugar-signalling pathway (Chi et al., 2010). All the earlier mentioned ... real-time qPCR analysis was the ABI PRISM7500 real-time PCR system. ... Construction of prokaryotic expression vector of Sc-GST gene. pET29a (+) ...

  6. Myopes show increased susceptibility to nearwork aftereffects.

    Science.gov (United States)

    Ciuffreda, K J; Wallis, D M

    1998-09-01

    Some aspects of accommodation may be slightly abnormal (or different) in myopes, compared with accommodation in emmetropes and hyperopes. For example, the initial magnitude of accommodative adaptation in the dark after nearwork is greatest in myopes. However, the critical test is to assess this initial accommodative aftereffect and its subsequent decay in the light under more natural viewing conditions with blur-related visual feedback present, if a possible link between this phenomenon and clinical myopia is to be considered. Subjects consisted of adult late- (n = 11) and early-onset (n = 13) myopes, emmetropes (n = 11), and hyperopes (n = 9). The distance-refractive state was assessed objectively using an autorefractor immediately before and after a 10-minute binocular near task at 20 cm (5 diopters [D]). Group results showed that myopes were most susceptible to the nearwork aftereffect. It averaged 0.35 D in initial magnitude, with considerably faster posttask decay to baseline in the early-onset (35 seconds) versus late-onset (63 seconds) myopes. There was no myopic aftereffect in the remaining two refractive groups. The myopes showed particularly striking accommodatively related nearwork aftereffect susceptibility. As has been speculated and found by many others, transient pseudomyopia may cause or be a precursor to permanent myopia or myopic progression. Time-integrated increased retinal defocus causing axial elongation is proposed as a possible mechanism.

  7. Ancient bacteria show evidence of DNA repair

    DEFF Research Database (Denmark)

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

    2007-01-01

    -term survival of bacteria sealed in frozen conditions for up to one million years. Our results show evidence of bacterial survival in samples up to half a million years in age, making this the oldest independently authenticated DNA to date obtained from viable cells. Additionally, we find strong evidence...... geological timescales. There has been no direct evidence in ancient microbes for the most likely mechanism, active DNA repair, or for the metabolic activity necessary to sustain it. In this paper, we couple PCR and enzymatic treatment of DNA with direct respiration measurements to investigate long...... that this long-term survival is closely tied to cellular metabolic activity and DNA repair that over time proves to be superior to dormancy as a mechanism in sustaining bacteria viability....

  8. Microbiological and environmental issues in show caves.

    Science.gov (United States)

    Saiz-Jimenez, Cesareo

    2012-07-01

    Cultural tourism expanded in the last half of the twentieth century, and the interest of visitors has come to include caves containing archaeological remains. Some show caves attracted mass tourism, and economical interests prevailed over conservation, which led to a deterioration of the subterranean environment and the rock art. The presence and the role of microorganisms in caves is a topic that is often ignored in cave management. Knowledge of the colonisation patterns, the dispersion mechanisms, and the effect on human health and, when present, over rock art paintings of these microorganisms is of the utmost importance. In this review the most recent advances in the study of microorganisms in caves are presented, together with the environmental implications of the findings.

  9. Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome.

    Science.gov (United States)

    Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette

    2009-06-01

    Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

  10. NASA GIBS Use in Live Planetarium Shows

    Science.gov (United States)

    Emmart, C. B.

    2015-12-01

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

  11. Assessing chlorinated ethene degradation in a large scale contaminant plume by dual carbon–chlorine isotope analysis and quantitative PCR

    DEFF Research Database (Denmark)

    Hunkeler, D.; Abe, Y.; Broholm, Mette Martina

    2011-01-01

    The fate of chlorinated ethenes in a large contaminant plume originating from a tetrachloroethene (PCE) source in a sandy aquifer in Denmark was investigated using novel methods including compound-specific carbon and chlorine isotope analysis and quantitative real-time polymerase chain reaction (q...... reduction by pyrite as indicated by the formation of cDCE and stable carbon isotope data. TCE and cDCE showed carbon isotope trends typical for reductive dechlorination with an initial depletion of 13C in the daughter products followed by an enrichment of 13C as degradation proceeded. At 1000 m downgradient......DCE. The significant enrichment of 13C in VC indicates that VC was transformed further, although the mechanismcould not be determined. The transformation of cDCEwas the rate limiting step as no accumulation of VC occurred. In summary, the study demonstrates that carbon–chlorine isotope analysis and qPCR combinedwith...

  12. Geoscience is Important? Show Me Why

    Science.gov (United States)

    Boland, M. A.

    2017-12-01

    "The public" is not homogenous and no single message or form of messaging will connect the entire public with the geosciences. One approach to promoting trust in, and engagement with, the geosciences is to identify specific sectors of the public and then develop interactions and communication products that are immediately relevant to that sector's interests. If the content and delivery are appropriate, this approach empowers people to connect with the geosciences on their own terms and to understand the relevance of the geosciences to their own situation. Federal policy makers are a distinct and influential subgroup of the general public. In preparation for the 2016 presidential election, the American Geosciences Institute (AGI) in collaboration with its 51 member societies prepared Geoscience for America's Critical Needs: Invitation to a National Dialogue, a document that identified major geoscience policy issues that should be addressed in a national policy platform. Following the election, AGI worked with eight other geoscience societies to develop Geoscience Policy Recommendations for the New Administration and the 115th Congress, which outlines specific policy actions to address national issues. State and local decision makers are another important subgroup of the public. AGI has developed online content, factsheets, and case studies with different levels of technical complexity so people can explore societally-relevant geoscience topics at their level of technical proficiency. A related webinar series is attracting a growing worldwide audience from many employment sectors. Partnering with government agencies and other scientific and professional societies has increased the visibility and credibility of these information products with our target audience. Surveys and other feedback show that these products are raising awareness of the geosciences and helping to build reciprocal relationships between geoscientists and decision makers. The core message of all

  13. Bacteriophages show promise as antimicrobial agents.

    Science.gov (United States)

    Alisky, J; Iczkowski, K; Rapoport, A; Troitsky, N

    1998-01-01

    The emergence of antibiotic-resistant bacteria has prompted interest in alternatives to conventional drugs. One possible option is to use bacteriophages (phage) as antimicrobial agents. We have conducted a literature review of all Medline citations from 1966-1996 that dealt with the therapeutic use of phage. There were 27 papers from Poland, the Soviet Union, Britain and the U.S.A. The Polish and Soviets administered phage orally, topically or systemically to treat a wide variety of antibiotic-resistant pathogens in both adults and children. Infections included suppurative wound infections, gastroenteritis, sepsis, osteomyelitis, dermatitis, empyemas and pneumonia; pathogens included Staphylococcus, Streptococcus, Klebsiella, Escherichia, Proteus, Pseudomonas, Shigella and Salmonella spp. Overall, the Polish and Soviets reported success rates of 80-95% for phage therapy, with rare, reversible gastrointestinal or allergic side effects. However, efficacy of phage was determined almost exclusively by qualitative clinical assessment of patients, and details of dosages and clinical criteria were very sketchy. There were also six British reports describing controlled trials of phage in animal models (mice, guinea pigs and livestock), measuring survival rates and other objective criteria. All of the British studies raised phage against specific pathogens then used to create experimental infections. Demonstrable efficacy against Escherichia, Acinetobacter, Pseudomonas and Staphylococcus spp. was noted in these model systems. Two U.S. papers dealt with improving the bioavailability of phage. Phage is sequestered in the spleen and removed from circulation. This can be overcome by serial passage of phage through mice to isolate mutants that resist sequestration. In conclusion, bacteriophages may show promise for treating antibiotic resistant pathogens. To facilitate further progress, directions for future research are discussed and a directory of authors from the reviewed

  14. Improved Culture Medium (TiKa) for Mycobacterium avium Subspecies Paratuberculosis (MAP) Matches qPCR Sensitivity and Reveals Significant Proportions of Non-viable MAP in Lymphoid Tissue of Vaccinated MAP Challenged Animals

    DEFF Research Database (Denmark)

    Bull, Tim J.; Munshil, Tulika; Melvang, Heidi Mikkelsen

    2017-01-01

    The quantitative detection of viable pathogen load is an important tool in determining the degree of infection in animals and contamination of foodstuffs. Current conventional culture methods are limited in their ability to determine these levels in Mycobacterium avium subspecies paratuberculosis......Ka culture equates well with qPCR and provides important evidence that accuracy in estimating viable MAP load using DNA tests alone may vary significantly between samples of mucosal and lymphatic origin....... (MAP) due to slow growth, clumping and low recoverability issues. The principle goal of this study was to evaluate a novel culturing process (TiKa) with unique ability to stimulate MAP growth from low sample loads and dilutions. We demonstrate it was able to stimulate a mean 29-fold increase...

  15. Genome-Wide Identification and Evaluation of Reference Genes for Quantitative RT-PCR Analysis during Tomato Fruit Development.

    Science.gov (United States)

    Cheng, Yuan; Bian, Wuying; Pang, Xin; Yu, Jiahong; Ahammed, Golam J; Zhou, Guozhi; Wang, Rongqing; Ruan, Meiying; Li, Zhimiao; Ye, Qingjing; Yao, Zhuping; Yang, Yuejian; Wan, Hongjian

    2017-01-01

    Gene expression analysis in tomato fruit has drawn increasing attention nowadays. Quantitative real-time PCR (qPCR) is a routine technique for gene expression analysis. In qPCR operation, reliability of results largely depends on the choice of appropriate reference genes (RGs). Although tomato is a model for fruit biology study, few RGs for qPCR analysis in tomato fruit had yet been developed. In this study, we initially identified 38 most stably expressed genes based on tomato transcriptome data set, and their expression stabilities were further determined in a set of tomato fruit samples of four different fruit developmental stages (Immature, mature green, breaker, mature red) using qPCR analysis. Two statistical algorithms, geNorm and Normfinder, concordantly determined the superiority of these identified putative RGs. Notably, SlFRG05 (Solyc01g104170), SlFRG12 (Solyc04g009770), SlFRG16 (Solyc10g081190), SlFRG27 (Solyc06g007510), and SlFRG37 (Solyc11g005330) were proved to be suitable RGs for tomato fruit development study. Further analysis using geNorm indicate that the combined use of SlFRG03 (Solyc02g063070) and SlFRG27 would provide more reliable normalization results in qPCR experiments. The identified RGs in this study will be beneficial for future qPCR analysis of tomato fruit developmental study, as well as for the potential identification of optimal normalization controls in other plant species.

  16. Genome-Wide Identification and Evaluation of Reference Genes for Quantitative RT-PCR Analysis during Tomato Fruit Development

    Directory of Open Access Journals (Sweden)

    Yuan Cheng

    2017-08-01

    Full Text Available Gene expression analysis in tomato fruit has drawn increasing attention nowadays. Quantitative real-time PCR (qPCR is a routine technique for gene expression analysis. In qPCR operation, reliability of results largely depends on the choice of appropriate reference genes (RGs. Although tomato is a model for fruit biology study, few RGs for qPCR analysis in tomato fruit had yet been developed. In this study, we initially identified 38 most stably expressed genes based on tomato transcriptome data set, and their expression stabilities were further determined in a set of tomato fruit samples of four different fruit developmental stages (Immature, mature green, breaker, mature red using qPCR analysis. Two statistical algorithms, geNorm and Normfinder, concordantly determined the superiority of these identified putative RGs. Notably, SlFRG05 (Solyc01g104170, SlFRG12 (Solyc04g009770, SlFRG16 (Solyc10g081190, SlFRG27 (Solyc06g007510, and SlFRG37 (Solyc11g005330 were proved to be suitable RGs for tomato fruit development study. Further analysis using geNorm indicate that the combined use of SlFRG03 (Solyc02g063070 and SlFRG27 would provide more reliable normalization results in qPCR experiments. The identified RGs in this study will be beneficial for future qPCR analysis of tomato fruit developmental study, as well as for the potential identification of optimal normalization controls in other plant species.

  17. Application of a qPCR assay in the investigation of susceptibility to malaria infection of the M and S molecular forms of An. gambiae s.s. in Cameroon.

    Directory of Open Access Journals (Sweden)

    Anne Boissière

    Full Text Available Plasmodium falciparum is the causative agent of malaria, a disease that kills almost one million persons each year, mainly in sub-Saharan Africa. P. falciparum is transmitted to the human host by the bite of an Anopheles female mosquito, and Anopheles gambiae sensus stricto is the most tremendous malaria vector in Africa, widespread throughout the afro-tropical belt. An. gambiae s.s. is subdivided into two distinct molecular forms, namely M and S forms. The two molecular forms are morphologically identical but they are distinct genetically, and differ by their distribution and their ecological preferences. The epidemiological importance of the two molecular forms in malaria transmission has been poorly investigated so far and gave distinct results in different areas. We have developed a real-time quantitative PCR (qPCR assay, and used it to detect P. falciparum at the oocyst stage in wild An. gambiae s.s. mosquitoes experimentally infected with natural isolates of parasites. Mosquitoes were collected at immature stages in sympatric and allopatric breeding sites and further infected at the adult stage. We next measured the infection prevalence and intensity in female mosquitoes using the qPCR assay and correlated the infection success with the mosquito molecular forms. Our results revealed different prevalence of infection between the M and S molecular forms of An. gambiae s.s. in Cameroon, for both sympatric and allopatric populations of mosquitoes. However, no difference in the infection intensity was observed. Thus, the distribution of the molecular forms of An. gambiae s.s. may impact on the malaria epidemiology, and it will be important to monitor the efficiency of malaria control interventions on the two M and S forms.

  18. Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests.

    Science.gov (United States)

    McKernan, Kevin; Spangler, Jessica; Helbert, Yvonne; Lynch, Ryan C; Devitt-Lee, Adrian; Zhang, Lei; Orphe, Wendell; Warner, Jason; Foss, Theodore; Hudalla, Christopher J; Silva, Matthew; Smith, Douglas R

    2016-01-01

    Background : The presence of bacteria and fungi in medicinal or recreational Cannabis poses a potential threat to consumers if those microbes include pathogenic or toxigenic species. This study evaluated two widely used culture-based platforms for total yeast and mold (TYM) testing marketed by 3M Corporation and Biomérieux, in comparison with a quantitative PCR (qPCR) approach marketed by Medicinal Genomics Corporation. Methods : A set of 15 medicinal Cannabis samples were analyzed using 3M and Biomérieux culture-based platforms and by qPCR to quantify microbial DNA. All samples were then subjected to next-generation sequencing and metagenomics analysis to enumerate the bacteria and fungi present before and after growth on culture-based media. Results : Several pathogenic or toxigenic bacterial and fungal species were identified in proportions of >5% of classified reads on the samples, including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Ralstonia pickettii, Salmonella enterica, Stenotrophomonas maltophilia, Aspergillus ostianus, Aspergillus sydowii, Penicillium citrinum and Penicillium steckii. Samples subjected to culture showed substantial shifts in the number and diversity of species present, including the failure of Aspergillus species to grow well on either platform. Substantial growth of Clostridium botulinum and other bacteria were frequently observed on one or both of the culture-based TYM platforms. The presence of plant growth promoting (beneficial) fungal species further influenced the differential growth of species in the microbiome of each sample. Conclusions : These findings have important implications for the Cannabis and food safety testing industries.

  19. Evaluation of Candidate Reference Genes for Quantitative Gene Expression Analysis in Spodoptera exigu a after Long-time Exposure to Cadmium.

    Science.gov (United States)

    Płachetka-Bożek, Anna; Augustyniak, Maria

    2017-08-21

    Studies on the transcriptional control of gene expression play an important role in many areas of biology. Reference genes, which are often referred to as housekeeping genes, such as GAPDH, G3PDH, EF2, RpL7A, RpL10, TUBα and Actin, have traditionally been assumed to be stably expressed in all conditions, and they are frequently used to normalize mRNA levels between different samples in qPCR analysis. However, it is known that the expression of these genes is influenced by numerous factors, such as experimental conditions. The difference in gene expression underlies a range of biological processes, including development, reproduction and behavior. The aim of this study was to show the problems associated with using reference genes in the qPCR technique, in a study on inbred strains of Spodoptera exigua selected toward cadmium resistance. We present and discuss our results and observations, and give some recommendations concerning the use and limitations of housekeeping genes as internal standards, especially in research on insects. Our results suggest that holometabolism and poikilothermia, as well as time since metamorphosis and the level of exposure to the selective factor (cadmium in this case), have a significant effect on the expression of reference genes.

  20. DNA Electrochemistry Shows DNMT1 Methyltransferase Hyperactivity in Colorectal Tumors.

    Science.gov (United States)

    Furst, Ariel L; Barton, Jacqueline K

    2015-07-23

    DNMT1, the most abundant human methyltransferase, is responsible for translating the correct methylation pattern during DNA replication, and aberrant methylation by DNMT1 has been linked to tumorigenesis. We have developed a sensitive signal-on electrochemical assay for the measurement of DNMT1 activity in crude tissue lysates. We have further analyzed ten tumor sets and have found a direct correlation between DNMT1 hyperactivity and tumorous tissue. In the majority of samples analyzed, the tumorous tissue has significantly higher DNMT1 activity than the healthy adjacent tissue. No such correlation is observed in measurements of DNMT1 expression by qPCR, DNMT1 protein abundance by western blotting, or DNMT1 activity using a radiometric DNA labeling assay. DNMT1 hyperactivity can result from both protein overexpression and enzyme hyperactivity. DNMT1 activity measured electrochemically provides a direct measure of activity in cell lysates and, as a result, provides a sensitive and early indication of cancerous transformation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Comparative proteomic analysis provides new insights into cadmium accumulation in rice grain under cadmium stress

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Dawei, E-mail: dwxue@hznu.edu.cn [College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036 (China); State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006 (China); Jiang, Hua [State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Zhejiang Academy of Agricultural Science, Hangzhou 310021 (China); Deng, Xiangxiong; Zhang, Xiaoqin [College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036 (China); Wang, Hua [State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Zhejiang Academy of Agricultural Science, Hangzhou 310021 (China); Xu, Xiangbin [College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036 (China); Hu, Jiang; Zeng, Dali [State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006 (China); Guo, Longbiao, E-mail: guolongbiao@caas.cn [State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006 (China); Qian, Qian, E-mail: qianqian188@hotmail.com [College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036 (China); State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006 (China)

    2014-09-15

    Graphical abstract: - Highlights: • Cd is the most toxic heavy metal and is a major pollutant in rice grains. • The mechanism of Cd accumulation in rice grains has not been well demonstrated. • Proteomics analysis is carried out and the verification is implemented by QPCR. • Proteins associated with ROS and photosynthesis showed large variation in expression. - Abstract: Rice is one of the most important staple crops. During the growth season, rice plants are inevitably subjected to numerous stresses, among which heavy metal stress represented by cadmium contamination not only hindering the yield of rice but also affecting the food safety by Cd accumulating in rice grains. The mechanism of Cd accumulation in rice grains has not been well elucidated. In this study, we compare the proteomic difference between two genotypes with different Cd accumulation ability in grains. Verification of differentially expressed protein-encoding genes was analyzing by quantitative PCR (QPCR) and reanalysis of microarray expression data. Forty-seven proteins in total were successfully identified through proteomic screening. GO and KEGG enrichment analysis showed Cd accumulation triggered stress-related pathways in the cells, and strongly affecting metabolic pathways. Many proteins associated with nutrient reservoir and starch-related enzyme were identified in this study suggesting that a considerably damage on grain quality was caused. The results also implied stress response was initiated by the abnormal cells and the transmission of signals may mediated by reactive oxygen species (ROS). Our research will provide new insights into Cd accumulation in rice grain under Cd stress.

  2. Identification of chloroquine resistance Pfcrt-K76T and determination of Pfmdr1-N86Y copy number by SYBR Green I qPCR

    Directory of Open Access Journals (Sweden)

    Addimas Tajebe

    2015-03-01

    Conclusions: The study showed high prevalence level and fixation of Pfcrt, 76T mutation after chloroquine withdrawal. The prevalence of Pfmdr1 copy number variant suggested that the presence of modulating factor for emergence of Plasmodium falciparum strains with higher copy numbers. However, the prevalence level was not statistically significant.

  3. Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency

    International Nuclear Information System (INIS)

    Ruijter, Jan M.; Hoff, Maurice J. B. van den; Lorenz, Peter; Tuomi, Jari M.; Hecker, Michael

    2014-01-01

    The analysis of quantitative PCR data usually does not take into account the fact that the increase in fluorescence depends on the monitoring chemistry, the input of ds-DNA or ss-cDNA, and the directionality of the targeting of probes or primers. The monitoring chemistries currently available can be categorized into six groups: (A) DNA-binding dyes; (B) hybridization probes; (C) hydrolysis probes; (D) LUX primers; (E) hairpin primers; and (F) the QZyme system. We have determined the kinetics of the increase in fluorescence for each of these groups with respect to the input of both ds-DNA and ss-cDNA. For the latter, we also evaluated mRNA and cDNA targeting probes or primers. This analysis revealed three situations. Hydrolysis probes and LUX primers, compared to DNA-binding dyes, do not require a correction of the observed quantification cycle. Hybridization probes and hairpin primers require a correction of −1 cycle (dubbed C-lag), while the QZyme system requires the C-lag correction and an efficiency-dependent C-shift correction. A PCR efficiency value can be derived from the relative increase in fluorescence in the exponential phase of the amplification curve for all monitoring chemistries. In case of hydrolysis probes, LUX primers and hairpin primers, however, this should be performed after cycle 12, and for the QZyme system after cycle 19, to keep the overestimation of the PCR efficiency below 0.5 %. (author)

  4. Show Horse Welfare: Horse Show Competitors' Understanding, Awareness, and Perceptions of Equine Welfare.

    Science.gov (United States)

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

    2016-01-01

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

  5. Best in show but not best shape: a photographic assessment of show dog body condition.

    Science.gov (United States)

    Such, Z R; German, A J

    2015-08-01

    Previous studies suggest that owners often wrongly perceive overweight dogs to be in normal condition. The body shape of dogs attending shows might influence owners' perceptions, with online images of overweight show winners having a negative effect. This was an observational in silico study of canine body condition. 14 obese-prone breeds and 14 matched non-obese-probe breeds were first selected, and one operator then used an online search engine to identify 40 images, per breed, of dogs that had appeared at a major national UK show (Crufts). After images were anonymised and coded, a second observer subjectively assessed body condition, in a single sitting, using a previously validated method. Of 1120 photographs initially identified, 960 were suitable for assessing body condition, with all unsuitable images being from longhaired breeds. None of the dogs (0 per cent) were underweight, 708 (74 per cent) were in ideal condition and 252 (26 per cent) were overweight. Pugs, basset hounds and Labrador retrievers were most likely to be overweight, while standard poodles, Rhodesian ridgebacks, Hungarian vizslas and Dobermanns were least likely to be overweight. Given the proportion of show dogs from some breeds that are overweight, breed standards should be redefined to be consistent with a dog in optimal body condition. British Veterinary Association.

  6. Non-invasive prenatal diagnosis of monogenic disorders: an optimized protocol using MEMO qPCR with miniSTR as internal control.

    Science.gov (United States)

    Guissart, Claire; Debant, Vanessa; Desgeorges, Marie; Bareil, Corinne; Raynal, Caroline; Toga, Caroline; Pritchard, Victoria; Koenig, Michel; Claustres, Mireille; Vincent, Marie-Claire

    2015-02-01

    Analysis of circulating cell-free fetal DNA (cffDNA) in maternal plasma is very promising for early diagnosis of monogenic diseases. However, this approach is not yet available for routine use and remains technically challenging because of the low concentration of cffDNA, which is swamped by the overwhelming maternal DNA. To make clinical applications more readily accessible, we propose a new approach based on mutant enrichment with 3'-modified oligonucleotides (MEMO) PCR along with real-time PCR to selectively amplify from the maternal blood the paternally inherited fetal allele that is not present in the maternal genome. The first proof of concept of this strategy was displayed for cystic fibrosis by the accuracy of our detection of the p.Gly542* mutation used as the initial developmental model. Subsequently, a retrospective study of plasmas originating from two pregnant women carrying a fetus with private mutation confirmed the effectiveness of our method. We confirmed the presence of cffDNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. This new non-invasive prenatal diagnosis test offers numerous advantages over current methods: it is simple, cost effective, time efficient and does not require complex equipment or bioinformatics settings. Moreover, our assays for different private mutations demonstrate the viability of this approach in clinical settings for monogenic disorders.

  7. Quantitative microbial community analysis of three different sulfidic mine tailing dumps generating acid mine drainage.

    Science.gov (United States)

    Kock, Dagmar; Schippers, Axel

    2008-08-01

    The microbial communities of three different sulfidic and acidic mine waste tailing dumps located in Botswana, Germany, and Sweden were quantitatively analyzed using quantitative real-time PCR (Q-PCR), fluorescence in situ hybridization (FISH), catalyzed reporter deposition-FISH (CARD-FISH), Sybr green II direct counting, and the most probable number (MPN) cultivation technique. Depth profiles of cell numbers showed that the compositions of the microbial communities are greatly different at the three sites and also strongly varied between zones of oxidized and unoxidized tailings. Maximum cell numbers of up to 10(9) cells g(-1) dry weight were determined in the pyrite or pyrrhotite oxidation zones, whereas cell numbers in unoxidized tailings were significantly lower. Bacteria dominated over Archaea and Eukarya at all tailing sites. The acidophilic Fe(II)- and/or sulfur-oxidizing Acidithiobacillus spp. dominated over the acidophilic Fe(II)-oxidizing Leptospirillum spp. among the Bacteria at two sites. The two genera were equally abundant at the third site. The acidophilic Fe(II)- and sulfur-oxidizing Sulfobacillus spp. were generally less abundant. The acidophilic Fe(III)-reducing Acidiphilium spp. could be found at only one site. The neutrophilic Fe(III)-reducing Geobacteraceae as well as the dsrA gene of sulfate reducers were quantifiable at all three sites. FISH analysis provided reliable data only for tailing zones with high microbial activity, whereas CARD-FISH, Q-PCR, Sybr green II staining, and MPN were suitable methods for a quantitative microbial community analysis of tailings in general.

  8. Immunoflourescence and mRNA analysis of human embryonic stem cells (hESCs) grown under feeder-free conditions

    DEFF Research Database (Denmark)

    Awan, Aashir; Oliveri, Roberto S; Jensen, Pernille L

    2010-01-01

    onto 16-well glass chambers, and continuing with the general IF and qPCR steps will be provided. The techniques will be illustrated with new results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, and PDGF signaling pathways to primary cilia in stem cell......This chapter describes the procedures in order to do immunofluorescence (IF) microscopy and quantitative PCR (qPCR) analysis of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol outlining the steps from initially growing the cells, passaging...

  9. Pedagogical Techniques Employed by the Television Show "MythBusters"

    Science.gov (United States)

    Zavrel, Erik

    2016-11-01

    "MythBusters," the long-running though recently discontinued Discovery Channel science entertainment television program, has proven itself to be far more than just a highly rated show. While its focus is on entertainment, the show employs an array of pedagogical techniques to communicate scientific concepts to its audience. These techniques include: achieving active learning, avoiding jargon, employing repetition to ensure comprehension, using captivating demonstrations, cultivating an enthusiastic disposition, and increasing intrinsic motivation to learn. In this content analysis, episodes from the show's 10-year history were examined for these techniques. "MythBusters" represents an untapped source of pedagogical techniques, which science educators may consider availing themselves of in their tireless effort to better reach their students. Physics educators in particular may look to "MythBusters" for inspiration and guidance in how to incorporate these techniques into their own teaching and help their students in the learning process.

  10. Cloning and expression analysis of a transformer gene in Daphnia pulex during different reproduction stages.

    Science.gov (United States)

    Chen, Ping; Xu, Shan-Liang; Zhou, Wei; Guo, Xiao-Ge; Wang, Chun-Lin; Wang, Dan-Li; Zhao, Yun-Long

    2014-05-01

    The full-length cDNA of a transformer gene (Dptra) was cloned from the cladoceran Daphnia pulex using RACE. Dptra expression was assessed by qPCR and whole-mount in situ hybridization in different reproductive stages. The Dptra cDNA, 1652bp in length, has a 1158-bp open reading frame that encodes a 385 amino acid polypeptide containing one Sex determination protein N terminal (SDP_N) superfamily, eight putative phosphorylation sites, and an arginine-serine (RS)-rich domain at the N-terminus. Dptra showed 81%, 53%, 51% and 45% identity to orthologous genes in Daphnia magna, Apis mellifera, Apis cerana and Bombus terrestris, respectively. Phylogenetic analysis based on deduced amino acid sequences revealed that Dptra clustered in the hymenopteran clade and was most closely related to D. magna and A. mellifera. qPCR showed that Dptra expression increased significantly (P<0.05) in different reproductive stages in the following order: male, ephippial female, parthenogenetic female, resting egg and juvenile female. Dptra expression is significantly different between males and females and it is significantly greater in ephippial females and males than in parthenogenetic D. pulex (with summer eggs). Whole-mount in situ hybridization revealed that Dptra was expressed at different levels between males and females. In males, hybridization signals were found in the first antennae, second antennae and thoracic limb, whereas expression levels in the corresponding sites of parthenogenetic and ephippial females were relatively weak. This suggests that the Dptra gene plays significant roles in switching modes of reproduction and in sexual differentiation. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Cryptosporidium parvum infection in SCID mice infected with only one oocyst: qPCR assessment of parasite replication in tissues and development of digestive cancer.

    Directory of Open Access Journals (Sweden)

    Sadia Benamrouz

    Full Text Available Dexamethasone (Dex treated Severe Combined Immunodeficiency (SCID mice were previously described as developing digestive adenocarcinoma after massive infection with Cryptosporidium parvum as soon as 45 days post-infection (P.I.. We aimed to determine the minimum number of oocysts capable of inducing infection and thereby gastrointestinal tumors in this model. Mice were challenged with calibrated oocyst suspensions containing intended doses of: 1, 10, 100 or 10(5 oocysts of C. parvum Iowa strain. All administered doses were infective for animals but increasing the oocyst challenge lead to an increase in mice infectivity (P = 0.01. Oocyst shedding was detected at 7 days P.I. after inoculation with more than 10 oocysts, and after 15 days in mice challenged with one oocyst. In groups challenged with lower inocula, parasite growth phase was significantly higher (P = 0.005 compared to mice inoculated with higher doses. After 45 days P.I. all groups of mice had a mean of oocyst shedding superior to 10,000 oocyst/g of feces. The most impressive observation of this study was the demonstration that C. parvum-induced digestive adenocarcinoma could be caused by infection with low doses of Cryptosporidium, even with only one oocyst: in mice inoculated with low doses, neoplastic lesions were detected as early as 45 days P.I. both in the stomach and ileo-caecal region, and these lesions could evolve in an invasive adenocarcinoma. These findings show a great amplification effect of parasites in mouse tissues after challenge with low doses as confirmed by quantitative PCR. The ability of C. parvum to infect mice with one oocyst and to develop digestive adenocarcinoma suggests that other mammalian species including humans could be also susceptible to this process, especially when they are severely immunocompromised.

  12. Cryptosporidium parvum infection in SCID mice infected with only one oocyst: qPCR assessment of parasite replication in tissues and development of digestive cancer.

    Science.gov (United States)

    Benamrouz, Sadia; Guyot, Karine; Gazzola, Sophie; Mouray, Anthony; Chassat, Thierry; Delaire, Baptiste; Chabé, Magali; Gosset, Pierre; Viscogliosi, Eric; Dei-Cas, Eduardo; Creusy, Colette; Conseil, Valerie; Certad, Gabriela

    2012-01-01

    Dexamethasone (Dex) treated Severe Combined Immunodeficiency (SCID) mice were previously described as developing digestive adenocarcinoma after massive infection with Cryptosporidium parvum as soon as 45 days post-infection (P.I.). We aimed to determine the minimum number of oocysts capable of inducing infection and thereby gastrointestinal tumors in this model. Mice were challenged with calibrated oocyst suspensions containing intended doses of: 1, 10, 100 or 10(5) oocysts of C. parvum Iowa strain. All administered doses were infective for animals but increasing the oocyst challenge lead to an increase in mice infectivity (P = 0.01). Oocyst shedding was detected at 7 days P.I. after inoculation with more than 10 oocysts, and after 15 days in mice challenged with one oocyst. In groups challenged with lower inocula, parasite growth phase was significantly higher (P = 0.005) compared to mice inoculated with higher doses. After 45 days P.I. all groups of mice had a mean of oocyst shedding superior to 10,000 oocyst/g of feces. The most impressive observation of this study was the demonstration that C. parvum-induced digestive adenocarcinoma could be caused by infection with low doses of Cryptosporidium, even with only one oocyst: in mice inoculated with low doses, neoplastic lesions were detected as early as 45 days P.I. both in the stomach and ileo-caecal region, and these lesions could evolve in an invasive adenocarcinoma. These findings show a great amplification effect of parasites in mouse tissues after challenge with low doses as confirmed by quantitative PCR. The ability of C. parvum to infect mice with one oocyst and to develop digestive adenocarcinoma suggests that other mammalian species including humans could be also susceptible to this process, especially when they are severely immunocompromised.

  13. Q-PCR Based Culture-Independent Enumeration and Detection of Enterobacter: An Emerging Environmental Human Pathogen in Riverine Systems and Potable Water

    Science.gov (United States)

    Patel, Chandra B.; Shanker, Rishi; Gupta, Vijai K.; Upadhyay, Ram S.

    2016-01-01

    The availability of safe and pristine water is a global challenge when large numbers of natural and anthropogenic water resources are being depleted with faster rate. The remaining water resources are severely contaminated with various kinds of contaminants including microorganisms. Enterobacter is one of the fecal coliform bacteria of family Enterobacteriaceae. Enterobacter was earlier used as an indicator bacterium along with other fecal Coliforms namely Escherichia coli, Citrobacter, and Klebsiella, but it is now known to cause various diseases in human beings. In this study, we have collected 55 samples from potable water and riverine system and proved their presence using their conserved sequences of 16S rRNA and 23S rRNA genes with the help of SYBR green real-time PCR, which showed very high specificity for the detection of Enterobacter. The Enterobacter counts in potable water were found to 1290 ± 32.89 to 1460 ± 39.42 cfu/100 ml. The Enterobacter levels in surface water were 1.76 × 104 ± 492, 1.33 × 104 ± 334, 1.15 × 104 ± 308, 2.56 × 104 ± 802, 2.89 × 104 ± 962, 8.16 × 104 ± 3443 cfu/100 ml; the levels of Enterobacter contamination associated with hydrophytes were 4.80 × 104 ± 1804, 3.48 × 104 ± 856, 8.50 × 104 ± 2074, 8.09 × 104 ± 1724, 6.30 × 104 ± 1738, 3.68 × 104 ± 949 cfu/10 g and the Enterobacter counts in sediments of the river, were 2.36 × 104 ± 703, 1.98 × 104 ± 530, 9.92 × 104 ± 3839, 6.80 × 104 ± 2230, 8.76 × 104 ± 3066 and 2.34 × 104 ± 732 cfu/10 g at the sampling Site #1, Site #2, Site #3, Site #4, Site #5, and Site #6, respectively. The assay could be used for the regular monitoring of potable water and other water reservoirs to check waterborne outbreaks. PMID:26925044

  14. Development of a qPCR Method for the Identification and Quantification of Two Closely Related Tuna Species, Bigeye Tuna (Thunnus obesus) and Yellowfin Tuna (Thunnus albacares), in Canned Tuna.

    Science.gov (United States)

    Bojolly, Daline; Doyen, Périne; Le Fur, Bruno; Christaki, Urania; Verrez-Bagnis, Véronique; Grard, Thierry

    2017-02-01

    Bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) are among the most widely used tuna species for canning purposes. Not only substitution but also mixing of tuna species is prohibited by the European regulation for canned tuna products. However, as juveniles of bigeye and yellowfin tunas are very difficult to distinguish, unintentional substitutions may occur during the canning process. In this study, two mitochondrial markers from NADH dehydrogenase subunit 2 and cytochrome c oxidase subunit II genes were used to identify bigeye tuna and yellowfin tuna, respectively, utilizing TaqMan qPCR methodology. Two different qPCR-based methods were developed to quantify the percentage of flesh of each species used for can processing. The first one was based on absolute quantification using standard curves realized with these two markers; the second one was founded on relative quantification with the universal 12S rRNA gene as the endogenous gene. On the basis of our results, we conclude that our methodology could be applied to authenticate these two closely related tuna species when used in a binary mix in tuna cans.

  15. DNA barcoding of freshwater fishes and the development of a quantitative qPCR assay for the species-specific detection and quantification of fish larvae from plankton samples.

    Science.gov (United States)

    Loh, W K W; Bond, P; Ashton, K J; Roberts, D T; Tibbetts, I R

    2014-08-01

    The barcoding of mitochondrial cytochrome c oxidase subunit 1 (coI) gene was amplified and sequenced from 16 species of freshwater fishes found in Lake Wivenhoe (south-eastern Queensland, Australia) to support monitoring of reservoir fish populations, ecosystem function and water health. In this study, 630-650 bp sequences of the coI barcoding gene from 100 specimens representing 15 genera, 13 families and two subclasses of fishes allowed 14 of the 16 species to be identified and differentiated. The mean ± s.e. Kimura 2 parameter divergence within and between species was 0.52 ± 0.10 and 23.8 ± 2.20% respectively, indicating that barcodes can be used to discriminate most of the fish species accurately. The two terapontids, Amniataba percoides and Leiopotherapon unicolor, however, shared coI DNA sequences and could not be differentiated using this gene. A barcoding database was established and a qPCR assay was developed using coI sequences to identify and quantify proportional abundances of fish species in ichthyoplankton samples from Lake Wivenhoe. These methods provide a viable alternative to the time-consuming process of manually enumerating and identifying ichthyoplankton samples. © 2014 The Fisheries Society of the British Isles.

  16. Quantitative analysis of Terminal Restriction Fragment Length Polymorphism (T-RFLP microbial community profiles: peak height data showed to be more reproducible than peak area Análise quantitativa de perfis de T-RFLP de comunidades microbianas: dados de altura de picos mostraram-se mais reprodutíveis do que os de área

    Directory of Open Access Journals (Sweden)

    Roberto A. Caffaro-Filho

    2007-12-01

    Full Text Available Terminal Restriction Fragment Length Polymorphism (T-RFLP is a culture-independent fingerprinting method for microbial community analysis. Profiles generated by an automated electrophoresis system can be analysed quantitatively using either peak height or peak area data. Statistical testing demontrated that peak height data showed to be more reproducible than peak area data.Terminal Restriction Fragment Length Polymorphism (T-RFLP é um método molecular, independente de cultivo, para análise de comunidades microbianas. Perfis gerados por um sistema automatizado de eletroforese podem ser analisados quantitativamente usando dados de altura ou área dos picos. Os dados de altura mostraram-se mais reprodutíveis do que os de área.

  17. Comparative genomic analysis shows that avian pathogenic Escherichia coli isolate IMT5155 (O2:K1:H5; ST complex 95, ST140 shares close relationship with ST95 APEC O1:K1 and human ExPEC O18:K1 strains.

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    Xiangkai Zhu Ge

    Full Text Available Avian pathogenic E. coli and human extraintestinal pathogenic E. coli serotypes O1, O2 and O18 strains isolated from different hosts are generally located in phylogroup B2 and ST complex 95, and they share similar genetic characteristics and pathogenicity, with no or minimal host specificity. They are popular objects for the study of ExPEC genetic characteristics and pathogenesis in recent years. Here, we investigated the evolution and genetic blueprint of APEC pathotype by performing phylogenetic and comparative genome analysis of avian pathogenic E. coli strain IMT5155 (O2:K1:H5; ST complex 95, ST140 with other E. coli pathotypes. Phylogeny analyses indicated that IMT5155 has closest evolutionary relationship with APEC O1, IHE3034, and UTI89. Comparative genomic analysis showed that IMT5155 and APEC O1 shared significant genetic overlap/similarities with human ExPEC dominant O18:K1 strains (IHE3034 and UTI89. Furthermore, the unique PAI I5155 (GI-12 was identified and found to be conserved in APEC O2 serotype isolates. GI-7 and GI-16 encoding two typical T6SSs in IMT5155 might be useful markers for the identification of ExPEC dominant serotypes (O1, O2, and O18 strains. IMT5155 contained a ColV plasmid p1ColV5155, which defined the APEC pathotype. The distribution analysis of 10 sequenced ExPEC pan-genome virulence factors among 47 sequenced E. coli strains provided meaningful information for B2 APEC/ExPEC-specific virulence factors, including several adhesins, invasins, toxins, iron acquisition systems, and so on. The pathogenicity tests of IMT5155 and other APEC O1:K1 and O2:K1 serotypes strains (isolated in China through four animal models showed that they were highly virulent for avian colisepticemia and able to cause septicemia and meningitis in neonatal rats, suggesting zoonotic potential of these APEC O1:K1 and O2:K1 isolates.

  18. Airline Overbooking Problem with Uncertain No-Shows

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    Chunxiao Zhang

    2014-01-01

    Full Text Available This paper considers an airline overbooking problem of a new single-leg flight with discount fare. Due to the absence of historical data of no-shows for a new flight, and various uncertain human behaviors or unexpected events which causes that a few passengers cannot board their aircraft on time, we fail to obtain the probability distribution of no-shows. In this case, the airlines have to invite some domain experts to provide belief degree of no-shows to estimate its distribution. However, human beings often overestimate unlikely events, which makes the variance of belief degree much greater than that of the frequency. If we still regard the belief degree as a subjective probability, the derived results will exceed our expectations. In order to deal with this uncertainty, the number of no-shows of new flight is assumed to be an uncertain variable in this paper. Given the chance constraint of social reputation, an overbooking model with discount fares is developed to maximize the profit rate based on uncertain programming theory. Finally, the analytic expression of the optimal booking limit is obtained through a numerical example, and the results of sensitivity analysis indicate that the optimal booking limit is affected by flight capacity, discount, confidence level, and parameters of the uncertainty distribution significantly.

  19. High-Resolution Observations of a Filament showing Activated Barb

    Science.gov (United States)

    Joshi, Anand; Martin, Sara F.; Mathew, Shibu; Srivastava, Nandita

    2012-07-01

    Analysis of a filament showing an activated barb using observations from the Dutch Open Telescope (DOT) on 2010 August 20 are presented. The DOT takes Doppler images in Hα, among other wavelengths, in a region about 110 × 110 arcsec^{2} in area, at a cadence of 30~seconds. The offline image restoration technique of speckle reconstruction is applied to obtain diffraction limited images. The filament developed a new barb in 10~minutes, which disappeared within the next 35~minutes. Such a rapid formation and disappearance of a filament barb is unusual, and has not been reported earlier. Line-of-sight velocity maps were constructed from the Doppler images of the target filament. We observe flows in the filament spine towards the barb location prior to its formation, and flows in the barb towards the spine during its disappearance. Photospheric magnetograms from Heliospheric Magnetic Imager on board the Solar Dynamics Observatory, at a cadence of 45~seconds, were used to determine the changes in magnetic flux in the region surrounding the barb location. The variation of magnetic flux in this duration supports the view that barbs are rooted in minor magnetic polarity. Our analysis shows that barbs can be short-lived and formation and disappearance of the barb was associated with cancellation of magnetic flux.

  20. Evolutionary Analysis of Minor Histocompatibility Genes In Hydra

    KAUST Repository

    Aalismail, Nojood

    2016-05-01

    Hydra is a simple freshwater solitary polyp used as a model system to study evolutionary aspects. The immune response of this organism has not been studied extensively and the immune response genes have not been identified and characterized. On the other hand, immune response has been investigated and genetic analysis has been initiated in other lower invertebrates. In the present study we took initiative to study the self/nonself recognition in hydra and its relation to the immune response. Moreover, performing phylogenetic analysis to look for annotated immune genes in hydra gave us a potential to analyze the expression of minor histocompatibility genes that have been shown to play a major role in grafting and transplantation in mammals. Here we obtained the cDNA library that shows expression of minor histocompatibility genes and confirmed that the annotated sequences in databases are actually present. In addition, grafting experiments suggested, although still preliminary, that homograft showed less rejection response than in heterograft. Involvement of possible minor histocompatibility gene orthologous in immune response was examined by qPCR.

  1. Molecular Characterization and Expression Analysis of Equine ( Gene in Horse (

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    Ki-Duk Song

    2014-05-01

    Full Text Available The objective of this study was to determine the molecular characteristics of the horse vascular endothelial growth factor alpha gene (VEGFα by constructing a phylogenetic tree, and to investigate gene expression profiles in tissues and blood leukocytes after exercise for development of suitable biomarkers. Using published amino acid sequences of other vertebrate species (human, chimpanzee, mouse, rat, cow, pig, chicken and dog, we constructed a phylogenetic tree which showed that equine VEGFα belonged to the same clade of the pig VEGFα. Analysis for synonymous (Ks and non-synonymous substitution ratios (Ka revealed that the horse VEGFα underwent positive selection. RNA was extracted from blood samples before and after exercise and different tissue samples of three horses. Expression analyses using reverse transcription-polymerase chain reaction (RT-PCR and quantitative-polymerase chain reaction (qPCR showed ubiquitous expression of VEGFα mRNA in skeletal muscle, kidney, thyroid, lung, appendix, colon, spinal cord, and heart tissues. Analysis of differential expression of VEGFα gene in blood leukocytes after exercise indicated a unimodal pattern. These results will be useful in developing biomarkers that can predict the recovery capacity of racing horses.

  2. Genome-wide analysis identifies chickpea (Cicer arietinum) heat stress transcription factors (Hsfs) responsive to heat stress at the pod development stage.

    Science.gov (United States)

    Chidambaranathan, Parameswaran; Jagannadham, Prasanth Tej Kumar; Satheesh, Viswanathan; Kohli, Deshika; Basavarajappa, Santosh Halasabala; Chellapilla, Bharadwaj; Kumar, Jitendra; Jain, Pradeep Kumar; Srinivasan, R

    2018-05-01

    The heat stress transcription factors (Hsfs) play a prominent role in thermotolerance and eliciting the heat stress response in plants. Identification and expression analysis of Hsfs gene family members in chickpea would provide valuable information on heat stress responsive Hsfs. A genome-wide analysis of Hsfs gene family resulted in the identification of 22 Hsf genes in chickpea in both desi and kabuli genome. Phylogenetic analysis distinctly separated 12 A, 9 B, and 1 C class Hsfs, respectively. An analysis of cis-regulatory elements in the upstream region of the genes identified many stress responsive elements such as heat stress elements (HSE), abscisic acid responsive element (ABRE) etc. In silico expression analysis showed nine and three Hsfs were also expressed in drought and salinity stresses, respectively. Q-PCR expression analysis of Hsfs under heat stress at pod development and at 15 days old seedling stage showed that CarHsfA2, A6, and B2 were significantly upregulated in both the stages of crop growth and other four Hsfs (CarHsfA2, A6a, A6c, B2a) showed early transcriptional upregulation for heat stress at seedling stage of chickpea. These subclasses of Hsfs identified in this study can be further evaluated as candidate genes in the characterization of heat stress response in chickpea.

  3. Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription.

    Science.gov (United States)

    Johnston, Stephen; Gallaher, Zachary; Czaja, Krzysztof

    2012-05-15

    Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2(-∆∆Ct) normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxin selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference β-III tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

  4. A Microbiomic Analysis in African Americans with Colonic Lesions Reveals Streptococcus sp.VT162 as a Marker of Neoplastic Transformation

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    Hassan Brim

    2017-11-01

    Full Text Available Increasing evidence suggests a role of the gut microbiota in colorectal carcinogenesis (CRC. To detect bacterial markers of colorectal cancer in African Americans a metabolomic analysis was performed on fecal water extracts. DNA from stool samples of adenoma and healthy subjects and from colon cancer and matched normal tissues was analyzed to determine the microbiota composition (using 16S rDNA and genomic content (metagenomics. Metagenomic functions with discriminative power between healthy and neoplastic specimens were established. Quantitative Polymerase Chain Reaction (q-PCR using primers and probes specific to Streptococcus sp. VT_162 were used to validate this bacterium association with neoplastic transformation in stool samples from two independent cohorts of African Americans and Chinese patients with colorectal lesions. The metabolomic analysis of adenomas revealed low amino acids content. The microbiota in both cancer vs. normal tissues and adenoma vs. normal stool samples were different at the 16S rRNA gene level. Cross-mapping of metagenomic data led to 9 markers with significant discriminative power between normal and diseased specimens. These markers identified with Streptococcus sp. VT_162. Q-PCR data showed a statistically significant presence of this bacterium in advanced adenoma and cancer samples in an independent cohort of CRC patients. We defined metagenomic functions from Streptococcus sp. VT_162 with discriminative power among cancers vs. matched normal and adenomas vs. healthy subjects’ stools. Streptococcus sp. VT_162 specific 16S rDNA was validated in an independent cohort. These findings might facilitate non-invasive screening for colorectal cancer.

  5. Validation of RNAi Silencing Efficiency Using Gene Array Data shows 18.5% Failure Rate across 429 Independent Experiments

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    Gyöngyi Munkácsy

    2016-01-01

    Full Text Available No independent cross-validation of success rate for studies utilizing small interfering RNA (siRNA for gene silencing has been completed before. To assess the influence of experimental parameters like cell line, transfection technique, validation method, and type of control, we have to validate these in a large set of studies. We utilized gene chip data published for siRNA experiments to assess success rate and to compare methods used in these experiments. We searched NCBI GEO for samples with whole transcriptome analysis before and after gene silencing and evaluated the efficiency for the target and off-target genes using the array-based expression data. Wilcoxon signed-rank test was used to assess silencing efficacy and Kruskal–Wallis tests and Spearman rank correlation were used to evaluate study parameters. All together 1,643 samples representing 429 experiments published in 207 studies were evaluated. The fold change (FC of down-regulation of the target gene was above 0.7 in 18.5% and was above 0.5 in 38.7% of experiments. Silencing efficiency was lowest in MCF7 and highest in SW480 cells (FC = 0.59 and FC = 0.30, respectively, P = 9.3E−06. Studies utilizing Western blot for validation performed better than those with quantitative polymerase chain reaction (qPCR or microarray (FC = 0.43, FC = 0.47, and FC = 0.55, respectively, P = 2.8E−04. There was no correlation between type of control, transfection method, publication year, and silencing efficiency. Although gene silencing is a robust feature successfully cross-validated in the majority of experiments, efficiency remained insufficient in a significant proportion of studies. Selection of cell line model and validation method had the highest influence on silencing proficiency.

  6. Intracerebral metastasis showing restricted diffusion: Correlation with histopathologic findings

    Energy Technology Data Exchange (ETDEWEB)

    Duygulu, G. [Radiology Department, Ege University Medicine School, Izmir (Turkey); Ovali, G. Yilmaz [Radiology Department, Celal Bayar University Medicine School, Manisa (Turkey)], E-mail: gulgun.yilmaz@bayar.edu.tr; Calli, C.; Kitis, O.; Yuenten, N. [Radiology Department, Ege University Medicine School, Izmir (Turkey); Akalin, T. [Pathology Department, Ege University Medicine School, Izmir (Turkey); Islekel, S. [Neurosurgery Department, Ege University Medicine School, Izmir (Turkey)

    2010-04-15

    Objective: We aimed to detect the frequency of restricted diffusion in intracerebral metastases and to find whether there is correlation between the primary tumor pathology and diffusion-weighted MR imaging (DWI) findings of these metastases. Material and methods: 87 patients with intracerebral metastases were examined with routine MR imaging and DWI. 11 hemorrhagic metastatic lesions were excluded. The routine MR imaging included three plans before and after contrast enhancement. The DWI was performed with spin-echo EPI sequence with three b values (0, 500 and 1000), and ADC maps were calculated. 76 patients with metastases were grouped according to primary tumor histology and the ratios of restricted diffusion were calculated according to these groups. ADCmin values were measured within the solid components of the tumors and the ratio of metastases with restricted diffusion to that which do not show restricted diffusion were calculated. Fisher's exact and Mann-Whitney U tests were used for the statistical analysis. Results: Restricted diffusion was observed in a total of 15 metastatic lesions (19, 7%). Primary malignancy was lung carcinoma in 10 of these cases (66, 6%) (5 small cell carcinoma, 5 non-small cell carcinoma), and breast carcinoma in three cases (20%). Colon carcinoma and testicular teratocarcinoma were the other two primary tumors in which restricted diffusion in metastasis was detected. There was no statistical significant difference between the primary pathology groups which showed restricted diffusion (p > 0.05). ADCmin values of solid components of the metastasis with restricted diffusion and other metastasis without restricted diffusion also showed no significant statistical difference (0.72 {+-} 0.16 x 10{sup -3} mm{sup 2}/s and 0.78 {+-} 21 x 10{sup -3} mm{sup 2}/s respectively) (p = 0.325). Conclusion: Detection of restricted diffusion on DWI in intracerebral metastasis is not rare, particularly if the primary tumor is lung or breast

  7. Real-time PCR quantification and diversity analysis of the functional genes aprA and dsrA of sulfate-reducing bacteria in marine sediments of the Peru continental margin and the Black Sea

    OpenAIRE

    Axel eSchippers; Anna eBlazejak

    2011-01-01

    A quantitative, real-time PCR (Q-PCR) assay for the functional gene adenosine 5´-phosphosulfate reductase (aprA) of sulfate-reducing bacteria (SRB) was designed. This assay was applied together with described Q-PCR assays for dissimilatory sulfite reductase (dsrA) and the 16S rRNA gene of total Bacteria to marine sediments from the Peru margin (0 – 121 meters below seafloor (mbsf)) and the Black Sea (0 – 6 mbsf). Clone libraries of aprA show that all isolated sequences originate from SRB...

  8. An Analysis of Thaumarchaeota Populations from the Northern Gulf of Mexico

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    Bradley B. Tolar

    2013-04-01

    Full Text Available We sampled Thaumarchaeota populations in the northern Gulf of Mexico, including shelf waters under the Mississippi River outflow plume that are subject to recurrent hypoxia. Data from this study allowed us to: 1 test the hypothesis that Thaumarchaeota would be abundant in this region; 2 assess phylogenetic composition of these populations for comparison with other regions; 3 compare the efficacy of quantitative PCR (qPCR based on primers for 16S rRNA genes (rrs with primers for genes in the ammonia oxidation (amoA and carbon fixation (accA, hcd pathways; 4 compare distributions obtained by qPCR with the relative abundance of Thaumarchaeota rrs in pyrosequenced libraries; 5 compare Thaumarchaeota distributions with environmental variables to help us elucidate the factors responsible for the distributions; 6 compare the distribution of Thaumarchaeota with Nitrite-Oxidizing Bacteria (NOB to gain insight into the coupling between ammonia and nitrite oxidation. We found up to 108 copies L-1 of Thaumarchaeota rrs in our samples (up to 40% of prokaryotes by qPCR, with maximum abundance in slope waters at 200-800 m. Thaumarchaeota rrs were also abundant in pyrosequenced libraries and their relative abundance correlated well with values determined by qPCR (r2=0.82. Thaumarchaeota populations were strongly stratified by depth. Canonical correspondence analysis using a suite of environmental variables explained 92% of the variance in qPCR-estimated gene abundances. Thaumarchaeota rrs abundance was correlated with salinity and depth, while accA abundance correlated with fluorescence and pH. Correlations of Archaeal amoA abundance with environmental variables were primer-dependent, suggesting differential responses of sub-populations to environmental variables. Bacterial amoA was at the limit of qPCR detection in most samples. NOB and Euryarchaeota rrs were found in the pyrosequenced libraries; NOB distribution was correlated with that of Thaumarchaeota (r2=0.49

  9. Prognostic Value of microRNA-9 in Various Cancers: a Meta-analysis.

    Science.gov (United States)

    Zhang, Yunyuan; Zhou, Jun; Sun, Meiling; Sun, Guirong; Cao, Yongxian; Zhang, Haiping; Tian, Runhua; Zhou, Lan; Duan, Liang; Chen, Xian; Lun, Limin

    2017-07-01

    Recently, there are more and more evidences from studies have revealed the association between microRNA-9 (miR-9) expression and outcome in multiple cancers, but inconsistent results have also been reported. It is necessary to rationalize a meta analysis of all available data to clarify the prognostic role of miR-9. Eligible studies were selected through multiple search strategies and the quality was assessed by MOOSE. Data was extracted from studies according to the key statistics index. All analyses were performed using STATA software. Twenty studies were selected in the meta-analysis to evaluate the prognostic role of miR-9 in multiple tumors. MiR-9 expression level was an independent prognostic biomarker for OS in tumor patients using multivariate and univariate analyses. High expression levels of miR-9 was demonstrated to associated with poor overall survival (OS) (HR = 2.23, 95 % CI: 1.56-3.17, P analysis showed that residence region (China and Japan), sample size, cancer type (solid or leukemia), follow-up months and analysis method (qPCR) did not alter the predictive value of miR-9 on OS in various cancers. Furthermore, no significant associations were detected for miR-9 expression and lymph node metastasis or distant metastasis. The present results suggest that promoted miR-9 expression is associated with poor OS in patients with general cancers.

  10. Human-directed social behaviour in dogs shows significant heritability.

    Science.gov (United States)

    Persson, M E; Roth, L S V; Johnsson, M; Wright, D; Jensen, P

    2015-04-01

    Through domestication and co-evolution with humans, dogs have developed abilities to attract human attention, e.g. in a manner of seeking assistance when faced with a problem solving task. The aims of this study were to investigate within breed variation in human-directed contact seeking in dogs and to estimate its genetic basis. To do this, 498 research beagles, bred and kept under standardized conditions, were tested in an unsolvable problem task. Contact seeking behaviours recorded included both eye contact and physical interactions. Behavioural data was summarized through a principal component analysis, resulting in four components: test interactions, social interactions, eye contact and physical contact. Females scored significantly higher on social interactions and physical contact and age had an effect on eye contact scores. Narrow sense heritabilities (h(2) ) of the two largest components were estimated at 0.32 and 0.23 but were not significant for the last two components. These results show that within the studied dog population, behavioural variation in human-directed social behaviours was sex dependent and that the utilization of eye contact seeking increased with age and experience. Hence, heritability estimates indicate a significant genetic contribution to the variation found in human-directed social interactions, suggesting that social skills in dogs have a genetic basis, but can also be shaped and enhanced through individual experiences. This research gives the opportunity to further investigate the genetics behind dogs' social skills, which could also play a significant part into research on human social disorders such as autism. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  11. iTRAQ Protein Profile Differential Analysis of Dormant and Germinated Grassbur Twin Seeds Reveals that Ribosomal Synthesis and Carbohydrate Metabolism Promote Germination Possibly Through the PI3K Pathway.

    Science.gov (United States)

    Zhang, Guo-Liang; Zhu, Yue; Fu, Wei-Dong; Wang, Peng; Zhang, Rui-Hai; Zhang, Yan-Lei; Song, Zhen; Xia, Gui-Xian; Wu, Jia-He

    2016-06-01

    Grassbur is a destructive and invasive weed in pastures, and its burs can cause gastric damage to animals. The strong adaptability and reproductive potential of grassbur are partly due to a unique germination mechanism whereby twin seeds develop in a single bur: one seed germinates, but the other remains dormant. To investigate the molecular mechanism of seed germination in twin seeds, we used isobaric tags for relative and absolute quantitation (iTRAQ) to perform a dynamic proteomic analysis of germination and dormancy. A total of 1,984 proteins were identified, 161 of which were considered to be differentially accumulated. The differentially accumulated proteins comprised 102 up-regulated and 59 down-regulated proteins. These proteins were grouped into seven functional categories, ribosomal proteins being the predominant group. The authenticity and accuracy of the results were confirmed by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time reverse transcription-PCR (qPCR). A dynamic proteomic analysis revealed that ribosome synthesis and carbohydrate metabolism affect seed germination possibly through the phosphoinositide 3-kinase (PI3K) pathway. As the PI3K pathway is generally activated by insulin, analyses of seeds treated with exogenous insulin by qPCR, ELISA and iTRAQ confirmed that the PI3K pathway can be activated, which suppresses dormancy and promotes germination in twin grassbur seeds. Together, these results show that the PI3K pathway may play roles in stimulating seed germination in grassbur by modulating ribosomal synthesis and carbohydrate metabolism. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  12. Quantitative analysis of bone morphogenetic protein 15 (BMP15 and growth differentiation factor 9 (GDF9 gene expression in calf and adult bovine ovaries

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    Hayashi Ken-go

    2011-03-01

    Full Text Available Abstract Background It has been reported that calf oocytes are less developmentally competent than oocytes obtained from adult cows. Bone morphogenetic protein 15 (BMP15 and growth and differentiation factor 9 (GDF9 play critical roles in folliculogenesis, follicular development and ovulation in mammalian ovaries. In the present study, we attempted to compare the expression patterns of BMP15 and GDF9 in the cells of calf and cow ovaries to determine a relationship between the level of these genes and the low developmental competence of calf oocytes. Methods Bovine tissues were collected from 9-11 months-old calves and from 4-6 years-old cows. We characterized the gene expression of BMP15 and GDF9 in calf and adult bovine oocytes and cumulus cells using quantitative real-time reverse transcriptase polymerase chain reaction (QPCR and in situ hybridization. Immunohistochemical analysis was also performed. Results The expression of BMP15 and GDF9 in cumulus cells of adult ovaries was significantly higher than that in calf ovaries, as revealed by QPCR. GDF9 expression in the oocytes of calf ovaries was significantly higher than in those of the adult ovaries. In contrast, BMP15 expression in the oocytes of calf and adult ovaries was not significantly different. The localization of gene expression and protein were ascertained by histochemistry. Conclusions Our result showed for the first time BMP15 and GDF9 expression in bovine cumulus cells. BMP15 and GDF9 mRNA expression in oocytes and cumulus cells was different in calves and cows.

  13. Specific genes involved in synthesis and editing of heparan sulfate proteoglycans show altered expression patterns in breast cancer

    International Nuclear Information System (INIS)

    Fernández-Vega, Iván; García, Olivia; Crespo, Ainara; Castañón, Sonia; Menéndez, Primitiva; Astudillo, Aurora; Quirós, Luis M

    2013-01-01

    The expression of a specific set of genes controls the different structures of heparan sulfate proteoglycans (HSPGs), which are involved in the growth, invasion and metastatic properties of cancerous cells. The purpose of this study is to increase knowledge of HSPG alterations in breast cancer. Twenty-three infiltrating ductal adenocarcinomas (IDCs), both metastatic and non-metastatic were studied. A transcriptomic approach to the structure of heparan sulfate (HS) chains was used, employing qPCR to analyze both the expression of the enzymes involved in their biosynthesis and editing, as well as the proteoglycan core proteins. Since some of these proteoglycans can also carry chondroitin sulfate chains, we extended the study to include the genes involved in the biosynthesis of these glycosaminoglycans. Histochemical techniques were also used to analyze tissular expression of particular genes showing significant expression differences, of potential interest. No significant change in transcription was detected in approximately 70% of analyzed genes. However, 13 demonstrated changes in both tumor types (40% showing more intense deregulation in the metastatic), while 5 genes showed changes only in non-metastatic tumors. Changes were related to 3 core proteins: overexpression of syndecan-1 and underexpression of glypican-3 and perlecan. HS synthesis was affected by lower levels of some 3-O-sulfotransferase transcripts, the expression of NDST4 and, only in non metastatic tumors, higher levels of extracellular sulfatases. Furthermore, the expression of chondroitin sulfate also was considerably affected, involving both the synthesis of the saccharidic chains and sulfations at all locations. However, the pro-metastatic enzyme heparanase did not exhibit significant changes in mRNA expression, although in metastatic tumors it appeared related to increased levels of the most stable form of mRNA. Finally, the expression of heparanase 2, which displays anti-metastatic features

  14. Payload specialist Reinhard Furrer show evidence of previous blood sampling

    Science.gov (United States)

    1985-01-01

    Payload specialist Reinhard Furrer shows evidence of previous blood sampling while Wubbo J. Ockels, Dutch payload specialist (only partially visible), extends his right arm after a sample has been taken. Both men show bruises on their arms.

  15. Uudised : Otsman taas Riias show'l. Rokkstaarist ministriks

    Index Scriptorium Estoniae

    2007-01-01

    Drag-kabareeartist Erkki Otsman esineb detsembris Riias "Sapnu Fabrikas" toimuval jõulu-show'l. Austraalia rokkansambli Midnight Oil endine laulja Peter Garrett nimetati valitsuse keskkonnaministriks

  16. Time dependent patient no-show predictive modelling development.

    Science.gov (United States)

    Huang, Yu-Li; Hanauer, David A

    2016-05-09

    Purpose - The purpose of this paper is to develop evident-based predictive no-show models considering patients' each past appointment status, a time-dependent component, as an independent predictor to improve predictability. Design/methodology/approach - A ten-year retrospective data set was extracted from a pediatric clinic. It consisted of 7,291 distinct patients who had at least two visits along with their appointment characteristics, patient demographics, and insurance information. Logistic regression was adopted to develop no-show models using two-thirds of the data for training and the remaining data for validation. The no-show threshold was then determined based on minimizing the misclassification of show/no-show assignments. There were a total of 26 predictive model developed based on the number of available past appointments. Simulation was employed to test the effective of each model on costs of patient wait time, physician idle time, and overtime. Findings - The results demonstrated the misclassification rate and the area under the curve of the receiver operating characteristic gradually improved as more appointment history was included until around the 20th predictive model. The overbooking method with no-show predictive models suggested incorporating up to the 16th model and outperformed other overbooking methods by as much as 9.4 per cent in the cost per patient while allowing two additional patients in a clinic day. Research limitations/implications - The challenge now is to actually implement the no-show predictive model systematically to further demonstrate its robustness and simplicity in various scheduling systems. Originality/value - This paper provides examples of how to build the no-show predictive models with time-dependent components to improve the overbooking policy. Accurately identifying scheduled patients' show/no-show status allows clinics to proactively schedule patients to reduce the negative impact of patient no-shows.

  17. Study shows women fail to land top grants

    Science.gov (United States)

    Jenner, Nicola

    2014-05-01

    Women in the UK are less successful than men at securing research council funding at almost every stage of their careers, according to an analysis published by Research Councils UK (RCUK) - the umbrella organization for the country's seven research councils.

  18. Using multiple PCR and CE with chemiluminescence detection for simultaneous qualitative and quantitative analysis of genetically modified organism.

    Science.gov (United States)

    Guo, Longhua; Qiu, Bin; Chi, Yuwu; Chen, Guonan

    2008-09-01

    In this paper, an ultrasensitive CE-CL detection system coupled with a novel double-on-column coaxial flow detection interface was developed for the detection of PCR products. A reliable procedure based on this system had been demonstrated for qualitative and quantitative analysis of genetically modified organism-the detection of Roundup Ready Soy (RRS) samples was presented as an example. The promoter, terminator, function and two reference genes of RRS were amplified with multiplex PCR simultaneously. After that, the multiplex PCR products were labeled with acridinium ester at the 5'-terminal through an amino modification and then analyzed by the proposed CE-CL system. Reproducibility of analysis times and peak heights for the CE-CL analysis were determined to be better than 0.91 and 3.07% (RSD, n=15), respectively, for three consecutive days. It was shown that this method could accurately and qualitatively detect RRS standards and the simulative samples. The evaluation in terms of quantitative analysis of RRS provided by this new method was confirmed by comparing our assay results with those of the standard real-time quantitative PCR (RT-QPCR) using SYBR Green I dyes. The results showed a good coherence between the two methods. This approach demonstrated the possibility for accurate qualitative and quantitative detection of GM plants in a single run.

  19. Entertaining politics, seriously?! : How talk show formats blur conceptual boundaries

    NARCIS (Netherlands)

    Schohaus, Birte

    2017-01-01

    What happens behind the scenes of a talk show? Why do some politicians seem to appear on every show while others are hardly ever seen? Birte Schohaus conducted a multi-layered research in which she conducted interviews with journalists, producers, PR advisors and (former) politicians and combined

  20. Effects of TV Crime Shows on Behavioural Development of Children

    Directory of Open Access Journals (Sweden)

    Abdullah Mudassar

    2017-01-01

    Full Text Available Television crime dramas and shows are very popular all over the world. This popularity is not bound to a certain age group, rather all the TV viewers like these shows very much. Like other countries, dozens of TV channels are telecasting these crime shows in Pakistan. Furthermore, few of the channels telecast crime shows at prime time which attests the popularity of such genre. Some of the media contents behave in morally disputed ways. The crime depictions as re-enactments of TV crime shows are questionable in the field of research signifying diverse cultural contexts. A large number of people are habitual to watch these shows, which may probably come out with negative behavioural outcomes. Especially the children who are at their behavioural developmental phase; are more susceptible to adopt negative behavioural leanings. In this research effort, introduction and detail of TV crime shows in Pakistan are provided, the literature concerning “media as risk factor“ in children development is discussed, and relevant theories inferences are deliberated.it was found that media has powerful role in behaviour formulating of children and violence media portrayal (TV crime shows may appear with grave concerns. Previous scientific literature was reviewed to find and discuss the problem in hand. In the research effort, the literature review provides research propositions to explore further dimensions to TV crime shows’ effects and possible negative or positive behavioural outcomes in children behaviour.

  1. The Presentation of Science in Everyday Life: The Science Show

    Science.gov (United States)

    Watermeyer, Richard

    2013-01-01

    This paper constitutes a case-study of the "science show" model of public engagement employed by a company of science communicators focused on the popularization of science, technology, engineering and mathematics (STEM) subject disciplines with learner constituencies. It examines the potential of the science show to foster the interest…

  2. "The Daily Show with Jon Stewart": Part 1

    Science.gov (United States)

    Trier, James

    2008-01-01

    Comedy Central's popular program "The Daily Show With Jon Stewart" is the best critical media literacy program on television, and it can be used in valuable ways in the classroom as part of a media literacy pedagogy. This Media Literacy column provides an overview of the show and its accompanying website and considers ways it might be used in the…

  3. The Daily Show with Jon Stewart: Part 2

    Science.gov (United States)

    Trier, James

    2008-01-01

    "The Daily Show With Jon Stewart" is one of the best critical literacy programs on television, and in this Media Literacy column the author suggests ways that teachers can use video clips from the show in their classrooms. (For Part 1, see EJ784683.)

  4. 16 CFR 5.57 - Order to show cause.

    Science.gov (United States)

    2010-01-01

    ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Order to show cause. 5.57 Section 5.57 Commercial Practices FEDERAL TRADE COMMISSION ORGANIZATION, PROCEDURES AND RULES OF PRACTICE STANDARDS OF CONDUCT Disciplinary Actions Concerning Postemployment Conflict of Interest § 5.57 Order to show cause. (a...

  5. Davedan Show Di Amphi Theatre Nusa Dua Bali

    Directory of Open Access Journals (Sweden)

    Ni Made Ruastiti

    2018-05-01

    Full Text Available Artikel ini disusun dari hasil penelitian yang bertujuan untuk dapat memahami pertunjukan Davedan Show di Amphi Theatre Nusa Dua Bali. Penelitian ini dilakukan karena adanya ketimpangan antara asumsi dan kenyataan di lapangan. Pada umumnya wisatawan yang datang ke Bali hanya senang dan antusias menonton seni pertunjukan pariwisata berbasis seni budaya lokal saja. Tetapi kenyataan ini berbeda. Walaupun Davedan Show tidak dibangun dari seni budaya lokal saja, tetapi kenyataannya wisatawan sangat senang menonton pertunjukan tersebut. Pertanyaannya: bagaimanakah bentuk pertunjukan Davedan Show tersebut?; mengapa wisatawan senang menonton pertunjukan itu?; apa implikasinya bagi pelaku, masyarakat, dan industri pariwisata di Nusa Dua, Bali?. Penelitian ini menggunakan metode penelitian kualitatif, khususnya implementatif partisipatoris yang mengutamakan kerjasama antara periset dengan para informan terkait. Sumber data penelitian ini adalah pertunjukan Davedan itu sendiri, pihak manajemen, para penari, penonton, hasil-hasil penelitian yang telah ada sebelumnya. Seluruh data yang telah dikumpulkan dengan teknik observasi, wawancara, FGD, dan studi kepustakaan itu dianalisis secara kritis dengan menggunakan teori estetika postmodern, teori praktik, dan teori relasi kuasa pengetahuan. Hasil penelitian menunjukan bahwa: (1 Davedan Show disajikan dalam bentuk oratorium. Hal itu dapat dilihat dari cara penyajian, koreografi, dan iringan pertunjukannya. Davedan Show yang menampilkan tema Treasure of The Archipelago, membuka gerbang petualangan baru itu diiringi musik rekaman etnik Nusantara secara medley, berkelanjutan dengan struktur pertunjukan: seni budaya Bali, Sumatra, Sunda, Solo, Kalimantan, dan seni budaya Papua; (2 Davedan Show banyak diminati wisatawan manca negara karena penciptaan pertunjukan itu dilatari oleh ideologi pasar, ideologi estetika, dan ideologi budaya Nusantara; (3 Hingga kini Davedan Show berkembang secara berkelanjutan di Nusa Dua

  6. QLab 3 show control projects for live performances & installations

    CERN Document Server

    Hopgood, Jeromy

    2013-01-01

    Used from Broadway to Britain's West End, QLab software is the tool of choice for many of the world's most prominent sound, projection, and integrated media designers. QLab 3 Show Control: Projects for Live Performances & Installations is a project-based book on QLab software covering sound, video, and show control. With information on both sound and video system basics and the more advanced functions of QLab such as MIDI show control, new OSC capabilities, networking, video effects, and microphone integration, each chapter's specific projects will allow you to learn the software's capabilitie

  7. marker development for two novel rice genes showing differential ...

    Indian Academy of Sciences (India)

    2014-08-19

    Aug 19, 2014 ... School of Crop Improvement, College of PostGraduate Studies, Central Agricultural University, ... from the root transcriptome data for tolerance to low P. .... Values show a representative result of three independent experiments ...

  8. Do men and women show love differently in marriage?

    Science.gov (United States)

    Schoenfeld, Elizabeth A; Bredow, Carrie A; Huston, Ted L

    2012-11-01

    In Western societies, women are considered more adept than men at expressing love in romantic relationships. Although scholars have argued that this view of love gives short shrift to men's ways of showing love (e.g., Cancian, 1986; Noller, 1996), the widely embraced premise that men and women "love differently" has rarely been examined empirically. Using data collected at four time points over 13 years of marriage, the authors examined whether love is associated with different behaviors for husbands and wives. Multilevel analyses revealed that, counter to theoretical expectations, both genders were equally likely to show love through affection. But whereas wives expressed love by enacting fewer negative or antagonistic behaviors, husbands showed love by initiating sex, sharing leisure activities, and doing household work together with their wives. Overall, the findings indicate that men and women show their love in more nuanced ways than cultural stereotypes suggest.

  9. Army Study Shows Decline In Behavioral Health Stigma

    Science.gov (United States)

    2012-01-01

    Army Study Shows Decline in Behavioral Health Stigma By Rob McIlvaine Army News Service WASHINGTON, Jan. 20, 2012 - A newly released Army study on...conference yesterday. The three-year study outlines the problem of suicide in the Army and related issues of substance abuse, spouse abuse and child abuse...REPORT TYPE 3. DATES COVERED 00-00-2012 to 00-00-2012 4. TITLE AND SUBTITLE Army Study Shows Decline In Behavioral Health Stigma 5a. CONTRACT

  10. International trade shows: Structure, strategy and performance of exhibitors at individual booths vs. joint booths

    DEFF Research Database (Denmark)

    Hansen, Kåre

    2000-01-01

    This paper examines differences in exhibitors who participate at international trade shows at joint booths and those who participate at individual booths. The structure, strategy, and trade show performance of exhibitors at joint booths and those at individual booths are analysed. The analysis...... implications for exhibitors at interna-tional trade shows and export marketing programmes and other marketing programmes offering services to international trade show exhibitors....... of exhibitors at the international food shows SIAL (Paris) and ANUGA (Cologne) showed several significant differences with regard to structure and strategy. However, no significant differences in the performance assessments between the two partici-pation modes were found. The findings have important...

  11. Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests [version 1; referees: 2 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Kevin McKernan

    2016-10-01

    Full Text Available Background: The presence of bacteria and fungi in medicinal or recreational Cannabis poses a potential threat to consumers if those microbes include pathogenic or toxigenic species. This study evaluated two widely used culture-based platforms for total yeast and mold (TYM testing marketed by 3M Corporation and Biomérieux, in comparison with a quantitative PCR (qPCR approach marketed by Medicinal Genomics Corporation. Methods: A set of 15 medicinal Cannabis samples were analyzed using 3M and Biomérieux culture-based platforms and by qPCR to quantify microbial DNA. All samples were then subjected to next-generation sequencing and metagenomics analysis to enumerate the bacteria and fungi present before and after growth on culture-based media. Results: Several pathogenic or toxigenic bacterial and fungal species were identified in proportions of >5% of classified reads on the samples, including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Ralstonia pickettii, Salmonella enterica, Stenotrophomonas maltophilia, Aspergillus ostianus, Aspergillus sydowii, Penicillium citrinum and Penicillium steckii. Samples subjected to culture showed substantial shifts in the number and diversity of species present, including the failure of Aspergillus species to grow well on either platform. Substantial growth of Clostridium botulinum and other bacteria were frequently observed on one or both of the culture-based TYM platforms. The presence of plant growth promoting (beneficial fungal species further influenced the differential growth of species in the microbiome of each sample. Conclusions: These findings have important implications for the Cannabis and food safety testing industries.

  12. Microbial Communities Show Parallels at Sites with Distinct

    Czech Academy of Sciences Publication Activity Database

    Ságová-Marečková, M.; Omelka, M.; Čermák, L.; Kameník, Zdeněk; Olšovská, J.; Hackl, E.; Kopecký, J.; Hadacek, F.

    2011-01-01

    Roč. 77, č. 21 (2011), s. 7560-7567 ISSN 0099-2240 Institutional research plan: CEZ:AV0Z50200510 Keywords : 16S RIBOSOMAL-RNA * LENGTH-POLYMORPHISM ANALYSIS * MULTIPLE SEQUENCE ALIGNMENT Subject RIV: EE - Microbiology, Virology Impact factor: 3.829, year: 2011

  13. Dolphin shows and interaction programs: benefits for conservation education?

    Science.gov (United States)

    Miller, L J; Zeigler-Hill, V; Mellen, J; Koeppel, J; Greer, T; Kuczaj, S

    2013-01-01

    Dolphin shows and dolphin interaction programs are two types of education programs within zoological institutions used to educate visitors about dolphins and the marine environment. The current study examined the short- and long-term effects of these programs on visitors' conservation-related knowledge, attitude, and behavior. Participants of both dolphin shows and interaction programs demonstrated a significant short-term increase in knowledge, attitudes, and behavioral intentions. Three months following the experience, participants of both dolphin shows and interaction programs retained the knowledge learned during their experience and reported engaging in more conservation-related behaviors. Additionally, the number of dolphin shows attended in the past was a significant predictor of recent conservation-related behavior suggesting that repetition of these types of experiences may be important in inspiring people to conservation action. These results suggest that both dolphin shows and dolphin interaction programs can be an important part of a conservation education program for visitors of zoological facilities. © 2012 Wiley Periodicals, Inc.

  14. The Last Gender Picture Show to Closure Buffy the Vampire Slayer

    Directory of Open Access Journals (Sweden)

    Sven Grampp

    2016-03-01

    Full Text Available This work presents in (and especially on one detail what it means to practice gender analysis as formal image analysis in the context of television shows. The chosen example for that is the closing scene of “Buffy the Vampire Slayer” (USA 1997-2003. This television show engages in a highly self-reflexive manner with common pop cultural representations of gender, which becomes particularly prevalent and radically condensed in the series finale.

  15. Implications of the Goal Theory on air show programs planning

    Directory of Open Access Journals (Sweden)

    Dewald Venter

    2014-01-01

    Full Text Available Events have long played an important role in human society (Shone & Parry, 2010: 3. The toils and efforts of daily lives have often been broken up by events of all kinds as humans seek an escape from the harsh reality of existence and events provide the outlet. Events are classified into four categories according to Shone and Parry (2010: 5 namely leisure (sport, recreation, personal (weddings, birthdays, cultural (art, folklore and organizational (politics, commercial. Successful events either match or exceed visitor motives and goals. It is critical that data be collected from visitors to determine their motives and goals in order to satisfy them and thereby encouraging repeat visits. One such event is the annual air show held at the Zwartkop Air Force Base (AFB in Pretoria, South Africa. Zwartkop AFB is also home to the South African Air Force (SAAF museum that also the hosts of the air show. Much of the museum‟s funds are generated through hosting the air show and sponsor contributions. Visitor goal satisfaction should therefore be of critically importance to the program planners. Military hardware has long held a fascination for those who used them and inspired the imagination of young and old. Such hardware often serves as a remembrance of times passed and as a testament to those who perished. For many visiting museums and air shows, curiosity plays a big role. The particular focus of this article will be on how the goal theory of leisure travel can be utilized by the air show organizers to enhance visitor experience to an air show.

  16. CERN cars drive by the Geneva Motor Show

    CERN Multimedia

    CERN Bulletin

    2011-01-01

    One of CERN's new gas-fuelled cars was a special guest at the press days of the Geneva motor show this year. The car enjoyed a prominent position on the Gazmobil stand, right next to the latest Mazeratis and Ferraris. Journalists previewing the motor show could discover CERN's support for green technologies and also find out more about the lab - home to the fastest racetrack on the planet, with protons in the LHC running at 99.9999991% of the speed of light.    

  17. The Biochemistry Show: a new and fun tool for learning

    Directory of Open Access Journals (Sweden)

    A.H Ono

    2006-07-01

    Full Text Available The traditional methods to teach biochemistry in most universities are based on the memorization of chemical structures,  biochemical  pathways  and  reagent  names,  which  is  many  times  dismotivating  for  the  students.  We presently describe an innovative, interactive and alternative method for teaching biochemistry to medical and nutrition undergraduate students, called the Biochemistry Show (BioBio Show.The Biobio show is based on active participation of the students. They are divided in groups and the groups face each other. One group faces another one group at a time, in a game based on true or false questions that involve subjects of applied biochemistry (exercise, obesity, diabetes, cholesterol, free radicals, among others. The questions of the Show are previously elaborated by senior students. The Biobio Show has four phases, the first one is a selection exam, and from the second to the fourth phase, eliminatory confrontations happen. On a confrontation, the first group must select a certain quantity of questions for the opponent to answer.  The group who choses the questions must know how to answer and justify the selected questions. This procedure is repeated on all phases of the show. On the last phase, the questions used are taken from an exam previously performed by the students: either the 9-hour biochemistry exam (Sé et al. A 9-hour biochemistry exam. An iron man competition or a good way of evaluating undergraduate students? SBBq 2005, abstract K-6 or the True-or-False exam (TFE (Sé et al. Are tutor-students capable of writing good biochemistry exams? SBBq 2004, abstract K-18. The winner group receives an extra 0,5 point on the final grade. Over 70% of the students informed on a questionnaire that the Biobio Show is a valuable tool for learning biochemistry.    That is a new way to enrich the discussion of biochemistry in the classroom without the students getting bored. Moreover, learning

  18. Comparative miRNA Analysis of Urine Extracellular Vesicles Isolated through Five Different Methods

    Directory of Open Access Journals (Sweden)

    Felix Royo

    2016-12-01

    Full Text Available Urine extracellular vesicles are a valuable low-invasive source of information, especially for the cells of the genitourinary tract. In the search for biomarkers, different techniques have been developed to isolate and characterize the cargo of these vesicles. In the present work, we compare five of these different isolation methods (three commercial isolation kits, ultracentrifugation, and lectin-based purification and perform miRNA profiling using a multiplex miRNA assay. The results showed high correlation through all isolation techniques, and 48 out of 68 miRNAs were detected above the detection limit at least 10 times. The results obtained by multiplex assay were validated through Taqman qPCR. In addition, using this technique combined with a clinically friendly extracellular vesicle (uEV-enrichment method, we performed the analysis of selected miRNAs in urine from patients affected with bladder cancer, benign prostate hyperplasia, or prostate cancer. Importantly, we found that those miRNAs could be detected in almost 100% of the samples, and no significant differences were observed between groups. Our results support the feasibility of analyzing exosomes-associated miRNAs using a methodology that requires a small volume of urine and is compatible with a clinical environment and high-throughput analysis.

  19. An autopsied case of tuberculous meningitis showing interesting CT findings

    International Nuclear Information System (INIS)

    Abiko, Takashi; Higuchi, Hiroshi; Imada, Ryuichi; Nagai, Kenichi

    1983-01-01

    A 61-year-old female patient died of a neurological disorder of unknown origin one month after the first visit and was found to have had tuberculous meningitis at autopsy. CT revealed a low density area showing an enlargement of the cerebral ventricle but did not reveal contrast enhancement in the basal cistern peculiar to tuberculous meningitis. (Namekawa, K.)

  20. Auditory temporal-order thresholds show no gender differences

    NARCIS (Netherlands)

    van Kesteren, Marlieke T. R.; Wierslnca-Post, J. Esther C.

    2007-01-01

    Purpose: Several studies on auditory temporal-order processing showed gender differences. Women needed longer inter-stimulus intervals than men when indicating the temporal order of two clicks presented to the left and right ear. In this study, we examined whether we could reproduce these results in

  1. Auditory temporal-order thresholds show no gender differences

    NARCIS (Netherlands)

    van Kesteren, Marlieke T R; Wiersinga-Post, J Esther C

    2007-01-01

    PURPOSE: Several studies on auditory temporal-order processing showed gender differences. Women needed longer inter-stimulus intervals than men when indicating the temporal order of two clicks presented to the left and right ear. In this study, we examined whether we could reproduce these results in

  2. An Easy Way to Show Memory Color Effects.

    Science.gov (United States)

    Witzel, Christoph

    2016-01-01

    This study proposes and evaluates a simple stimulus display that allows one to measure memory color effects (the effect of object knowledge and memory on color perception). The proposed approach is fast and easy and does not require running an extensive experiment. It shows that memory color effects are robust to minor variations due to a lack of color calibration.

  3. Polypyridyl iron(II) complexes showing remarkable photocytotoxicity ...

    Indian Academy of Sciences (India)

    aditya

    Polypyridyl iron(II) complexes showing remarkable photocytotoxicity in visible light. ADITYA GARAI a. , UTTARA BASU a. , ILA PANT b. , PATURU KONDAIAH*. ,b. AND. AKHIL R. CHAKRAVARTY*. ,a a. Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore. 560012, India. E-mail: ...

  4. Manumycin from a new Streptomyces strain shows antagonistic ...

    African Journals Online (AJOL)

    Manumycin from a new Streptomyces strain shows antagonistic effect against methicillin-resistant Staphylococcus aureus (MRSA)/vancomycin-resistant enterococci (VRE) strains from Korean Hospitals. Yun Hee Choi, Seung Sik Cho, Jaya Ram Simkhada, Chi Nam Seong, Hyo Jeong Lee, Hong Seop Moon, Jin Cheol Yoo ...

  5. Five kepler target stars that show multiple transiting exoplanet candidates

    DEFF Research Database (Denmark)

    Steffen..[], Jason H.; Batalha, N. M.; Broucki, W J.

    2010-01-01

    We present and discuss five candidate exoplanetary systems identified with the Kepler spacecraft. These five systems show transits from multiple exoplanet candidates. Should these objects prove to be planetary in nature, then these five systems open new opportunities for the field of exoplanets a...

  6. Your Town Television Show: SMART Program (Part 1) [video

    OpenAIRE

    Naval Postgraduate School, (U.S.); Sanders, John; Millsaps, Knox; Shifflett, Deborah

    2010-01-01

    From "Your Town" television show. SMART Scholarship Program featured on Your Town television program in Monterey, California. Host John Sanders, Special Collections Manager of the Naval Postgraduate School's Dudley Knox Library, interviews Dr. Knox Millsaps, Executive Agent for the SMART Program, and Deborah Shifflett, SMART Program Manager.

  7. Your Town Television Show: SMART Program (Part 3) [video

    OpenAIRE

    Naval Postgraduate School, (U.S.); Sanders, John; Millsaps, Knox; Shifflett, Deborah

    2010-01-01

    From "Your Town" television show. SMART Scholarship Program featured on Your Town television program in Monterey, California. Host John Sanders, Special Collections Manager of the Naval Postgraduate School's Dudley Knox Library, interviews Dr. Knox Millsaps, Executive Agent for the SMART Program, and Deborah Shifflett, SMART Program Manager.

  8. A Progress Evaluation of Four Bilingual Children's Television Shows.

    Science.gov (United States)

    Klein, Stephen P.; And Others

    An evaluation of a bilingual education TV series was conducted involving 6-year-old English speaking, Spanish speaking, and bilingual children at four sites. Children were assigned to control and experimental groups with the latter group seeing four 30 minute shows. A pretest-posttest design was employed with the pretest serving as the covariate…

  9. The neonicotinoid imidachloprid shows high chronic toxicity to mayfly nymphs

    NARCIS (Netherlands)

    Roessink, I.; Merga, L.B.; Zweers, A.J.; Brink, van den P.J.

    2013-01-01

    The present study evaluated the acute and chronic toxicity of imidacloprid to a range of freshwater arthropods. Mayfly and caddisfly species were most sensitive to short-term imidacloprid exposures (10 tests), whereas the mayflies showed by far the most sensitive response to long-term exposure of

  10. An Easy Way to Show Memory Color Effects

    OpenAIRE

    Witzel, Christoph

    2016-01-01

    This study proposes and evaluates a simple stimulus display that allows one to measure memory color effects (the effect of object knowledge and memory on color perception). The proposed approach is fast and easy and does not require running an extensive experiment. It shows that memory color effects are robust to minor variations due to a lack of color calibration.

  11. 36 CFR 14.24 - Showing as to citizenship required.

    Science.gov (United States)

    2010-07-01

    ... INTERIOR RIGHTS-OF-WAY Procedures § 14.24 Showing as to citizenship required. (a) Individuals. An individual applicant applying for a right-of-way under any right-of-way act, except the Act of March 3, 1891... applicant resided in the United States thereafter while a minor, should be furnished. Where the husband and...

  12. Mice lacking neuropeptide Y show increased sensitivity to cocaine

    DEFF Research Database (Denmark)

    Sørensen, Gunnar; Woldbye, David Paul Drucker

    2012-01-01

    There is increasing data implicating neuropeptide Y (NPY) in the neurobiology of addiction. This study explored the possible role of NPY in cocaine-induced behavior using NPY knockout mice. The transgenic mice showed a hypersensitive response to cocaine in three animal models of cocaine addiction...

  13. Television Judge Shows: Nordic and U.S. Perspectives

    DEFF Research Database (Denmark)

    Porsdam, Helle

    2017-01-01

    Legal discourse is language that people use in a globalizing and multicultural society to negotiate acceptable behaviors and values. We see this played out in popular cultural forums such as judicial television dramas. In the American context, television judge shows are virtually synonymous...

  14. Mixed cultures of Kimchi lactic acid bacteria show increased cell ...

    African Journals Online (AJOL)

    ufuoma

    anaerobic organisms that are highly resistant to salts. Probiotic cultures for use in ... kimchi have a superior ability to decompose and utilize nutrients, and show ... citrate, 5 g sodium acetate, 1 g Tween, 2 g K2HPO4, 0.2 g. MgSO4•7H2O, 0.2 g ...

  15. Teaching Job Interviewing Skills with the Help of Television Shows

    Science.gov (United States)

    Bloch, Janel

    2011-01-01

    Because of its potential for humor and drama, job interviewing is frequently portrayed on television. This article discusses how scenes from popular television series such as "Everybody Loves Raymond," "Friends," and "The Mary Tyler Moore Show" can be used to teach effective job interview skills in business communication courses. Television…

  16. Triphala, a formulation of traditional Ayurvedic medicine, shows

    Indian Academy of Sciences (India)

    Triphala, a formulation of traditional Ayurvedic medicine, shows protective effect against X-radiation in HeLa cells. YUKI TAKAUJI KENSUKE ... with the cellscultured in vitro. The simple bioassay system with human cultured cells would facilitate the understanding of themolecular basis for the beneficial effects of Triphala.

  17. Bilinguals Show Weaker Lexical Access during Spoken Sentence Comprehension

    Science.gov (United States)

    Shook, Anthony; Goldrick, Matthew; Engstler, Caroline; Marian, Viorica

    2015-01-01

    When bilinguals process written language, they show delays in accessing lexical items relative to monolinguals. The present study investigated whether this effect extended to spoken language comprehension, examining the processing of sentences with either low or high semantic constraint in both first and second languages. English-German…

  18. Soil bacteria show different tolerance ranges to an unprecedented disturbance

    DEFF Research Database (Denmark)

    Nunes, Ines Marques; Jurburg, Stephanie; Jacquiod, Samuel Jehan Auguste

    2018-01-01

    stress doses. FRG1, the most sensitive group, was dominated by Actinobacteria. FRG2 and FRG3, with intermediate tolerance, displayed prevalence of Proteobacteria, while FRG4, the most resistant group, was driven by Firmicutes. While the most sensitive FRGs showed predictable responses linked to changes...

  19. Genoa Boat Show – Good Example of Event Management

    Directory of Open Access Journals (Sweden)

    Dunja Demirović

    2012-07-01

    Full Text Available International Boat Show, a business and tourist event, has been held annually in Italian city of Genoa since 1962. The fair is one of the oldest, largest and best known in the field of boating industry worldwide, primarily due to good management of the event and it can serve as case study for domestic fair organizers to improve the quality of their business and services. Since Belgrade is the city of fairs, but compared to Genoa still underdeveloped in terms of trade shows, the following tasks imposed naturally in this study: to determine the relationship of the organizers of Genoa Boat Show in the sector of preparation and fair offer, in the sector of selection and communication with specific target groups (especially visitors, services during the fair and functioning of the city during the fair. During the research the authors have mostly used historical method, comparison, synthesis and the interview method. The results of theoretical research, in addition, may help not only managers of fair shows and of exhibitions, but also to organizers of other events in our country

  20. Recurrent and multiple bladder tumors show conserved expression profiles

    International Nuclear Information System (INIS)

    Lindgren, David; Fioretos, Thoas; Månsson, Wiking; Höglund, Mattias; Gudjonsson, Sigurdur; Jee, Kowan Ja; Liedberg, Fredrik; Aits, Sonja; Andersson, Anna; Chebil, Gunilla; Borg, Åke; Knuutila, Sakari

    2008-01-01

    Urothelial carcinomas originate from the epithelial cells of the inner lining of the bladder and may appear as single or as multiple synchronous tumors. Patients with urothelial carcinomas frequently show recurrences after treatment making follow-up necessary. The leading hypothesis explaining the origin of meta- and synchronous tumors assumes a monoclonal origin. However, the genetic relationship among consecutive tumors has been shown to be complex in as much as the genetic evolution does not adhere to the chronological appearance of the metachronous tumors. Consequently, genetically less evolved tumors may appear chronologically later than genetically related but more evolved tumors. Forty-nine meta- or synchronous urothelial tumors from 22 patients were analyzed using expression profiling, conventional CGH, LOH, and mutation analyses. We show by CGH that partial chromosomal losses in the initial tumors may not be present in the recurring tumors, by LOH that different haplotypes may be lost and that detected regions of LOH may be smaller in recurring tumors, and that mutations present in the initial tumor may not be present in the recurring ones. In contrast we show that despite apparent genomic differences, the recurrent and multiple bladder tumors from the same patients display remarkably similar expression profiles. Our findings show that even though the vast majority of the analyzed meta- and synchronous tumors from the same patients are not likely to have originated directly from the preceding tumor they still show remarkably similar expressions profiles. The presented data suggests that an expression profile is established early in tumor development and that this profile is stable and maintained in recurring tumors

  1. Transcriptomic Analysis of Avocado Hass (Persea americana Mill) in the Interaction System Fruit-Chitosan-Colletotrichum.

    Science.gov (United States)

    Xoca-Orozco, Luis-Ángel; Cuellar-Torres, Esther Angélica; González-Morales, Sandra; Gutiérrez-Martínez, Porfirio; López-García, Ulises; Herrera-Estrella, Luis; Vega-Arreguín, Julio; Chacón-López, Alejandra

    2017-01-01

    Avocado ( Persea americana ) is one of the most important crops in Mexico as it is the main producer, consumer, and exporter of avocado fruit in the world. However, successful avocado commercialization is often reduced by large postharvest losses due to Colletotrichum sp., the causal agent of anthracnose. Chitosan is known to have a direct antifungal effect and acts also as an elicitor capable of stimulating a defense response in plants. However, there is little information regarding the genes that are either activated or repressed in fruits treated with chitosan. The aim of this study was to identify by RNA-seq the genes differentially regulated by the action of low molecular weight chitosan in the avocado-chitosan- Colletotrichum interaction system. The samples for RNA-seq were obtained from fruits treated with chitosan, fruits inoculated with Colletotrichum and fruits both treated with chitosan and inoculated with the fungus. Non-treated and non-inoculated fruits were also analyzed. Expression profiles showed that in short times, the fruit-chitosan system presented a greater number of differentially expressed genes, compared to the fruit-pathogen system. Gene Ontology analysis of differentially expressed genes showed a large number of metabolic processes regulated by chitosan, including those preventing the spread of Colletotrichum . It was also found that there is a high correlation between the expression of genes in silico and qPCR of several genes involved in different metabolic pathways.

  2. New Inspiring Planetarium Show Introduces ALMA to the Public

    Science.gov (United States)

    2009-03-01

    As part of a wide range of education and public outreach activities for the International Year of Astronomy 2009 (IYA2009), ESO, together with the Association of French Language Planetariums (APLF), has produced a 30-minute planetarium show, In Search of our Cosmic Origins. It is centred on the global ground-based astronomical Atacama Large Millimeter/submillimeter Array (ALMA) project and represents a unique chance for planetariums to be associated with the IYA2009. ESO PR Photo 09a/09 Logo of the ALMA Planetarium Show ESO PR Photo 09b/09 Galileo's first observations with a telescope ESO PR Photo 09c/09 The ALMA Observatory ESO PR Photo 09d/09 The Milky Way band ESO PR Video 09a/09 Trailer in English ALMA is the leading telescope for observing the cool Universe -- the relic radiation of the Big Bang, and the molecular gas and dust that constitute the building blocks of stars, planetary systems, galaxies and life itself. It is currently being built in the extremely arid environment of the Chajnantor plateau, at 5000 metres altitude in the Chilean Andes, and will start scientific observations around 2011. ALMA, the largest current astronomical project, is a revolutionary telescope, comprising a state-of-the-art array of 66 giant 12-metre and 7-metre diameter antennas observing at millimetre and submillimetre wavelengths. In Search of our Cosmic Origins highlights the unprecedented window on the Universe that this facility will open for astronomers. "The show gives viewers a fascinating tour of the highest observatory on Earth, and takes them from there out into our Milky Way, and beyond," says Douglas Pierce-Price, the ALMA Public Information Officer at ESO. Edited by world fulldome experts Mirage3D, the emphasis of the new planetarium show is on the incomparable scientific adventure of the ALMA project. A young female astronomer guides the audience through a story that includes unique animations and footage, leading the viewer from the first observations by Galileo

  3. HUBBLE VISION: A Planetarium Show About Hubble Space Telescope

    Science.gov (United States)

    Petersen, Carolyn Collins

    1995-05-01

    In 1991, a planetarium show called "Hubble: Report From Orbit" outlining the current achievements of the Hubble Space Telescope was produced by the independent planetarium production company Loch Ness Productions, for distribution to facilities around the world. The program was subsequently converted to video. In 1994, that program was updated and re-produced under the name "Hubble Vision" and offered to the planetarium community. It is periodically updated and remains a sought-after and valuable resource within the community. This paper describes the production of the program, and the role of the astronomical community in the show's production (and subsequent updates). The paper is accompanied by a video presentation of Hubble Vision.

  4. Political Show-Technology in the Post-Soviet Space

    Directory of Open Access Journals (Sweden)

    O. E. Grishin

    2015-01-01

    Full Text Available In the modern political process of Russia actively used technology show. With their help, political actors can influence public opinion and shape the public interest in certain issues. In Russia, these technologies are relevant, and are especially well developed. The pressing of the problem is due to a new round of information war on the territory of Ukraine. Inclusion in the information space, and discuss the pressing issues of modern Ukraine by media people such as C. Shuster and V. Solovyov, suggests that the political show has entertainment figures, actuality, the ambiguity of the proposed conclusions. At the same time it becomes part of the information war and political reality of the normal state.

  5. Migraine patients consistently show abnormal vestibular bedside tests

    Directory of Open Access Journals (Sweden)

    Eliana Teixeira Maranhão

    2015-01-01

    Full Text Available Migraine and vertigo are common disorders, with lifetime prevalences of 16% and 7% respectively, and co-morbidity around 3.2%. Vestibular syndromes and dizziness occur more frequently in migraine patients. We investigated bedside clinical signs indicative of vestibular dysfunction in migraineurs.Objective To test the hypothesis that vestibulo-ocular reflex, vestibulo-spinal reflex and fall risk (FR responses as measured by 14 bedside tests are abnormal in migraineurs without vertigo, as compared with controls.Method Cross-sectional study including sixty individuals – thirty migraineurs, 25 women, 19-60 y-o; and 30 gender/age healthy paired controls.Results Migraineurs showed a tendency to perform worse in almost all tests, albeit only the Romberg tandem test was statistically different from controls. A combination of four abnormal tests better discriminated the two groups (93.3% specificity.Conclusion Migraine patients consistently showed abnormal vestibular bedside tests when compared with controls.

  6. Migraine patients consistently show abnormal vestibular bedside tests.

    Science.gov (United States)

    Maranhão, Eliana Teixeira; Maranhão-Filho, Péricles; Luiz, Ronir Raggio; Vincent, Maurice Borges

    2016-01-01

    Migraine and vertigo are common disorders, with lifetime prevalences of 16% and 7% respectively, and co-morbidity around 3.2%. Vestibular syndromes and dizziness occur more frequently in migraine patients. We investigated bedside clinical signs indicative of vestibular dysfunction in migraineurs. To test the hypothesis that vestibulo-ocular reflex, vestibulo-spinal reflex and fall risk (FR) responses as measured by 14 bedside tests are abnormal in migraineurs without vertigo, as compared with controls. Cross-sectional study including sixty individuals - thirty migraineurs, 25 women, 19-60 y-o; and 30 gender/age healthy paired controls. Migraineurs showed a tendency to perform worse in almost all tests, albeit only the Romberg tandem test was statistically different from controls. A combination of four abnormal tests better discriminated the two groups (93.3% specificity). Migraine patients consistently showed abnormal vestibular bedside tests when compared with controls.

  7. CT findings of rectosigmoid carcinoma showing exophytic growth

    International Nuclear Information System (INIS)

    Ohgi, Kazuyuki; Kohno, Atsushi; Higuchi, Mutsumi

    1987-01-01

    CT findings of 7 rectosigmoid carcinomas showing exophytic growth were evaluated. All cases had bulky masses, ranging from 6.0 to 11.5 cm in maximum diameter. All masses were difficult to differentiate from the other pelvic masses on CT, presumably due to their exophytic growth and/or invasion to the surrounding organs. However, 3 out of 7 cases showed diffuse rectosigmoidal wall thickening adjacent to the primary tumor, and it is considered to be valuable in the determination of primary site. All female cases had gynecological symptom such as genital bleeding, due to uterine and/or vaginal invasion. When indeterminate pelvic mass is revealed by CT, rectosigmoid carcinoma should be considered into differential diagnosis. (author)

  8. Preschoolers show less trust in physically disabled or obese informants

    Directory of Open Access Journals (Sweden)

    Sara eJaffer

    2015-01-01

    Full Text Available This research examined whether preschool-aged children show less trust in physically disabled or obese informants. In Study 1, when learning about novel physical activities and facts, 4- and 5-year-olds preferred to endorse the testimony of a physically abled, non-obese informant rather than a physically disabled or obese one. In Study 2, after seeing that the physically disabled or obese informant was previously reliable whereas the physically abled, non-obese one was unreliable, 4- and 5-year-olds did not show a significant preference for either informant. We conclude that in line with the literature on children’s negative stereotypes of physically disabled or obese others, preschoolers are biased against these individuals as potential sources of new knowledge. This bias is robust in that past reliability might undermine its effect on children, but cannot reverse it.

  9. El reality show a la hora de la merienda

    Directory of Open Access Journals (Sweden)

    Lic. Rosa María Ganga Ganga

    2000-01-01

    Full Text Available Los programas de testimonio, inscritos dentro del género televisivo del Reality Show, son una variante del más amplio subgénero del Talk Show y tienen ya una cierta tradición en nuestro país. El presente trabajo se centrará en este tipo de programas de testimonio que basan su estrategia discursiva en la presentación y representación del relato autobiográfico del hombre o la mujer anónimos, integrándose de esta forma en las corrientes más recientes de la sociología y la historiografía, y persigue esclarecer algunas de sus características y funciones, especialmente su función socializadora, a través del mecanismo biográfico y del concepto de habitus tomado de Pierre Bourdieu.

  10. High-frequency parameters of magnetic films showing magnetization dispersion

    International Nuclear Information System (INIS)

    Sidorenkov, V.V.; Zimin, A.B.; Kornev, Yu.V.

    1988-01-01

    Magnetization dispersion leads to skewed resonance curves shifted towards higher magnetizing fields, together with considerable reduction in the resonant absorption, while the FMR line width is considerably increased. These effects increase considerably with frequency, in contrast to films showing magnetic-anisotropy dispersion, where they decrease. It is concluded that there may be anomalies in the frequency dependence of the resonance parameters for polycrystalline magnetic films

  11. Persistent cannabis users show neuropsychological decline from childhood to midlife

    OpenAIRE

    Meier, Madeline H.; Caspi, Avshalom; Ambler, Antony; Harrington, HonaLee; Houts, Renate; Keefe, Richard S. E.; McDonald, Kay; Ward, Aimee; Poulton, Richie; Moffitt, Terrie E.

    2012-01-01

    Recent reports show that fewer adolescents believe that regular cannabis use is harmful to health. Concomitantly, adolescents are initiating cannabis use at younger ages, and more adolescents are using cannabis on a daily basis. The purpose of the present study was to test the association between persistent cannabis use and neuropsychological decline and determine whether decline is concentrated among adolescent-onset cannabis users. Participants were members of the Dunedin Study, a prospecti...

  12. Bonobos (Pan paniscus) show an attentional bias toward conspecifics' emotions.

    Science.gov (United States)

    Kret, Mariska E; Jaasma, Linda; Bionda, Thomas; Wijnen, Jasper G

    2016-04-05

    In social animals, the fast detection of group members' emotional expressions promotes swift and adequate responses, which is crucial for the maintenance of social bonds and ultimately for group survival. The dot-probe task is a well-established paradigm in psychology, measuring emotional attention through reaction times. Humans tend to be biased toward emotional images, especially when the emotion is of a threatening nature. Bonobos have rich, social emotional lives and are known for their soft and friendly character. In the present study, we investigated (i) whether bonobos, similar to humans, have an attentional bias toward emotional scenes compared with conspecifics showing a neutral expression, and (ii) which emotional behaviors attract their attention the most. As predicted, results consistently showed that bonobos' attention was biased toward the location of the emotional versus neutral scene. Interestingly, their attention was grabbed most by images showing conspecifics such as sexual behavior, yawning, or grooming, and not as much-as is often observed in humans-by signs of distress or aggression. The results suggest that protective and affiliative behaviors are pivotal in bonobo society and therefore attract immediate attention in this species.

  13. An Undergraduate Endeavor: Assembling a Live Planetarium Show About Mars

    Science.gov (United States)

    McGraw, Allison M.

    2016-10-01

    Viewing the mysterious red planet Mars goes back thousands of years with just the human eye but in more recent years the growth of telescopes, satellites and lander missions unveil unrivaled detail of the Martian surface that tells a story worth listening to. This planetarium show will go through the observations starting with the ancients to current understandings of the Martian surface, atmosphere and inner-workings through past and current Mars missions. Visual animations of its planetary motions, display of high resolution images from the Hi-RISE (High Resolution Imaging Science Experiment) and CTX (Context Camera) data imagery aboard the MRO (Mars Reconnaissance Orbiter) as well as other datasets will be used to display the terrain detail and imagery of the planet Mars with a digital projection system. Local planetary scientists and Mars specialists from the Lunar and Planetary Lab at the University of Arizona (Tucson, AZ) will be interviewed and used in the show to highlight current technology and understandings of the red planet. This is an undergraduate project that is looking for collaborations and insight in order gain structure in script writing that will teach about this planetary body to all ages in the format of a live planetarium show.

  14. AirShow 1.0 CFD Software Users' Guide

    Science.gov (United States)

    Mohler, Stanley R., Jr.

    2005-01-01

    AirShow is visualization post-processing software for Computational Fluid Dynamics (CFD). Upon reading binary PLOT3D grid and solution files into AirShow, the engineer can quickly see how hundreds of complex 3-D structured blocks are arranged and numbered. Additionally, chosen grid planes can be displayed and colored according to various aerodynamic flow quantities such as Mach number and pressure. The user may interactively rotate and translate the graphical objects using the mouse. The software source code was written in cross-platform Java, C++, and OpenGL, and runs on Unix, Linux, and Windows. The graphical user interface (GUI) was written using Java Swing. Java also provides multiple synchronized threads. The Java Native Interface (JNI) provides a bridge between the Java code and the C++ code where the PLOT3D files are read, the OpenGL graphics are rendered, and numerical calculations are performed. AirShow is easy to learn and simple to use. The source code is available for free from the NASA Technology Transfer and Partnership Office.

  15. Five Kepler target stars that show multiple transiting exoplanet candidates

    Energy Technology Data Exchange (ETDEWEB)

    Steffen, Jason H.; /Fermilab; Batalha, Natalie M.; /San Jose State U.; Borucki, William J.; /NASA, Ames; Buchhave, Lars A.; /Harvard-Smithsonian Ctr. Astrophys. /Bohr Inst.; Caldwell, Douglas A.; /NASA, Ames /SETI Inst., Mtn. View; Cochran, William D.; /Texas U.; Endl, Michael; /Texas U.; Fabrycky, Daniel C.; /Harvard-Smithsonian Ctr. Astrophys.; Fressin, Francois; /Harvard-Smithsonian Ctr. Astrophys.; Ford, Eric B.; /Florida U.; Fortney, Jonathan J.; /UC, Santa Cruz, Phys. Dept. /NASA, Ames

    2010-06-01

    We present and discuss five candidate exoplanetary systems identified with the Kepler spacecraft. These five systems show transits from multiple exoplanet candidates. Should these objects prove to be planetary in nature, then these five systems open new opportunities for the field of exoplanets and provide new insights into the formation and dynamical evolution of planetary systems. We discuss the methods used to identify multiple transiting objects from the Kepler photometry as well as the false-positive rejection methods that have been applied to these data. One system shows transits from three distinct objects while the remaining four systems show transits from two objects. Three systems have planet candidates that are near mean motion commensurabilities - two near 2:1 and one just outside 5:2. We discuss the implications that multitransiting systems have on the distribution of orbital inclinations in planetary systems, and hence their dynamical histories; as well as their likely masses and chemical compositions. A Monte Carlo study indicates that, with additional data, most of these systems should exhibit detectable transit timing variations (TTV) due to gravitational interactions - though none are apparent in these data. We also discuss new challenges that arise in TTV analyses due to the presence of more than two planets in a system.

  16. Compliance With Recommended Food Safety Practices in Television Cooking Shows.

    Science.gov (United States)

    Cohen, Nancy L; Olson, Rita Brennan

    Examine compliance with recommended food safety practices in television cooking shows. Using a tool based on the Massachusetts Food Establishment Inspection Report, raters examined 39 episodes from 10 television cooking shows. Chefs demonstrated conformance with good retail practices for proper use and storage of utensils in 78% of episodes; preventing contamination (62%), and fingernail care (82%). However, 50% to 88% of episodes were found to be out of compliance with other personal hygiene practices, proper use of gloves and barriers (85% to 100%), and maintaining proper time and temperature controls (93%). Over 90% failed to conform to recommendations regarding preventing contamination through wiping cloths and washing produce. In only 13% of episodes were food safety practices mentioned. There appears to be little attention to food safety during most cooking shows. Celebrity and competing chefs have the opportunity to model and teach good food safety practices for millions of viewers. Copyright © 2016 Society for Nutrition Education and Behavior. Published by Elsevier Inc. All rights reserved.

  17. Architectures for Quantum Simulation Showing a Quantum Speedup

    Science.gov (United States)

    Bermejo-Vega, Juan; Hangleiter, Dominik; Schwarz, Martin; Raussendorf, Robert; Eisert, Jens

    2018-04-01

    One of the main aims in the field of quantum simulation is to achieve a quantum speedup, often referred to as "quantum computational supremacy," referring to the experimental realization of a quantum device that computationally outperforms classical computers. In this work, we show that one can devise versatile and feasible schemes of two-dimensional, dynamical, quantum simulators showing such a quantum speedup, building on intermediate problems involving nonadaptive, measurement-based, quantum computation. In each of the schemes, an initial product state is prepared, potentially involving an element of randomness as in disordered models, followed by a short-time evolution under a basic translationally invariant Hamiltonian with simple nearest-neighbor interactions and a mere sampling measurement in a fixed basis. The correctness of the final-state preparation in each scheme is fully efficiently certifiable. We discuss experimental necessities and possible physical architectures, inspired by platforms of cold atoms in optical lattices and a number of others, as well as specific assumptions that enter the complexity-theoretic arguments. This work shows that benchmark settings exhibiting a quantum speedup may require little control, in contrast to universal quantum computing. Thus, our proposal puts a convincing experimental demonstration of a quantum speedup within reach in the near term.

  18. Eccentric muscle challenge shows osteopontin polymorphism modulation of muscle damage.

    Science.gov (United States)

    Barfield, Whitney L; Uaesoontrachoon, Kitipong; Wu, Chung-Sheih; Lin, Stephen; Chen, Yue; Wang, Paul C; Kanaan, Yasmine; Bond, Vernon; Hoffman, Eric P

    2014-08-01

    A promoter polymorphism of the osteopontin (OPN) gene (rs28357094) has been associated with multiple inflammatory states, severity of Duchenne muscular dystrophy (DMD) and muscle size in healthy young adults. We sought to define the mechanism of action of the polymorphism, using allele-specific in vitro reporter assays in muscle cells, and a genotype-stratified intervention in healthy controls. In vitro reporter constructs showed the G allele to respond to estrogen treatment, whereas the T allele showed no transcriptional response. Young adult volunteers (n = 187) were enrolled into a baseline study, and subjects with specific rs28357094 genotypes enrolled into an eccentric muscle challenge intervention [n = 3 TT; n = 3 GG/GT (dominant inheritance model)]. Female volunteers carrying the G allele showed significantly greater inflammation and increased muscle volume change as determined by magnetic resonance imaging T1- and T2-weighted images after eccentric challenge, as well as greater decrement in biceps muscle force. Our data suggest a model where the G allele enables enhanced activities of upstream enhancer elements due to loss of Sp1 binding at the polymorphic site. This results in significantly greater expression of the pro-inflammatory OPN cytokine during tissue remodeling in response to challenge in G allele carriers, promoting muscle hypertrophy in normal females, but increased damage in DMD patients. © The Author 2014. Published by Oxford University Press.

  19. Radon in Austrian tourist mines and show caves

    International Nuclear Information System (INIS)

    Ringer, W.; Graeser, J.

    2009-01-01

    The radon situation in tourist mines and show caves is barely investigated in Austria. This paper investigates the influence of its determining factors, such as climate, structure and geology. For this purpose, long-term time-resolved measurements over 6 to 12 months in 4 tourist mines and 2 show caves - with 5 to 9 measuring points each - have been carried out to obtain the course of radon concentration throughout the year. In addition, temperature and air-pressure were measured and compared to the data outside where available. Results suggest that the dominating factors of the average radon concentration are structure and location (geology) of the tunnel-system, whereas the diurnal and annual variation is mainly caused by the changing airflow, which is driven by the difference in temperature inside and outside. Downcast air is connected with very low radon concentrations, upcast air with high concentrations. In some locations the maximum values appear when the airflow ceases. But airflow can be different in different parts of mines and caves. Systems close to the surface show generally lower radon levels than the ones located deeper underground. Due to variation of structure, geology and local climate, the radon situation in mines and caves can only be described by simultaneous measurements at several measuring points. (orig.)

  20. Paks shows the way towards good operating practices

    International Nuclear Information System (INIS)

    Anon.

    1989-01-01

    The Paks-3 unit in Hungary was the first VVER (Soviet designed Pressurized Water Reactor) to be scrutinized by an International Atomic Energy Agency Operational Safety Analysis Review Team. A number of examples of good operational practice were noted. Those reported here include the cleanliness of the plant, the management attitude to training, early detection of and action to correct problems as they arise, an accident avoidance policy, a back-up research and development programme, and the provision of computer-based assistance to the operator to present operational data in an easily comprehensible form. (U.K.)

  1. Showing/Sharing: Analysing Visual Communication from a Praxeological Perspective

    Directory of Open Access Journals (Sweden)

    Maria Schreiber

    2017-12-01

    Full Text Available This contribution proposes a methodological framework for empirical research into visual practices on social media. The framework identifies practices, pictures and platforms as relevant dimensions of analysis. It is mainly developed within, and is compatible with qualitative, interpretive approaches which focus on visual communication as part of everyday personal communicative practices. Two screenshots from Instagram and Facebook are introduced as empirical examples to investigate collaborative practices of meaning-making relating to pictures on social media. While social media seems to augment reflexive, processual practices of negotiating identities, visual media, in particular, amps up aesthetic, ambivalent and embodied dimensions within these practices.

  2. Remembering Operación Triunfo: a Latin Music Reality Show in the Era of Talent Shows

    NARCIS (Netherlands)

    Savini, Paola

    2016-01-01

    abstractThe music format Operación Triunfo (2001–2011), which aired on RTVE for the first time in 2001, started as a television (TV) and musical success in Spain and today is one of the most famous shows around the world as well as an incredible socio-economic phenomenon in Spanish TV. This paper

  3. Comportamento agressivo em shows musicais: analisando notícias de jornal impresso

    Directory of Open Access Journals (Sweden)

    Carlos Eduardo Pimentel

    2011-08-01

    Full Text Available This paper reports a content analysis of the newspaper articles dealing with aggressive behavior in music shows. This analysis permits an understanding of the characteristics of these situations, the aggressive behaviors in shows, and the way in which the print media treat the subject. Despite of the newspapers’ influence on public opinion formation, that is, the transmission of attitudes and behavior repertoires, this kind of analysis is rarely reported in Brazilian social psychological literature. Some 31 newspaper articles were content analyzed as to aggressive behavior during music shows. Results indicate that aggressive behaviors occur in shows of most varied musical styles. Results are discussed on the basis of social cognitive theory.

  4. Integrative Analysis of Disease Signatures Shows Inflammation Disrupts Juvenile Experience-Dependent Cortical Plasticity

    Science.gov (United States)

    Smith, Milo R.; Burman, Poromendro

    2016-01-01

    Throughout childhood and adolescence, periods of heightened neuroplasticity are critical for the development of healthy brain function and behavior. Given the high prevalence of neurodevelopmental disorders, such as autism, identifying disruptors of developmental plasticity represents an essential step for developing strategies for prevention and intervention. Applying a novel computational approach that systematically assessed connections between 436 transcriptional signatures of disease and multiple signatures of neuroplasticity, we identified inflammation as a common pathological process central to a diverse set of diseases predicted to dysregulate plasticity signatures. We tested the hypothesis that inflammation disrupts developmental cortical plasticity in vivo using the mouse ocular dominance model of experience-dependent plasticity in primary visual cortex. We found that the administration of systemic lipopolysaccharide suppressed plasticity during juvenile critical period with accompanying transcriptional changes in a particular set of molecular regulators within primary visual cortex. These findings suggest that inflammation may have unrecognized adverse consequences on the postnatal developmental trajectory and indicate that treating inflammation may reduce the burden of neurodevelopmental disorders. PMID:28101530

  5. A micro-epidemiological analysis of febrile malaria in Coastal Kenya showing hotspots within hotspots

    NARCIS (Netherlands)

    Bejon, P.; Williams, T.N.; Nyundo, C.; Hay, S.I.; Benz, D.; Gething, P.W.; Otiende, M.; Peshu, J.; Bashraheil, M.; Greenhouse, B.; Bousema, T.; Bauni, E.; Marsh, K.; Smith, D.L.; Borrmann, S.

    2014-01-01

    Malaria transmission is spatially heterogeneous. This reduces the efficacy of control strategies, but focusing control strategies on clusters or 'hotspots' of transmission may be highly effective. Among 1500 homesteads in coastal Kenya we calculated (a) the fraction of febrile children with positive

  6. Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water

    Energy Technology Data Exchange (ETDEWEB)

    Riback, Joshua A.; Bowman, Micayla A.; Zmyslowski, Adam M.; Knoverek, Catherine R.; Jumper, John M.; Hinshaw, James R.; Kaye, Emily B.; Freed, Karl F.; Clark, Patricia L.; Sosnick, Tobin R.

    2017-10-12

    A substantial fraction of the proteome is intrinsically disordered, and even well-folded proteins adopt non-native geometries during synthesis, folding, transport, and turnover. Characterization of intrinsically disordered proteins (IDPs) is challenging, in part because of a lack of accurate physical models and the difficulty of interpreting experimental results. We have developed a general method to extract the dimensions and solvent quality (self-interactions) of IDPs from a single small-angle x-ray scattering measurement. We applied this procedure to a variety of IDPs and found that even IDPs with low net charge and high hydrophobicity remain highly expanded in water, contrary to the general expectation that protein-like sequences collapse in water. Our results suggest that the unfolded state of most foldable sequences is expanded; we conjecture that this property was selected by evolution to minimize misfolding and aggregation.

  7. Gene-based interaction analysis shows GABAergic genes interacting with parenting in adolescent depressive symptoms

    NARCIS (Netherlands)

    Van Assche, Evelien; Moons, Tim; Cinar, Ozan; Viechtbauer, Wolfgang; Oldehinkel, Albertine J.; Van Leeuwen, Karla; Verschueren, Karine; Colpin, Hilde; Lambrechts, Diether; Van den Noortgate, Wim; Goossens, Luc; Claes, Stephan; van Winkel, Ruud

    2017-01-01

    BACKGROUND: Most gene-environment interaction studies (G × E) have focused on single candidate genes. This approach is criticized for its expectations of large effect sizes and occurrence of spurious results. We describe an approach that accounts for the polygenic nature of most psychiatric

  8. Latent class analysis shows strong heritability of the Child Behavior Checklist-Juvenile Bipolar Phenotype.

    NARCIS (Netherlands)

    Althoff, R.; Rettew, D.C.; Faraone, S.V.; Boomsma, D.I.; Hudziak, J.

    2006-01-01

    Background: The Child Behavior Checklist (CBCL) has been used to provide a quantitative description of childhood bipolar disorder (BPAD). Many have reported that children in the clinical range on the Attention Problems (AP), Aggressive Behavior (AGG), and Anxious-Depressed (A/D) syndromes

  9. Gene-set analysis shows association between FMRP targets and autism spectrum disorder

    NARCIS (Netherlands)

    Jansen, Arija; Dieleman, Gwen C; Smit, August B; Verhage, Matthijs; Verhulst, Frank C; Polderman, Tinca J C; Posthuma, Danielle

    Autism spectrum disorder (ASD) is a heterogeneous group of disorders characterized by problems with social interaction, communication, and repetitive and restricted behavior. Despite its high heritability and the substantial progress made in elucidating genetic associations, the corresponding

  10. ye movement parameters while reading show cognitive processes of structural analysis of written speech

    Directory of Open Access Journals (Sweden)

    Latanov, Alexander V.

    2016-06-01

    Full Text Available This paper gives an overview of the published data on eye movement parameters while reading sentences in different languages with both local and global syntactic ambiguity. A locally ambiguous sentence contains a syntactically problematic phrase that leads to only one interpretation, while a globally ambiguous sentence has more than one distinct interpretation. In the first case the ambiguity persists only to the end of the sentence, when it is successfully resolved; in the second case the ambiguity is still present after reading the whole sentence. The obvious difficulty in analyzing the structure of locally and globally ambiguous sentences leads to increased reading time compared with unambiguous sentences. The syntactic ambiguity increases two major parameters: the fixation duration when reading words critical for interpreting the sentence, and the frequency of regressive saccades to reread those words. The reading time for critical words, disambiguating the local ambiguity, depends on the principle of early/late closure (i.e., high/low attachment: preferring a recurrent pattern to associate the critical word with a distant or closer word, respectively (as determined by its position in the sentence, and differs across languages. The first study of eye movement parameters in reading globally syntactic ambiguous sentences in the Russian language is reported in this paper. Our findings open up the prospects of quantitative studies of syntactic disambiguation in Slavonic and Romano-Germanic languages.

  11. "Let's Get This Party Started!": An Analysis of Health Risk Behavior on MTV Reality Television Shows.

    Science.gov (United States)

    Flynn, Mark A; Morin, David; Park, Sung-Yeon; Stana, Alexandru

    2015-01-01

    Past research has examined portrayals of risk behavior in various media, including television, advertising, and film. To address an underexplored area, this study analyzed drinking, smoking, and sexual activities in MTV reality programming popular among adolescent viewers from 2004 to 2011. Cast members' demographic attributes were also examined in relation to their risk behaviors. Results demonstrated that drinking and casual sexual behaviors were pervasive among cast members. Smoking and more intense sexual behaviors were also present, but to a smaller degree. Men and young adult cast members were more likely to engage in risk behaviors than women and teenage cast members. Also, ethnic/racial minority characters were shown drinking more often than were White cast members. Interpretations of these findings are discussed based in social cognitive theory and the concept of super peers. Implications for future research are provided.

  12. Perforating pilomatrixoma showing atypical presentation: A rare clinical variant

    Directory of Open Access Journals (Sweden)

    Nevra Seyhan

    2018-03-01

    Full Text Available Pilomatrixoma, also known as calcifying epithelioma of Malherbe, is a rare benign skin tumor arising from hair follicle stem cells. The most common localization is the head and neck region. Female/male ratio is 3/2. It shows deep subcutaneous placement and occurs in the first two decades of life. Its diameter ranges from 0.5 cm to 3 cm. Multiple lesions are rarely seen. Histopathologically it is characterized by basoloid and ghost cells. Perforating type is a rare clinical variant. Treatment is surgical excision. Our case is presented to draw attention to a rare clinical variant of pilomatrixioma.

  13. PROTOTIPE VIDEO EDITOR DENGAN MENGGUNAKAN DIRECT X DAN DIRECT SHOW

    Directory of Open Access Journals (Sweden)

    Djoni Haryadi Setiabudi

    2004-01-01

    Full Text Available Technology development had given people the chance to capture their memorable moments in video format. A high quality digital video is a result of a good editing process. Which in turn, arise the new need of an editor application. In accordance to the problem, here the process of making a simple application for video editing needs. The application development use the programming techniques often applied in multimedia applications, especially video. First part of the application will begin with the video file compression and decompression, then we'll step into the editing part of the digital video file. Furthermore, the application also equipped with the facilities needed for the editing processes. The application made with Microsoft Visual C++ with DirectX technology, particularly DirectShow. The application provides basic facilities that will help the editing process of a digital video file. The application will produce an AVI format file after the editing process is finished. Through the testing process of this application shows the ability of this application to do the 'cut' and 'insert' of video files in AVI, MPEG, MPG and DAT formats. The 'cut' and 'insert' process only can be done in static order. Further, the aplication also provide the effects facility for transition process in each clip. Lastly, the process of saving the new edited video file in AVI format from the application. Abstract in Bahasa Indonesia : Perkembangan teknologi memberi kesempatan masyarakat untuk mengabadikan saat - saat yang penting menggunakan video. Pembentukan video digital yang baik membutuhkan proses editing yang baik pula. Untuk melakukan proses editing video digital dibutuhkan program editor. Berdasarkan permasalahan diatas maka pada penelitian ini dibuat prototipe editor sederhana untuk video digital. Pembuatan aplikasi memakai teknik pemrograman di bidang multimedia, khususnya video. Perencanaan dalam pembuatan aplikasi tersebut dimulai dengan pembentukan

  14. Pseudo Random Coins Show More Heads Than Tails

    OpenAIRE

    Bauke, Heiko; Mertens, Stephan

    2003-01-01

    Tossing a coin is the most elementary Monte Carlo experiment. In a computer the coin is replaced by a pseudo random number generator. It can be shown analytically and by exact enumerations that popular random number generators are not capable of imitating a fair coin: pseudo random coins show more heads than tails. This bias explains the empirically observed failure of some random number generators in random walk experiments. It can be traced down to the special role of the value zero in the ...

  15. Model shows future cut in U.S. ozone levels

    International Nuclear Information System (INIS)

    Anon.

    1991-01-01

    A joint U.S. auto-oil industry research program says modeling shows that changing gasoline composition can reduce ozone levels for Los Angeles in 2010 and for New York City and Dallas-Fort Worth in 2005. The air quality modeling was based on vehicle emissions research data released late last year (OGJ, Dec. 24, 1990, p. 20). The effort is sponsored by the big three auto manufacturers and 14 oil companies. Sponsors the cars and small trucks account for about one third of ozone generated in the three cities studied but by 2005-10 will account for only 5-9%

  16. The use of computerized tomography in patients showing tardive dyskinesia

    International Nuclear Information System (INIS)

    Themelis, I.

    1983-01-01

    29 patients showing moderate to markedly pronounced tardive dyskinesia (TD) and a further 29 control patients (C) under a similar long-term medication with neuroleptics that had been so chosen as to match the age and sex distributions of the former group were subjected to computered tomography, neurological examination and psychological testing. The results did not point to any correlations between the structural changes and duration of treatment and the clinical signs or symptoms of extrapyramidal disorder. This was taken as further evidence in support of the theory that the initial damage in tardive dyskinesia mainly is at the level of the basal ganglia. (orig./MG) [de

  17. DIME Students Show Off their Lego(TM) Challenge Creation

    Science.gov (United States)

    2002-01-01

    Two students show the Lego (TM) Challenge device they designed and built to operate in the portable drop tower demonstrator as part of the second Dropping in a Microgravity Environment (DIME) competition held April 23-25, 2002, at NASA's Glenn Research Center. Competitors included two teams from Sycamore High School, Cincinnati, OH, and one each from Bay High School, Bay Village, OH, and COSI Academy, Columbus, OH. DIME is part of NASA's education and outreach activities. Details are on line at http://microgravity.grc.nasa.gov/DIME_2002.html.

  18. Economics show CO2 EOR potential in central Kansas

    Science.gov (United States)

    Dubois, M.K.; Byrnes, A.P.; Pancake, R.E.; Willhite, G.P.; Schoeling, L.G.

    2000-01-01

    Carbon dioxide (CO2) enhanced oil recovery (EOR) may be the key to recovering hundreds of millions of bbl of trapped oil from the mature fields in central Kansas. Preliminary economic analysis indicates that CO2 EOR should provide an internal rate of return (IRR) greater than 20%, before income tax, assuming oil sells for \\$20/bbl, CO2 costs \\$1/Mcf, and gross utilization is 10 Mcf of CO2/bbl of oil recovered. If the CO2 cost is reduced to \\$0.75/Mcf, an oil price of $17/bbl yields an IRR of 20%. Reservoir and economic modeling indicates that IRR is most sensitive to oil price and CO2 cost. A project requires a minimum recovery of 1,500 net bbl/acre (about 1 million net bbl/1-mile section) under a best-case scenario. Less important variables to the economics are capital costs and non-CO2 related lease operating expenses.

  19. Herbarium specimens show contrasting phenological responses to Himalayan climate.

    Science.gov (United States)

    Hart, Robbie; Salick, Jan; Ranjitkar, Sailesh; Xu, Jianchu

    2014-07-22

    Responses by flowering plants to climate change are complex and only beginning to be understood. Through analyses of 10,295 herbarium specimens of Himalayan Rhododendron collected by plant hunters and botanists since 1884, we were able to separate these responses into significant components. We found a lack of directional change in mean flowering time over the past 45 y of rapid warming. However, over the full 125 y of collections, mean flowering time shows a significant response to year-to-year changes in temperature, and this response varies with season of warming. Mean flowering advances with annual warming (2.27 d earlier per 1 °C warming), and also is delayed with fall warming (2.54 d later per 1 °C warming). Annual warming may advance flowering through positive effects on overwintering bud formation, whereas fall warming may delay flowering through an impact on chilling requirements. The lack of a directional response suggests that contrasting phenological responses to temperature changes may obscure temperature sensitivity in plants. By drawing on large collections from multiple herbaria, made over more than a century, we show how these data may inform studies even of remote localities, and we highlight the increasing value of these and other natural history collections in understanding long-term change.

  20. [How children show positive and negative relationships on their drawings].

    Science.gov (United States)

    Gramel, Sabine

    2005-01-01

    This study analyses, whether pictures of children showing a positive relationship are significantly different from those showing a negative one with respect to several criteria. The study involved a random selection of 45 children aged 4;6 to 11;6 years. The children painted a picture with themselves and a person they liked and a picture of themselves with someone they disliked. For the most part, the children drew pictures of themselves with peers both with respect to positive as well as negative images. In an interview afterwards, the children specified the criteria in their drawings by which the quality of the particular relationship can be identified. Positive and negative relationship paintings differ in the character of activity described. The sun as an element in children's paintings is painted not more frequent on positive compared to negative pictures. The colour black is used more often in the drawings signifying negative relationships. While girls used more colour in negative relationship drawings, boys used more colour in the positive ones. There was no significant difference in the use of favourite colours and decorative elements between the two groups. Only in negative relationship drawings people were looking away from each other. Smiling individuals were more common in the positive relationship pictures and in pictures painted by the 6 to 8 year olds. A greater distance between the individuals emerged on negative relationship drawings of the girls.

  1. Double polymer sheathed carbon nanotube supercapacitors show enhanced cycling stability

    Science.gov (United States)

    Zhao, Wenqi; Wang, Shanshan; Wang, Chunhui; Wu, Shiting; Xu, Wenjing; Zou, Mingchu; Ouyang, An; Cao, Anyuan; Li, Yibin

    2015-12-01

    Pseudo-materials are effective in boosting the specific capacitance of supercapacitors, but during service their degradation may also be very strong, causing reduced cycling stability. Here, we show that a carbon nanotube sponge grafted by two conventional pseudo-polymer layers in sequence can serve as a porous supercapacitor electrode with significantly enhanced cycling stability compared with single polymer grafting. Creating conformal polymer coatings on the nanotube surface and the resulting double-sheath configuration are important structural factors leading to the enhanced performance. Combining different polymers as double sheaths as reported here might be a potential route to circumvent the dilemma of pseudo-materials, and to simultaneously improve the capacitance and stability for various energy storage devices.Pseudo-materials are effective in boosting the specific capacitance of supercapacitors, but during service their degradation may also be very strong, causing reduced cycling stability. Here, we show that a carbon nanotube sponge grafted by two conventional pseudo-polymer layers in sequence can serve as a porous supercapacitor electrode with significantly enhanced cycling stability compared with single polymer grafting. Creating conformal polymer coatings on the nanotube surface and the resulting double-sheath configuration are important structural factors leading to the enhanced performance. Combining different polymers as double sheaths as reported here might be a potential route to circumvent the dilemma of pseudo-materials, and to simultaneously improve the capacitance and stability for various energy storage devices. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05978j

  2. Ovalbumin with Glycated Carboxyl Groups Shows Membrane-Damaging Activity

    Directory of Open Access Journals (Sweden)

    Ching-Chia Tang

    2017-02-01

    Full Text Available The aim of the present study was to investigate whether glycated ovalbumin (OVA showed novel activity at the lipid-water interface. Mannosylated OVA (Man-OVA was prepared by modification of the carboxyl groups with p-aminophenyl α-dextro (d-mannopyranoside. An increase in the number of modified carboxyl groups increased the membrane-damaging activity of Man-OVA on cell membrane-mimicking vesicles, whereas OVA did not induce membrane permeability in the tested phospholipid vesicles. The glycation of carboxyl groups caused a notable change in the gross conformation of OVA. Moreover, owing to their spatial positions, the Trp residues in Man-OVA were more exposed, unlike those in OVA. Fluorescence quenching studies suggested that the Trp residues in Man-OVA were located on the interface binds with the lipid vesicles, and their microenvironment was abundant in positively charged residues. Although OVA and Man-OVA showed a similar binding affinity for lipid vesicles, the lipid-interacting feature of Man-OVA was distinct from that of OVA. Chemical modification studies revealed that Lys and Arg residues, but not Trp residues, played a crucial role in the membrane-damaging activity of Man-OVA. Taken together, our data suggest that glycation of carboxyl groups causes changes in the structural properties and membrane-interacting features of OVA, generating OVA with membrane-perturbing activities at the lipid-water interface.

  3. Fish kidney cells show higher tolerance to hyperosmolality than amphibian

    Directory of Open Access Journals (Sweden)

    Lang Gui

    2018-05-01

    Full Text Available In contrast to fish, amphibians inhabit both aquatic and terrestrial environments. To better understand osmoregulation in fish and amphibian, we have investigated the morphological changes in kidney cells to osmotic stress. To address this, kidney cell line isolated from the freshwater grass carp (CIK and Chinese giant salamander (GSK were challenged to different mediums with distinct osmotic pressures (100, 300 and 700 mOsm. Morphological alterations of the fish and amphibian cells were compared by optical and electron microscopy. Following hyposmotic treatment (100 mOsm, both CIK and GSK cells became unhealthy and show condensed chromatin, swollen mitochondria and cytoplasmic vacuole. Meanwhile, after hyperosmotic treatment (700 mOsm, shrunken CIK cells with multipolar shape, pale or lightly stained cytoplasm, condensed chromatin, vacuoles and swollen mitochondria were detected. GSK cells were seriously damaged and most were completely lysed. The results suggest that fish kidney cells show a higher degree of tolerance to hyperosmoticity by comparing to amphibians and provide novel insights on the osmoregulatory capacity and adaptability of kidney cells between the two animal groups.

  4. Phytoceramide Shows Neuroprotection and Ameliorates Scopolamine-Induced Memory Impairment

    Directory of Open Access Journals (Sweden)

    Seikwan Oh

    2011-10-01

    Full Text Available The function and the role phytoceramide (PCER and phytosphingosine (PSO in the central nervous system has not been well studied. This study was aimed at investigating the possible roles of PCER and PSO in glutamate-induced neurotoxicity in cultured neuronal cells and memory function in mice. Phytoceramide showed neuro-protective activity in the glutamate-induced toxicity in cultured cortical neuronal cells. Neither phytosphingosine nor tetraacetylphytosphingosine (TAPS showed neuroproective effects in neuronal cells. PCER (50 mg/kg, p.o. recovered the scopolamine-induced reduction in step-through latency in the passive avoidance test; however, PSO did not modulate memory function on this task. The ameliorating effects of PCER on spatial memory were confirmed by the Morris water maze test. In conclusion, through behavioral and neurochemical experimental results, it was demonstrated that central administration of PCER produces amelioration of memory impairment. These results suggest that PCER plays an important role in neuroprotection and memory enhancement and PCER could be a potential new therapeutic agent for the treatment of neurodegenerative diseases such as Alzheimer’s disease.

  5. Face and body recognition show similar improvement during childhood.

    Science.gov (United States)

    Bank, Samantha; Rhodes, Gillian; Read, Ainsley; Jeffery, Linda

    2015-09-01

    Adults are proficient in extracting identity cues from faces. This proficiency develops slowly during childhood, with performance not reaching adult levels until adolescence. Bodies are similar to faces in that they convey identity cues and rely on specialized perceptual mechanisms. However, it is currently unclear whether body recognition mirrors the slow development of face recognition during childhood. Recent evidence suggests that body recognition develops faster than face recognition. Here we measured body and face recognition in 6- and 10-year-old children and adults to determine whether these two skills show different amounts of improvement during childhood. We found no evidence that they do. Face and body recognition showed similar improvement with age, and children, like adults, were better at recognizing faces than bodies. These results suggest that the mechanisms of face and body memory mature at a similar rate or that improvement of more general cognitive and perceptual skills underlies improvement of both face and body recognition. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Talk shows, fascinación o rechazo

    Directory of Open Access Journals (Sweden)

    Jorge Acevedo Rojas

    2015-01-01

    Full Text Available Infidelidades, odio, violencia, alcoholismo, mentira y traiciones, entre otras debilidades humanas, han pasado a formar parte de las programaciones de televisión en América Latina, durante la década de los 90 a través de los programas de Talk Shows. Cristina, El Padre Alberto, Laura en América, Mary Tere, entre los más vistos. Da cuenta de lo que hay detrás de estos programas; por qué se muestra la vida íntima; por qué se los dirige a sectores socioeconómicos bajos; por qué los excesos y degradación humana. Será que esta televisión basura está al servicio del poder.

  7. Machine-Learning-Based No Show Prediction in Outpatient Visits

    Directory of Open Access Journals (Sweden)

    Carlos Elvira

    2018-03-01

    Full Text Available A recurring problem in healthcare is the high percentage of patients who miss their appointment, be it a consultation or a hospital test. The present study seeks patient’s behavioural patterns that allow predicting the probability of no- shows. We explore the convenience of using Big Data Machine Learning models to accomplish this task. To begin with, a predictive model based only on variables associated with the target appointment is built. Then the model is improved by considering the patient’s history of appointments. In both cases, the Gradient Boosting algorithm was the predictor of choice. Our numerical results are considered promising given the small amount of information available. However, there seems to be plenty of room to improve the model if we manage to collect additional data for both patients and appointments.

  8. Showing Off in Humans: Male Generosity as a Mating Signal

    Directory of Open Access Journals (Sweden)

    Wendy Iredale

    2008-07-01

    Full Text Available We examined people's charity contributions while in the presence of an observer of the same sex, opposite sex, or no observer. Inspired by costly signaling theory, we hypothesized that men would be more generous in the presence of a potential mate. Men and women played a number of experimental games in which they could earn money. On completion of these games participants were asked what percentage of their earned money they would be willing to donate to charity. Our results show that men contribute more to charity when observed by a member of the opposite sex than by a member of the same sex or no observer. Conversely, female charity donations did not significantly vary across the three observer conditions. Findings support the notion that men's generosity might have evolved as a mating signal.

  9. KAKO PREDŠKOLSKA DJECA VIDE "BIG BROTHER" SHOW?

    OpenAIRE

    Janković, Andrea; Sindik, Joško; Jaman, Kamea

    2007-01-01

    Cilj istraživanja bio je ispitivanje nekih karakteristika gledanosti Big Brother show-a kod djece predškolske dobi, razloga gledanja te emisije, s aspekta preferiranih sadržaja emisije te ličnosti (kandidata) koje preferiraju djeca. Ispitan je prigodni uzorak djece srednje i starije vrtićke dobi iz 3 dječja vrtića iz Zagreba (Dječjeg vrtića Grigora Viteza i Dječjeg vrtića "Maksimir" i "Trnoružica", N = 142). Utvrđene su čestine razloga preferencije pojedinih sudionika show–a te pojedinih s...

  10. SuperFormLab: showing SuperFormLab

    DEFF Research Database (Denmark)

    2013-01-01

    bachelor program, followed by two years of master studies. The courses are offered equally to students from other design disciplines, e.g. industrial design. Teaching is mainly in English as the program is attended by a relatively large group of non-Danish students, who seek exactly this combination......3D-printing in clay and ceramic objects shaped by your own sounds and movements! Digital form transferred via CNC-milling to ornamental ceramic wall-cladding. Brave New World… Students and their teacher at SuperFormLab, the new ceramic workshop of the School of Design at the Royal Danish Academy...... of Fine Arts in Copenhagen, will be showing results of their investigations into the potential of combining digital technologies with ceramic materials. It is now possible to shape the most complex mathematical, virtual 3D objects through the use of advanced software-programs. And more than that – you can...

  11. Bex1 knock out mice show altered skeletal muscle regeneration

    International Nuclear Information System (INIS)

    Koo, Jae Hyung; Smiley, Mark A.; Lovering, Richard M.; Margolis, Frank L.

    2007-01-01

    Bex1 and Calmodulin (CaM) are upregulated during skeletal muscle regeneration. We confirm this finding and demonstrate the novel finding that they interact in a calcium-dependent manner. To study the role of Bex1 and its interaction with CaM in skeletal muscle regeneration, we generated Bex1 knock out (Bex1-KO) mice. These mice appeared to develop normally and are fertile, but displayed a functional deficit in exercise performance compared to wild type (WT) mice. After intramuscular injection of cardiotoxin, which causes extensive and reproducible myotrauma followed by recovery, regenerating muscles of Bex1-KO mice exhibited elevated and prolonged cell proliferation, as well as delayed cell differentiation, compared to WT mice. Thus, our results provide the first evidence that Bex1-KO mice show altered muscle regeneration, and allow us to propose that the interaction of Bex1 with Ca 2+ /CaM may be involved in skeletal muscle regeneration

  12. Measuring political polarization: Twitter shows the two sides of Venezuela

    Science.gov (United States)

    Morales, A. J.; Borondo, J.; Losada, J. C.; Benito, R. M.

    2015-03-01

    We say that a population is perfectly polarized when divided in two groups of the same size and opposite opinions. In this paper, we propose a methodology to study and measure the emergence of polarization from social interactions. We begin by proposing a model to estimate opinions in which a minority of influential individuals propagate their opinions through a social network. The result of the model is an opinion probability density function. Next, we propose an index to quantify the extent to which the resulting distribution is polarized. Finally, we apply the proposed methodology to a Twitter conversation about the late Venezuelan president, Hugo Chávez, finding a good agreement between our results and offline data. Hence, we show that our methodology can detect different degrees of polarization, depending on the structure of the network.

  13. Prestin shows divergent evolution between constant frequency echolocating bats.

    Science.gov (United States)

    Shen, Bin; Avila-Flores, Rafael; Liu, Yang; Rossiter, Stephen J; Zhang, Shuyi

    2011-10-01

    The gene Prestin encodes a motor protein that is thought to confer the high-frequency sensitivity and selectivity that characterizes the mammalian auditory system. Recent research shows that the Prestin gene has undergone a burst of positive selection on the ancestral branch of the Old World horseshoe and leaf-nosed bats (Rhinolophidae and Hipposideridae, respectively), and also on the branch leading to echolocating cetaceans. Moreover, these two groups share a large number of convergent amino acid sequence replacements. Horseshoe and leaf-nosed bats exhibit narrowband echolocation, in which the emitted calls are based on the second harmonic of a predominantly constant frequency (CF) component, the frequency of which is also over-represented in the cochlea. This highly specialized form of echolocation has also evolved independently in the neotropical Parnell's mustached bat (Pteronotus parnellii). To test whether the convergent evolution of CF echolocation between lineages has arisen from common changes in the Prestin gene, we sequenced the Prestin coding region (~2,212 bp, >99% coverage) in P. parnellii and several related species that use broadband echolocation calls. Our reconstructed Prestin gene tree and amino acid tree showed that P. parnellii did not group together with Old World horseshoe and leaf-nosed bats, but rather clustered within its true sister species. Comparisons of sequences confirmed that P. parnellii shared most amino acid changes with its congeners, and we found no evidence of positive selection in the branch leading to the genus of Pteronotus. Our result suggests that the adaptive changes seen in Prestin in horseshoe and leaf-nosed bats are not necessary for CF echolocation in P. parnellii.

  14. Patterned basal seismicity shows sub-ice stream bedforms

    Science.gov (United States)

    Barcheck, C. G.; Tulaczyk, S. M.; Schwartz, S. Y.

    2017-12-01

    Patterns in seismicity emanating from the bottom of fast-moving ice streams and glaciers may indicate localized patches of higher basal resistance— sometimes called 'sticky spots', or otherwise varying basal properties. These seismogenic basal areas resist an unknown portion of the total driving stress of the Whillans Ice Plain (WIP), in West Antarctica, but may play an important role in the WIP stick-slip cycle and ice stream slowdown. To better understand the mechanism and importance of basal seismicity beneath the WIP, we analyze seismic data collected by a small aperture (micro-earthquakes in Dec 2014, and we compare the resulting map of seismicity to ice bottom depth measured by airborne radar. The number of basal earthquakes per area within the network is spatially heterogeneous, but a pattern of two 400m wide streaks of high seismicity rates is evident, with >50-500 earthquakes detected per 50x50m grid cell in 2 weeks. These seismically active streaks are elongated approximately in the ice flow direction with a spacing of 750m. Independent airborne radar measurements of ice bottom depth from Jan 2013 show a low-amplitude ( 5m) undulation in the basal topography superposed on a regional gradient in ice bottom depth. The flow-perpendicular wavelength of these low-amplitude undulations is comparable to the spacing of the high seismicity bands, and the streaks of high seismicity intersect local lows in the undulating basal topography. We interpret these seismic and radar observations as showing seismically active sub-ice stream bedforms that are low amplitude and elongated in the direction of ice flow, comparable to the morphology of mega scale glacial lineations (MSGLs), with high basal seismicity rates observed in the MSGL troughs. These results have implications for understanding the formation mechanism of MSGLS and well as understanding the interplay between basal topographic roughness, spatially varying basal till and hydrologic properties, basal

  15. Low LET protons focused to submicrometer shows enhanced radiobiological effectiveness

    International Nuclear Information System (INIS)

    Schmid, T E; Zlobinskaya, O; Michalski, D; Molls, M; Multhoff, G; Greubel, C; Hable, V; Girst, S; Siebenwirth, C; Dollinger, G; Schmid, E

    2012-01-01

    This study shows that enhanced radiobiological effectiveness (RBE) values can be generated focusing low linear energy transfer (LET) radiation and thus changing the microdose distribution. 20 MeV protons (LET = 2.65 keV µm −1 ) are focused to submicrometer diameter at the ion microprobe superconducting nanoprobe for applied nuclear (Kern) physics experiments of the Munich tandem accelerator. The RBE values, as determined by measuring micronuclei (RBE MN = 1.48 ± 0.07) and dicentrics (RBE D = 1.92 ± 0.15), in human–hamster hybrid (A L ) cells are significantly higher when 117 protons were focused to a submicrometer irradiation field within a 5.4 × 5.4 µm 2 matrix compared to quasi homogeneous in a 1 × 1 µm 2 matrix applied protons (RBE MN = 1.28 ± 0.07; RBE D = 1.41 ± 0.14) at the same average dose of 1.7 Gy. The RBE values are normalized to standard 70 kV (dicentrics) or 200 kV (micronuclei) x-ray irradiation. The 117 protons applied per point deposit the same amount of energy like a 12 C ion with 55 MeV total energy (4.48 MeV u −1 ). The enhancements are about half of that obtained for 12 C ions (RBE MN = 2.20 ± 0.06 and RBE D = 3.21 ± 0.10) and they are attributed to intertrack interactions of the induced damages. The measured RBE values show differences from predictions of the local effect model (LEM III) that is used to calculate RBE values for irradiation plans to treat tumors with high LET particles. (paper)

  16. Low LET protons focused to submicrometer shows enhanced radiobiological effectiveness

    Science.gov (United States)

    Schmid, T. E.; Greubel, C.; Hable, V.; Zlobinskaya, O.; Michalski, D.; Girst, S.; Siebenwirth, C.; Schmid, E.; Molls, M.; Multhoff, G.; Dollinger, G.

    2012-10-01

    This study shows that enhanced radiobiological effectiveness (RBE) values can be generated focusing low linear energy transfer (LET) radiation and thus changing the microdose distribution. 20 MeV protons (LET = 2.65 keV µm-1) are focused to submicrometer diameter at the ion microprobe superconducting nanoprobe for applied nuclear (Kern) physics experiments of the Munich tandem accelerator. The RBE values, as determined by measuring micronuclei (RBEMN = 1.48 ± 0.07) and dicentrics (RBED = 1.92 ± 0.15), in human-hamster hybrid (AL) cells are significantly higher when 117 protons were focused to a submicrometer irradiation field within a 5.4 × 5.4 µm2 matrix compared to quasi homogeneous in a 1 × 1 µm2 matrix applied protons (RBEMN = 1.28 ± 0.07; RBED = 1.41 ± 0.14) at the same average dose of 1.7 Gy. The RBE values are normalized to standard 70 kV (dicentrics) or 200 kV (micronuclei) x-ray irradiation. The 117 protons applied per point deposit the same amount of energy like a 12C ion with 55 MeV total energy (4.48 MeV u-1). The enhancements are about half of that obtained for 12C ions (RBEMN = 2.20 ± 0.06 and RBED = 3.21 ± 0.10) and they are attributed to intertrack interactions of the induced damages. The measured RBE values show differences from predictions of the local effect model (LEM III) that is used to calculate RBE values for irradiation plans to treat tumors with high LET particles.

  17. Pumilio2-deficient mice show a predisposition for epilepsy

    Directory of Open Access Journals (Sweden)

    Philipp Follwaczny

    2017-11-01

    Full Text Available Epilepsy is a neurological disease that is caused by abnormal hypersynchronous activities of neuronal ensembles leading to recurrent and spontaneous seizures in human patients. Enhanced neuronal excitability and a high level of synchrony between neurons seem to trigger these spontaneous seizures. The molecular mechanisms, however, regarding the development of neuronal hyperexcitability and maintenance of epilepsy are still poorly understood. Here, we show that pumilio RNA-binding family member 2 (Pumilio2; Pum2 plays a role in the regulation of excitability in hippocampal neurons of weaned and 5-month-old male mice. Almost complete deficiency of Pum2 in adult Pum2 gene-trap mice (Pum2 GT causes misregulation of genes involved in neuronal excitability control. Interestingly, this finding is accompanied by the development of spontaneous epileptic seizures in Pum2 GT mice. Furthermore, we detect an age-dependent increase in Scn1a (Nav1.1 and Scn8a (Nav1.6 mRNA levels together with a decrease in Scn2a (Nav1.2 transcript levels in weaned Pum2 GT that is absent in older mice. Moreover, field recordings of CA1 pyramidal neurons show a tendency towards a reduced paired-pulse inhibition after stimulation of the Schaffer-collateral-commissural pathway in Pum2 GT mice, indicating a predisposition to the development of spontaneous seizures at later stages. With the onset of spontaneous seizures at the age of 5 months, we detect increased protein levels of Nav1.1 and Nav1.2 as well as decreased protein levels of Nav1.6 in those mice. In addition, GABA receptor subunit alpha-2 (Gabra2 mRNA levels are increased in weaned and adult mice. Furthermore, we observe an enhanced GABRA2 protein level in the dendritic field of the CA1 subregion in the Pum2 GT hippocampus. We conclude that altered expression levels of known epileptic risk factors such as Nav1.1, Nav1.2, Nav1.6 and GABRA2 result in enhanced seizure susceptibility and manifestation of epilepsy in the

  18. Different Achilles Tendon Pathologies Show Distinct Histological and Molecular Characteristics

    Directory of Open Access Journals (Sweden)

    Franka Klatte-Schulz

    2018-01-01

    Full Text Available Reasons for the development of chronic tendon pathologies are still under debate and more basic knowledge is needed about the different diseases. The aim of the present study was therefore to characterize different acute and chronic Achilles tendon disorders. Achilles tendon samples from patients with chronic tendinopathy (n = 7, chronic ruptures (n = 6, acute ruptures (n = 13, and intact tendons (n = 4 were analyzed. The histological score investigating pathological changes was significantly increased in tendinopathy and chronic ruptures compared to acute ruptures. Inflammatory infiltration was detected by immunohistochemistry in all tendon pathology groups, but was significantly lower in tendinopathy compared to chronic ruptures. Quantitative real-time PCR (qRT-PCR analysis revealed significantly altered expression of genes related to collagens and matrix modeling/remodeling (matrix metalloproteinases, tissue inhibitors of metalloproteinases in tendinopathy and chronic ruptures compared to intact tendons and/or acute ruptures. In all three tendon pathology groups markers of inflammation (interleukin (IL 1β, tumor necrosis factor α, IL6, IL10, IL33, soluble ST2, transforming growth factor β1, cyclooxygenase 2, inflammatory cells (cluster of differentaition (CD 3, CD68, CD80, CD206, fat metabolism (fatty acid binding protein 4, peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α, adiponectin, and innervation (protein gene product 9.5, growth associated protein 43, macrophage migration inhibitory factor were detectable, but only in acute ruptures significantly regulated compared to intact tendons. The study gives an insight into structural and molecular changes of pathological processes in tendons and might be used to identify targets for future therapy of tendon pathologies.

  19. Young children show representational flexibility when interpreting drawings.

    Science.gov (United States)

    Allen, Melissa L; Nurmsoo, Erika; Freeman, Norman

    2016-02-01

    Drawings can be ambiguous and represent more than one entity. In three experiments, we examine whether young children show representational flexibility by allowing one picture to be called by a second name. We also evaluate the hypothesis that children who are representationally flexible see the artist's intention as binding, rather than changeable. In Experiment 1, an artist declared what she intended to draw (e.g. a balloon) but then produced an ambiguous drawing. Children were asked whether the drawings could be interpreted differently (e.g. 'could this be a lollipop?') in the presence of a perceptually similar or dissimilar distractor (e.g., lollipop or snake). Six-year-olds accepted two labels for drawings in both conditions, but four-year-olds only did so in the dissimilar condition. Experiment 2 probed each possible interpretation more deeply by asking property questions (e.g., 'does it float?, does it taste good?'). Preschoolers who understood that the ambiguous drawing could be given two interpretations nevertheless mostly endorsed only properties associated with the prior intent. Experiment 3 provided converging evidence that 4-year-olds were representationally flexible using a paradigm that did not rely upon modal questioning. Taken together, our results indicate that even 4-year-olds understand that pictures may denote more than one referent, they still think of the symbol as consistent with the artist's original intention. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Benthic communities under anthropogenic pressure show resilience across the Quaternary.

    Science.gov (United States)

    Martinelli, Julieta C; Soto, Luis P; González, Jorge; Rivadeneira, Marcelo M

    2017-09-01

    The Southeast Pacific is characterized by rich upwelling systems that have sustained and been impacted by human groups for at least 12 ka. Recent fishing and aquaculture practices have put a strain on productive coastal ecosystems from Tongoy Bay, in north-central Chile. We use a temporal baseline to determine whether potential changes to community structure and composition over time are due to anthropogenic factors, natural climatic variations or both. We compiled a database ( n  = 33 194) with mollusc species abundances from the Mid-Pleistocene, Late Pleistocene, Holocene, dead shell assemblages and live-sampled communities. Species richness was not significantly different, neither were diversity and evenness indices nor rank abundance distributions. There is, however, an increase in relative abundance for the cultured scallop Argopecten , while the previously dominant clam Mulinia is locally very rare. Results suggest that impacts from both natural and anthropogenic stressors need to be better understood if benthic resources are to be preserved. These findings provide the first Pleistocene temporal baseline for the south Pacific that shows that this highly productive system has had the ability to recover from past alterations, suggesting that if monitoring and management practices continue to be implemented, moderately exploited communities from today have hopes for recovery.

  1. Management in a neotropical show cave: planning for invertebrates conservation

    Directory of Open Access Journals (Sweden)

    Thais Giovannini Pellegrini

    2012-07-01

    Full Text Available Lapa Nova is a dolomitic cave about 4.5 km long located in northwestern Minas Gerais state, Brazil. The cave experiences intense tourism, concentrated over a single period of the year, during the Feast of Our Lady of Lapa. In order to evaluate the impacts felt by the invertebrate community from this tourism, a new methodology was proposed. Four types of areas (intense visitation area, outlying visitation areas, moderate visitation areas and no-visitation areas were sampled for invertebrates. There was one sampling prior and another on the last day of the 128th feast, to evaluate the effects of visitation on cave-dwelling invertebrates. Results show that invertebrate populations residing in more intensely visited areas of the cave undergo changes in distribution following the event. As a consequence of tourism, invertebrates shift to outlying locations from the visited area, which serve as refuges to the communities. Apparently, the fact that there are places inside Lapa Nova inaccessible to tourists reduces the impact suffered by the invertebrate community, as those sites serve as refuges for cave-dwelling organisms during the pilgrimage. A proper management plan was devised for the tourism/religious use of the cave. It consists basically of delimiting marked pathways for tourists, allowing invertebrates to seek shelter at locations outside visited areas and keeping no-visitation areas off-limits to tourism based on the results of the visitation effects on cave-dwelling invertebrates.

  2. Platelets from Asthmatic Individuals Show Less Reliance on Glycolysis.

    Directory of Open Access Journals (Sweden)

    Weiling Xu

    Full Text Available Asthma, a chronic inflammatory airway disease, is typified by high levels of TH2-cytokines and excessive generation of reactive nitrogen and oxygen species, which contribute to bronchial epithelial injury and airway remodeling. While immune function plays a major role in the pathogenesis of the disease, accumulating evidence suggests that altered cellular metabolism is a key determinant in the predisposition and disease progression of asthma. Further, several studies demonstrate altered mitochondrial function in asthmatic airways and suggest that these changes may be systemic. However, it is unknown whether systemic metabolic changes can be detected in circulating cells in asthmatic patients. Platelets are easily accessible blood cells that are known to propagate airway inflammation in asthma. Here we perform a bioenergetic screen of platelets from asthmatic and healthy individuals and demonstrate that asthmatic platelets show a decreased reliance on glycolytic processes and have increased tricarboxylic acid cycle activity. These data demonstrate a systemic alteration in asthma and are consistent with prior reports suggesting that oxidative phosphorylation is more efficient asthmatic individuals. The implications for this potential metabolic shift will be discussed in the context of increased oxidative stress and hypoxic adaptation of asthmatic patients. Further, these data suggest that platelets are potentially a good model for the monitoring of bioenergetic changes in asthma.

  3. Film showing - Higgs: into the heart of imagination

    CERN Multimedia

    CERN Bulletin

    2010-01-01

    On 29 April at 7pm Dutch filmmakers, Hannie van den Bergh and Jan van den Berg, will introduce their directorial debut, Higgs: into the heart of imagination in CERN’s Main Auditorium.   This documentary is about the curiousity, passion and imaginative powers of science. Featuring physicists working at CERN, in particular in ATLAS, and filmed over four years, the film-makers have created a cinematic journey into the heart of imagination. They follow Stan Bentvelsen, head of the Dutch research group at CERN, and watch as he prepares his team for the start of the LHC, as well as the scientific competition to find the elusive Higgs particle. The film also features Peter Higgs as he discusses his work from 1964. The directors have created theatre productions and other multimedia projects under the title The Imagination of Invisible Dimensions, which allow for adventurous dialogues between art and science. All are welcome to attend this showing and afterwards there will be a short question...

  4. Variation in the peacock's train shows a genetic component.

    Science.gov (United States)

    Petrie, Marion; Cotgreave, Peter; Pike, Thomas W

    2009-01-01

    Female peafowl (Pavo cristatus) show a strong mating preference for males with elaborate trains. This, however, poses something of a paradox because intense directional selection should erode genetic variation in the males' trains, so that females will no longer benefit by discriminating among males on the basis of these traits. This situation is known as the 'lek paradox', and leads to the theoretical expectation of low heritability in the peacock's train. We used two independent breeding experiments, involving a total of 42 sires and 86 of their male offspring, to estimate the narrow sense heritabilities of male ornaments and other morphometric traits. Contrary to expectation, we found significant levels of heritability in a trait known to be used by females during mate choice (train length), while no significant heritabilities were evident for other, non-fitness related morphological traits (tarsus length, body weight or spur length). This study adds to the building body of evidence that high levels of additive genetic variance can exist in secondary sexual traits under directional selection, but further emphasizes the main problem of what maintains this variation.

  5. Active explorers show low learning performance in a social insect

    Institute of Scientific and Technical Information of China (English)

    Eve UDINO; Margot PEREZ; Claudio CARERE; Patrizia d'ETTORRE

    2017-01-01

    An intriguing question in behavioral biology is whether consistent individual differences (called animal personalities) relate to variation in cognitive performance because commonly measured personality traits may be associated with risk-reward trade-offs.Social insects,whose learning abilities have been extensively characterized,show consistent behavioral variability,both at colony and at individual level.We investigated the possible link between personality traits and learning performance in the carpenter ant Camponotus aethiops.Exploratory activity,sociability,and aggression were assessed twice in ant foragers.Behaviors differed among individuals,they were partly repeatable across time and exploratory activity correlated positively with aggression.Learning abilities were quantified by differential conditioning of the maxilla-labium extension response,a task that requires cue perception and information storage.We found that exploratory activity of individual ants significantly predicted learning performance:"active-explorers" were slower in learning the task than "inactive-explorers".The results suggest for the first time a link between a personality trait and cognitive performance in eusocial insects,and that the underlying individual variability could affect colony performance and success.

  6. The Oral Antimalarial Drug Tafenoquine Shows Activity against Trypanosoma brucei.

    Science.gov (United States)

    Carvalho, Luis; Martínez-García, Marta; Pérez-Victoria, Ignacio; Manzano, José Ignacio; Yardley, Vanessa; Gamarro, Francisco; Pérez-Victoria, José M

    2015-10-01

    The protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, or sleeping sickness, a neglected tropical disease that requires new, safer, and more effective treatments. Repurposing oral drugs could reduce both the time and cost involved in sleeping sickness drug discovery. Tafenoquine (TFQ) is an oral antimalarial drug belonging to the 8-aminoquinoline family which is currently in clinical phase III. We show here that TFQ efficiently kills different T. brucei spp. in the submicromolar concentration range. Our results suggest that TFQ accumulates into acidic compartments and induces a necrotic process involving cell membrane disintegration and loss of cytoplasmic content, leading to parasite death. Cell lysis is preceded by a wide and multitarget drug action, affecting the lysosome, mitochondria, and acidocalcisomes and inducing a depolarization of the mitochondrial membrane potential, elevation of intracellular Ca(2+), and production of reactive oxygen species. This is the first report of an 8-aminoquinoline demonstrating significant in vitro activity against T. brucei. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. Show: Dr. H, the life and death of pancakes

    CERN Multimedia

    2012-01-01

    Dr. H, the life and death of pancakes, a comic theatrical creation, written and performed by Heiko Buchholz.   Monday 2 April 2012 in German Tuesday 3 April 2012 in English Wednesday 4 April 2012 in French 8:30 p.m. at the Globe of Science and Innovation. This production takes a comic look at scientific methods, as applied to a common object: the pancake. More specifically, Dr H. regales his audience with statistics, experiments and scientific data surrounding this egg-and-milk-based culinary delight. And although these zany sketches are nothing short of absurd, the audience is drawn in more often than you might expect… and taken on quite an unexpected journey into the behavioural disorders, personality quirks and psychoanalysis of the base pancake. This show playfully mocks scientific logic and discourse, forcing the audience to reflect on their gullibility in the face of science and its impenetrable jargon. It purports to be neither explanation nor illustration of scientific fact,...

  8. Insect immunity shows specificity in protection upon secondary pathogen exposure.

    Science.gov (United States)

    Sadd, Ben M; Schmid-Hempel, Paul

    2006-06-20

    Immunological memory in vertebrates, conferring lasting specific protection after an initial pathogen exposure, has implications for a broad spectrum of evolutionary, epidemiological, and medical phenomena . However, the existence of specificity in protection upon secondary pathogen exposure in invertebrates remains controversial . To separate this functional phenomenon from a particular mechanism, we refer to it as specific immune priming. We investigate the presence of specific immune priming in workers of the social insect Bombus terrestris. Using three bacterial pathogens, we test whether a prior homologous pathogen exposure gives a benefit in terms of long-term protection against a later challenge, over and above a heterologous combination. With a reciprocally designed initial and second-exposure protocol (i.e., all combinations of bacteria were tested), we demonstrate, even several weeks after the clearance of a first exposure, increased protection and narrow specificity upon secondary exposure. This demonstrates that the invertebrate immune system is functionally capable of unexpectedly specific and durable induced protection. Ultimately, despite general broad differences between vertebrates and invertebrates, the ability of both immune systems to show specificity in protection suggests that their immune defenses have found comparable solutions to similar selective pressures over evolutionary time.

  9. Roadside soils show low plant available zinc and copper concentrations.

    Science.gov (United States)

    Morse, Natalie; Walter, M Todd; Osmond, Deanna; Hunt, William

    2016-02-01

    Vehicle combustion and component wear are a major source of metal contamination in the environment, which could be especially concerning where road ditches are actively farmed. The objective of this study was to assess how site variables, namely age, traffic (vehicles day(-1)), and percent carbon (%C) affect metal accumulation in roadside soils. A soil chronosequence was established with sites ranging from 3 to 37 years old and bioavailable, or mobile, concentrations of Zinc (Zn) and Copper (Cu) were measured along major highways in North Carolina using a Mehlich III extraction. Mobile Zn and Cu concentrations were low overall, and when results were scaled via literature values to "total metal", the results were still generally lower than previous roadside studies. This could indicate farming on lands near roads would pose a low plant toxicity risk. Zinc and Cu were not correlated with annual average traffic count, but were positively correlated with lifetime traffic load (the product of site age and traffic count). This study shows an often overlooked variable, site age, should be included when considering roadside pollution accumulation. Zinc and Cu were more strongly associated with %C, than traffic load. Because vehicle combustion is also a carbon source, it is not obvious whether the metals and carbon are simply co-accumulating or whether the soil carbon in roadside soils may facilitate previously overlooked roles in sequestering metals on-site. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Limonene hydroperoxide analogues show specific patch test reactions.

    Science.gov (United States)

    Christensson, Johanna Bråred; Hellsén, Staffan; Börje, Anna; Karlberg, Ann-Therese

    2014-05-01

    The fragrance terpene R-limonene is a very weak sensitizer, but forms allergenic oxidation products upon contact with air. The primary oxidation products of oxidized limonene, the hydroperoxides, have an important impact on the sensitizing potency of the oxidation mixture. One analogue, limonene-1-hydroperoxide, was experimentally shown to be a significantly more potent sensitizer than limonene-2-hydroperoxide in the local lymph node assay with non-pooled lymph nodes. To investigate the pattern of reactivity among consecutive dermatitis patients to two structurally closely related limonene hydroperoxides, limonene-1-hydroperoxide and limonene-2-hydroperoxide. Limonene-1-hydroperoxide, limonene-2-hydroperoxide, at 0.5% in petrolatum, and oxidized limonene 3.0% pet. were tested in 763 consecutive dermatitis patients. Of the tested materials, limonene-1-hydroperoxide gave most reactions, with 2.4% of the patients showing positive patch test reactions. Limonene-2-hydroperoxide and oxidized R-limonene gave 1.7% and 1.2% positive patch test reactions, respectively. Concomitant positive patch test reactions to other fragrance markers in the baseline series were frequently noted. The results are in accordance with the experimental studies, as limonene-1-hydroperoxide gave more positive patch test reactions in the tested patients than limonene-2-hydroperoxide. Furthermore, the results support the specificity of the allergenic activity of the limonene hydroperoxide analogues and the importance of oxidized limonene as a cause of contact allergy. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Benthic communities under anthropogenic pressure show resilience across the Quaternary

    Science.gov (United States)

    Martinelli, Julieta C.; Soto, Luis P.; González, Jorge; Rivadeneira, Marcelo M.

    2017-09-01

    The Southeast Pacific is characterized by rich upwelling systems that have sustained and been impacted by human groups for at least 12 ka. Recent fishing and aquaculture practices have put a strain on productive coastal ecosystems from Tongoy Bay, in north-central Chile. We use a temporal baseline to determine whether potential changes to community structure and composition over time are due to anthropogenic factors, natural climatic variations or both. We compiled a database (n = 33 194) with mollusc species abundances from the Mid-Pleistocene, Late Pleistocene, Holocene, dead shell assemblages and live-sampled communities. Species richness was not significantly different, neither were diversity and evenness indices nor rank abundance distributions. There is, however, an increase in relative abundance for the cultured scallop Argopecten, while the previously dominant clam Mulinia is locally very rare. Results suggest that impacts from both natural and anthropogenic stressors need to be better understood if benthic resources are to be preserved. These findings provide the first Pleistocene temporal baseline for the south Pacific that shows that this highly productive system has had the ability to recover from past alterations, suggesting that if monitoring and management practices continue to be implemented, moderately exploited communities from today have hopes for recovery.

  12. Cold thyroid nodules show a marked increase in proliferation markers.

    Science.gov (United States)

    Krohn, Knut; Stricker, Ingo; Emmrich, Peter; Paschke, Ralf

    2003-06-01

    Thyroid follicular adenomas and adenomatous thyroid nodules are a frequent finding in geographical areas with iodine deficiency. They occur as hypofunctioning (scintigraphically cold) or hyperfunctioning (scintigraphically hot) nodules. Their predominant clonal origin suggests that they result from clonal expansion of a single cell, which is very likely the result of a prolonged increase in proliferation compared with non-affected surrounding cells. To test whether increased cell proliferation is detectable in cold thyroid nodules, we studied paraffin-embedded tissue from 40 cold thyroid nodules and their surrounding normal thyroid tissue for the occurrence of the proliferating cell nuclear antigen (PCNA) and Ki-67 (MIB-1 antibody) epitopes as markers for cell proliferation. All 40 thyroid nodules were histologically well characterized and have been studied for molecular characteristics before. The labeling index (number of labeled cells versus total cell number) for nodular and surrounding tissue was calculated. In 33 cold thyroid nodules a significant (p thyroid nodules a significant (p thyroid epithelial cell proliferation is a uniform feature common to most cold nodules. However, the increase of proliferation markers shows a heterogeneity that is not correlated with histopathologic, molecular, or clinical characteristics.

  13. Apocrine hidradenocarcinoma showing Paget's disease and mucinous metaplasia.

    Science.gov (United States)

    Wahl, Carter E; Todd, Douglas H; Binder, Scott W; Cassarino, David S

    2009-05-01

    A 54-year-old man presented with a solitary, erythematous, rapidly growing 1-cm nodule on his scalp that had arisen over the previous 3 months. He had no history of skin cancer. An excisional biopsy of the lesion showed a fairly well-circumscribed but focally invasive tumor consisting of areas of typical-appearing clear cell hidradenoma as well as areas with mucinous goblet-type cells and cells with eosinophilic cytoplasm and decapitation-type secretion. There was marked cellular atypia, numerous atypical mitotic figures and focal necrosis. The tumor cells focally involved the overlying epidermis (Paget's disease). Large areas of mucin were identified throughout the lesion. The tumor cells stained with markers for cytokeratin 7 and focally for EMA and CEA, confirming ductal differentiation. The goblet cells and mucinous areas stained with mucicarmine and PASD. The patient was diagnosed with hidradenocarcinoma with mucinous differentiation. Associated Paget's disease has only rarely been reported, and mucinous metaplasia is a previously unreported feature in hidradenocarcinoma.

  14. Neighbouring chimpanzee communities show different preferences in social grooming behaviour.

    Science.gov (United States)

    van Leeuwen, Edwin J C; Cronin, Katherine A; Haun, Daniel B M; Mundry, Roger; Bodamer, Mark D

    2012-11-07

    Grooming handclasp (GHC) behaviour was originally advocated as the first evidence of social culture in chimpanzees owing to the finding that some populations engaged in the behaviour and others do not. To date, however, the validity of this claim and the extent to which this social behaviour varies between groups is unclear. Here, we measured (i) variation, (ii) durability and (iii) expansion of the GHC behaviour in four chimpanzee communities that do not systematically differ in their genetic backgrounds and live in similar ecological environments. Ninety chimpanzees were studied for a total of 1029 h; 1394 GHC bouts were observed between 2010 and 2012. Critically, GHC style (defined by points of bodily contact) could be systematically linked to the chimpanzee's group identity, showed temporal consistency both within and between groups, and could not be accounted for by the arm-length differential between partners. GHC has been part of the behavioural repertoire of the chimpanzees under study for more than 9 years (surpassing durability criterion) and spread across generations (surpassing expansion criterion). These results strongly indicate that chimpanzees' social behaviour is not only motivated by innate predispositions and individual inclinations, but may also be partly cultural in nature.

  15. Giant pandas failed to show mirror self-recognition.

    Science.gov (United States)

    Ma, Xiaozan; Jin, Yuan; Luo, Bo; Zhang, Guiquan; Wei, Rongping; Liu, Dingzhen

    2015-05-01

    Mirror self-recognition (MSR), i.e., the ability to recognize oneself in a mirror, is considered a potential index of self-recognition and the foundation of individual development. A wealth of literature on MSR is available for social animals, such as chimpanzees, Asian elephants and dolphins, yet little is known about MSR in solitary mammalian species. We aimed to evaluate whether the giant panda can recognize itself in the mirror, and whether this capacity varies with age. Thirty-four captive giant pandas (F:M = 18:16; juveniles, sub-adults and adults) were subjected to four mirror tests: covered mirror tests, open mirror tests, water mark control tests, and mark tests. The results showed that, though adult, sub-adult and juvenile pandas exposed to mirrors spent similar amounts of time in social mirror-directed behaviors (χ(2) = 0.719, P = 0.698), none of them used the mirror to touch the mark on their head, a self-directed behavior suggesting MSR. Individuals of all age groups initially displayed attacking, threatening, foot scraping and backwards walking behaviors when exposed to their self-images in the mirror. Our data indicate that, regardless of age, the giant pandas did not recognize their self-image in the mirror, but instead considered the image to be a conspecific. Our results add to the available information on mirror self-recognition in large mammals, provide new information on a solitary species, and will be useful for enclosure design and captive animal management.

  16. Non-asthmatic patients show increased exhaled nitric oxide concentrations

    Directory of Open Access Journals (Sweden)

    Beatriz M. Saraiva-Romanholo

    2009-01-01

    Full Text Available OBJECTIVE: Evaluate whether exhaled nitric oxide may serve as a marker of intraoperative bronchospasm. INTRODUCTION: Intraoperative bronchospasm remains a challenging event during anesthesia. Previous studies in asthmatic patients suggest that exhaled nitric oxide may represent a noninvasive measure of airway inflammation. METHODS: A total of 146,358 anesthesia information forms, which were received during the period from 1999 to 2004, were reviewed. Bronchospasm was registered on 863 forms. From those, three groups were identified: 9 non-asthmatic patients (Bronchospasm group, 12 asthmatics (Asthma group and 10 subjects with no previous airway disease or symptoms (Control group. All subjects were submitted to exhaled nitric oxide measurements (parts/billion, spirometry and the induced sputum test. The data was compared by ANOVA followed by the Tukey test and Kruskal-Wallis followed by Dunn's test. RESULTS: The normal lung function test results for the Bronchospasm group were different from those of the asthma group (p <0.05. The median percentage of eosinophils in induced sputum was higher for the Asthma [2.46 (0.45-6.83] compared with either the Bronchospasm [0.55 (0-1.26] or the Control group [0.0 (0] (p <0.05; exhaled nitric oxide followed a similar pattern for the Asthma [81.55 (57.6-86.85], Bronchospasm [46.2 (42.0 -62.6] and Control group [18.7 (16.0-24.7] (p< 0.05. CONCLUSIONS: Non-asthmatic patients with intraoperative bronchospasm detected during anesthesia and endotracheal intubation showed increased expired nitric oxide.

  17. Can recurrence networks show small-world property?

    International Nuclear Information System (INIS)

    Jacob, Rinku; Harikrishnan, K.P.; Misra, R.; Ambika, G.

    2016-01-01

    Recurrence networks are complex networks, constructed from time series data, having several practical applications. Though their properties when constructed with the threshold value ϵ chosen at or just above the percolation threshold of the network are quite well understood, what happens as the threshold increases beyond the usual operational window is still not clear from a complex network perspective. The present Letter is focused mainly on the network properties at intermediate-to-large values of the recurrence threshold, for which no systematic study has been performed so far. We argue, with numerical support, that recurrence networks constructed from chaotic attractors with ϵ equal to the usual recurrence threshold or slightly above cannot, in general, show small-world property. However, if the threshold is further increased, the recurrence network topology initially changes to a small-world structure and finally to that of a classical random graph as the threshold approaches the size of the strange attractor. - Highlights: • Properties of recurrence networks at intermediate-to-large values of recurrence threshold are analyzed from a complex network perspective. • Using a combined plot of characteristic path length and clustering coefficient, it is shown that the recurrence network constructed with recurrence threshold equal to or just above the percolation threshold cannot, in general, display small-world property. • As the recurrence threshold is increased from its usual operational window, the resulting network makes a smooth transition initially to a small-world network for an intermediate range of thresholds and finally to the classical random graph as the threshold becomes comparable to the size of the attractor.

  18. Can recurrence networks show small-world property?

    Energy Technology Data Exchange (ETDEWEB)

    Jacob, Rinku, E-mail: rinku.jacob.vallanat@gmail.com [Department of Physics, The Cochin College, Cochin, 682002 (India); Harikrishnan, K.P., E-mail: kp_hk2002@yahoo.co.in [Department of Physics, The Cochin College, Cochin, 682002 (India); Misra, R., E-mail: rmisra@iucaa.in [Inter University Centre for Astronomy and Astrophysics, Pune, 411007 (India); Ambika, G., E-mail: g.ambika@iiserpune.ac.in [Indian Institute of Science Education and Research, Pune, 411008 (India)

    2016-08-12

    Recurrence networks are complex networks, constructed from time series data, having several practical applications. Though their properties when constructed with the threshold value ϵ chosen at or just above the percolation threshold of the network are quite well understood, what happens as the threshold increases beyond the usual operational window is still not clear from a complex network perspective. The present Letter is focused mainly on the network properties at intermediate-to-large values of the recurrence threshold, for which no systematic study has been performed so far. We argue, with numerical support, that recurrence networks constructed from chaotic attractors with ϵ equal to the usual recurrence threshold or slightly above cannot, in general, show small-world property. However, if the threshold is further increased, the recurrence network topology initially changes to a small-world structure and finally to that of a classical random graph as the threshold approaches the size of the strange attractor. - Highlights: • Properties of recurrence networks at intermediate-to-large values of recurrence threshold are analyzed from a complex network perspective. • Using a combined plot of characteristic path length and clustering coefficient, it is shown that the recurrence network constructed with recurrence threshold equal to or just above the percolation threshold cannot, in general, display small-world property. • As the recurrence threshold is increased from its usual operational window, the resulting network makes a smooth transition initially to a small-world network for an intermediate range of thresholds and finally to the classical random graph as the threshold becomes comparable to the size of the attractor.

  19. Roadside soils show low plant available zinc and copper concentrations

    International Nuclear Information System (INIS)

    Morse, Natalie; Walter, M. Todd; Osmond, Deanna; Hunt, William

    2016-01-01

    Vehicle combustion and component wear are a major source of metal contamination in the environment, which could be especially concerning where road ditches are actively farmed. The objective of this study was to assess how site variables, namely age, traffic (vehicles day"−"1), and percent carbon (%C) affect metal accumulation in roadside soils. A soil chronosequence was established with sites ranging from 3 to 37 years old and bioavailable, or mobile, concentrations of Zinc (Zn) and Copper (Cu) were measured along major highways in North Carolina using a Mehlich III extraction. Mobile Zn and Cu concentrations were low overall, and when results were scaled via literature values to “total metal”, the results were still generally lower than previous roadside studies. This could indicate farming on lands near roads would pose a low plant toxicity risk. Zinc and Cu were not correlated with annual average traffic count, but were positively correlated with lifetime traffic load (the product of site age and traffic count). This study shows an often overlooked variable, site age, should be included when considering roadside pollution accumulation. Zinc and Cu were more strongly associated with %C, than traffic load. Because vehicle combustion is also a carbon source, it is not obvious whether the metals and carbon are simply co-accumulating or whether the soil carbon in roadside soils may facilitate previously overlooked roles in sequestering metals on-site. - Highlights: • Low plant available zinc and copper concentrations in roadside soils of the southeast U.S. • Metals from vehicular traffic may not be adversely affecting plants in roadside environment. • Traffic volume and site age better predictor of metal pollution than traffic volume alone. - Mobile concentrations of Zn and Cu in roadside soils were below toxic levels. Zn and Cu concentrations were better correlated with lifetime vehicle load, as opposed to traffic volume.

  20. A case of osseous scintigraphy showing focuses of multifocal tuberculosis

    International Nuclear Information System (INIS)

    Faurous-Vanaud, R.; Faurous, P.; Kalfa, G.; Collet, H.; Couty, H.; Artus, J.C.

    1997-01-01

    Incidence of tuberculosis in developed countries presents in the last years a spectacular boost. However, the osteo-articular system is afflicted by tuberculosis only in a few percent of cases and the multifocal character represents less than 5% of osseous tuberculosis. It is presented here a case of multifocal osseous tuberculosis only to stress its rarity. A woman 28 years old from Djibouti presents since 8 months an asthenia and dorsal pains long thought as psychalgia. The examination has shown a major vertebral stiffness and limitation of right haunch. The radiographs showed a scoliosis centered on D8 with modifications. The biologic data are VS = 90, CRP = 124 and the hemogram is normal. The intradermal reaction (10 U of tuberculin) is phlyctenular. The TDM indicates lytic vertebral extension on D7 - D8, the osseous scintigraphy with MDP - 99 mTc indicates no anomaly in the vascular arrival time; a few minutes after injection: dorsal and upper and of right femur hyperactivity; 2 h after injection: hyper-fixation of D7 to D9 and of the upper end of right femur. The IRM indicates para-vertebral abscess of D7 to D9 with epidural abscess; abscess in the upper part of the loge of right adductors with osseous affliction of the femur's upper end; a bacillary infectious process was a priori evoked. The patient was then placed under quadruple antibiotic-therapy anti-tuberculous care. In case of anomalous multifocal osseous scintigraphy the tuberculous affliction is part of the differential diagnosis, particularly in the immigrant populations or in HIV+ persons

  1. Proceedings of CanWEA's 2006 conference and trade show

    International Nuclear Information System (INIS)

    2006-01-01

    generated by the Wind Atlas Analysis and Application Program (WAsP). A new tool to monitor regional climatological wind resource anomalies was also discussed along with issues concerning wind power in cold climates. The conference featured 47 presentations, of which 46 were catalogued separately for inclusion in this database

  2. Transcriptional profiling of whole blood and serum protein analysis of mice exposed to the neurotoxin Pacific Ciguatoxin-1.

    Science.gov (United States)

    Ryan, James C; Bottein Dechraoui, Marie-Yasmine; Morey, Jeanine S; Rezvani, Amir; Levin, Edward D; Gordon, Christopher J; Ramsdell, John S; Van Dolah, Frances M

    2007-11-01

    Ciguatoxins (CTX) are a suite of cyclic polyether toxins produced by the marine dinoflagellate Gambierdiscus sp., are potent activators of voltage-gated sodium channels and a leading cause of human poisoning from food fish. This report characterizes the genomic and proteomic response in whole blood of adult male mice exposed i.p. to 264 ng/kg of the Pacific congener of CTX (P-CTX-1) at 1, 4 and 24h. Whole genome microarray expression data were filtered by tightness of fit between replicates, fold change (1.8) and p-value (10(-5)), resulting in 183 annotated genes used for trending analysis, K-means clustering and ontology classification. Genes involved with cytokine signaling, proteasome complex and ribosomal function were dominant. qPCR performed on 19 genes of interest had a correlation of 0.95 to array results by Pearson's correlation coefficient. Serum protein analysis showed small but significant changes in 6 of 60 proteins assayed: Ccl2, Ccl12, CD40, IL-10, leptin and M-CSF. In large part, the gene expression was consistent with a Th2 immune response with interesting similarities to expression seen in asthmatic models.

  3. Suitable Reference Genes for Accurate Gene Expression Analysis in Parsley (Petroselinum crispum) for Abiotic Stresses and Hormone Stimuli.

    Science.gov (United States)

    Li, Meng-Yao; Song, Xiong; Wang, Feng; Xiong, Ai-Sheng

    2016-01-01

    Parsley, one of the most important vegetables in the Apiaceae family, is widely used in the food, medicinal, and cosmetic industries. Recent studies on parsley mainly focus on its chemical composition, and further research involving the analysis of the plant's gene functions and expressions is required. qPCR is a powerful method for detecting very low quantities of target transcript levels and is widely used to study gene expression. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization. In this study, four software, namely geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stabilities of eight candidate reference genes of parsley ( GAPDH, ACTIN, eIF-4 α, SAND, UBC, TIP41, EF-1 α, and TUB ) under various conditions, including abiotic stresses (heat, cold, salt, and drought) and hormone stimuli treatments (GA, SA, MeJA, and ABA). Results showed that EF-1 α and TUB were the most stable genes for abiotic stresses, whereas EF-1 α, GAPDH , and TUB were the top three choices for hormone stimuli treatments. Moreover, EF-1 α and TUB were the most stable reference genes among all tested samples, and UBC was the least stable one. Expression analysis of PcDREB1 and PcDREB2 further verified that the selected stable reference genes were suitable for gene expression normalization. This study can guide the selection of suitable reference genes in gene expression in parsley.

  4. Suitable reference genes for accurate gene expression analysis in parsley (Petroselinum crispum for abiotic stresses and hormone stimuli

    Directory of Open Access Journals (Sweden)

    Meng-Yao Li

    2016-09-01

    Full Text Available Parsley is one of the most important vegetable in Apiaceae family and widely used in food industry, medicinal and cosmetic. The recent studies in parsley are mainly focus on chemical composition, further research involving the analysis of the gene functions and expressions will be required. qPCR is a powerful method for detecting very low quantities of target transcript levels and widely used for gene expression studies. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization. In this study, three software geNorm, NormFinder, and BestKeeper were used to evaluate the expression stabilities of eight candidate reference genes (GAPDH, ACTIN, eIF-4α, SAND, UBC, TIP41, EF-1α, and TUB under various conditions including abiotic stresses (heat, cold, salt, and drought and hormone stimuli treatments (GA, SA, MeJA, and ABA. The results showed that EF-1α and TUB were identified as the most stable genes for abiotic stresses, while EF-1α, GAPDH, and TUB were the top three choices for hormone stimuli treatments. Moreover, EF-1α and TUB were the most stable reference genes across all the tested samples, while UBC was the least stable one. The expression analysis of PcDREB1 and PcDREB2 further verified that the selected stable reference genes were suitable for gene expression normalization. This study provides a guideline for selection the suitable reference genes in gene expression in parsley.

  5. Grab That Mike: communicative issues in French Talk Show "Grab That Mike": questões comunicativas em um "Talk Show" Francês

    Directory of Open Access Journals (Sweden)

    Ludwina Van Son

    2004-12-01

    Full Text Available In this analysis we have chosen a recent French talk show to illustrate how communication is turned into some new kind of "ideology"nowadays: in other words, you have to communicate if you consider yourself a citizen of today's world. The main characteristic of issue-centered talk shows being the destabilization of the implicit rules and participation framework, we observe how the so-called democratic right to express ourselves is (misused by the talk show host to secure the dynamics of the show. In order to reveal the host's manipulations, we have examined the verbal interactions between host and guests on t