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Sample records for combining dna pooling

  1. Combination fence and solar heater for swimming pools

    SciTech Connect

    Divine, D.L.

    1981-07-28

    A combination fence and solar heater for swimming pools comprises a fence shaped for extending about the periphery of the pool to restrict ingress and egress therefrom. A tubular heat exchanger is formed in at least one section of the fence, includes an exterior surface adapted to absorb solar energy, and communicates with the water in the swimming pool. The number of heat exchanger fence sections can be varied in accordance with the climate in which the pool is located. A pump flows the water in the swimming pool through the heat exchanger fence sections during daylight hours, thereby simultaneously heating the water in the pool, and providing an attractive and protective safety barrier about the swimming pool.

  2. vipR: variant identification in pooled DNA using R

    PubMed Central

    Altmann, Andre; Weber, Peter; Quast, Carina; Rex-Haffner, Monika; Binder, Elisabeth B.; Müller-Myhsok, Bertram

    2011-01-01

    Motivation: High-throughput-sequencing (HTS) technologies are the method of choice for screening the human genome for rare sequence variants causing susceptibility to complex diseases. Unfortunately, preparation of samples for a large number of individuals is still very cost- and labor intensive. Thus, recently, screens for rare sequence variants were carried out in samples of pooled DNA, in which equimolar amounts of DNA from multiple individuals are mixed prior to sequencing with HTS. The resulting sequence data, however, poses a bioinformatics challenge: the discrimination of sequencing errors from real sequence variants present at a low frequency in the DNA pool. Results: Our method vipR uses data from multiple DNA pools in order to compensate for differences in sequencing error rates along the sequenced region. More precisely, instead of aiming at discriminating sequence variants from sequencing errors, vipR identifies sequence positions that exhibit significantly different minor allele frequencies in at least two DNA pools using the Skellam distribution. The performance of vipR was compared with three other models on data from a targeted resequencing study of the TMEM132D locus in 600 individuals distributed over four DNA pools. Performance of the methods was computed on SNPs that were also genotyped individually using a MALDI-TOF technique. On a set of 82 sequence variants, vipR achieved an average sensitivity of 0.80 at an average specificity of 0.92, thus outperforming the reference methods by at least 0.17 in specificity at comparable sensitivity. Availability: The code of vipR is freely available via: http://sourceforge.net/projects/htsvipr/ Contact: altmann@mpipsykl.mpg.de PMID:21685105

  3. Impaired DNA replication within progenitor cell pools promotes leukemogenesis.

    PubMed

    Bilousova, Ganna; Marusyk, Andriy; Porter, Christopher C; Cardiff, Robert D; DeGregori, James

    2005-12-01

    Impaired cell cycle progression can be paradoxically associated with increased rates of malignancies. Using retroviral transduction of bone marrow progenitors followed by transplantation into mice, we demonstrate that inhibition of hematopoietic progenitor cell proliferation impairs competition, promoting the expansion of progenitors that acquire oncogenic mutations which restore cell cycle progression. Conditions that impair DNA replication dramatically enhance the proliferative advantage provided by the expression of Bcr-Abl or mutant p53, which provide no apparent competitive advantage under conditions of healthy replication. Furthermore, for the Bcr-Abl oncogene the competitive advantage in contexts of impaired DNA replication dramatically increases leukemogenesis. Impaired replication within hematopoietic progenitor cell pools can select for oncogenic events and thereby promote leukemia, demonstrating the importance of replicative competence in the prevention of tumorigenesis. The demonstration that replication-impaired, poorly competitive progenitor cell pools can promote tumorigenesis provides a new rationale for links between tumorigenesis and common human conditions of impaired DNA replication such as dietary folate deficiency, chemotherapeutics targeting dNTP synthesis, and polymorphisms in genes important for DNA metabolism. PMID:16277552

  4. Association mapping of disease loci, by use of a pooled DNA genomic screen.

    PubMed Central

    Barcellos, L F; Klitz, W; Field, L L; Tobias, R; Bowcock, A M; Wilson, R; Nelson, M P; Nagatomi, J; Thomson, G

    1997-01-01

    Genomic screening to map disease loci by association requires automation, pooling of DNA samples, and 3,000-6,000 highly polymorphic, evenly spaced microsatellite markers. Case-control samples can be used in an initial screen, followed by family-based data to confirm marker associations. Association mapping is relevant to genetic studies of complex diseases in which linkage analysis may be less effective and to cases in which multigenerational data are difficult to obtain, including rare or late-onset conditions and infectious diseases. The method can also be used effectively to follow up and confirm regions identified in linkage studies or to investigate candidate disease loci. Study designs can incorporate disease heterogeneity and interaction effects by appropriate subdivision of samples before screening. Here we report use of pooled DNA amplifications-the accurate determination of marker-disease associations for both case-control and nuclear family-based data-including application of correction methods for stutter artifact and preferential amplification. These issues, combined with a discussion of both statistical power and experimental design to define the necessary requirements for detecting of disease loci while virtually eliminating false positives, suggest the feasibility and efficiency of association mapping using pooled DNA screening. PMID:9326338

  5. DNA Probe Pooling for Rapid Delineation of Chromosomal Breakpoints

    SciTech Connect

    Lu, Chun-Mei; Kwan, Johnson; Baumgartner, Adolf; Weier, Jingly F.; Wang, Mei; Escudero, Tomas; Munne', Santiago; Zitzelsberger, Horst F.; Weier, Heinz-Ulrich

    2009-01-30

    Structural chromosome aberrations are hallmarks of many human genetic diseases. The precise mapping of translocation breakpoints in tumors is important for identification of genes with altered levels of expression, prediction of tumor progression, therapy response, or length of disease-free survival as well as the preparation of probes for detection of tumor cells in peripheral blood. Similarly, in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for carriers of balanced, reciprocal translocations benefit from accurate breakpoint maps in the preparation of patient-specific DNA probes followed by a selection of normal or balanced oocytes or embryos. We expedited the process of breakpoint mapping and preparation of case-specific probes by utilizing physically mapped bacterial artificial chromosome (BAC) clones. Historically, breakpoint mapping is based on the definition of the smallest interval between proximal and distal probes. Thus, many of the DNA probes prepared for multi-clone and multi-color mapping experiments do not generate additional information. Our pooling protocol described here with examples from thyroid cancer research and PGD accelerates the delineation of translocation breakpoints without sacrificing resolution. The turnaround time from clone selection to mapping results using tumor or IVF patient samples can be as short as three to four days.

  6. Genetic linkage analysis using pooled DNA and infrared detection of tailed STRP primer patterns

    NASA Astrophysics Data System (ADS)

    Oetting, William S.; Wildenberg, Scott C.; King, Richard A.

    1996-04-01

    The mapping of a disease locus to a specific chromosomal region is an important step in the eventual isolation and analysis of a disease causing gene. Conventional mapping methods analyze large multiplex families and/or smaller nuclear families to find linkage between the disease and a chromosome marker that maps to a known chromosomal region. This analysis is time consuming and tedious, typically requiring the determination of 30,000 genotypes or more. For appropriate populations, we have instead utilized pooled DNA samples for gene mapping which greatly reduces the amount of time necessary for an initial chromosomal screen. This technique assumes a common founder for the disease locus of interest and searches for a region of a chromosome shared between affected individuals. Our analysis involves the PCR amplification of short tandem repeat polymorphisms (STRP) to detect these shared regions. In order to reduce the cost of genotyping, we have designed unlabeled tailed PCR primers which, when combined with a labeled universal primer, provides for an alternative to synthesizing custom labeled primers. The STRP pattern is visualized with an infrared fluorescence based automated DNA sequencer and the patterns quantitated by densitometric analysis of the allele pattern. Differences in the distribution of alleles between pools of affected and unaffected individuals, including a reduction in the number of alleles in the affected pool, indicate the sharing of a region of a chromosome. We have found this method effective for markers 10 - 15 cM away from the disease locus for a recessive genetic disease.

  7. Selective DNA pooling for determination of linkage between a molecular marker and a quantitative trait locus

    SciTech Connect

    Darvasi, A.; Soller, M.

    1994-12-01

    Selective genotyping is a method to reduce costs in marker-quantitative trait locus (QTL) linkage determination by genotyping only those individuals with extreme, and hence most informative, quantitative trait values. The DNA pooling strategy (termed: {open_quotes}selective DNA pooling{close_quotes}) takes this one step further by pooling DNA from the selected individuals at each of the two phenotypic extremes, and basing the test for linkage on marker allele frequencies as estimated from the pooled samples only. This can reduce genotyping costs of marker-QTL linkage determination by up to two orders of magnitude. Theoretical analysis of selective DNA pooling shows that for experiments involving backcross, F{sub 2} and half-sib designs, the power of selective DNA pooling for detecting genes with large effect can be the same as that obtained by individual selective genotyping. Power for detecting genes with small effect, however, was found to decrease strongly with increase in the technical error of estimating allele frequencies in the pooled samples. The effect of technical error, however, can be markedly reduced by replication of technical procedures. It is also shown that a proportion selected of 0.1 at each tail will be appropriate for a wide range of experimental conditions. 25 refs., 3 figs., 3 tabs.

  8. Selective DNA Pooling for Determination of Linkage between a Molecular Marker and a Quantitative Trait Locus

    PubMed Central

    Darvasi, A.; Soller, M.

    1994-01-01

    Selective genotyping is a method to reduce costs in marker-quantitative trait locus (QTL) linkage determination by genotyping only those individuals with extreme, and hence most informative, quantitative trait values. The DNA pooling strategy (termed: ``selective DNA pooling'') takes this one step further by pooling DNA from the selected individuals at each of the two phenotypic extremes, and basing the test for linkage on marker allele frequencies as estimated from the pooled samples only. This can reduce genotyping costs of marker-QTL linkage determination by up to two orders of magnitude. Theoretical analysis of selective DNA pooling shows that for experiments involving backcross, F(2) and half-sib designs, the power of selective DNA pooling for detecting genes with large effect, can be the same as that obtained by individual selective genotyping. Power for detecting genes with small effect, however, was found to decrease strongly with increase in the technical error of estimating allele frequencies in the pooled samples. The effect of technical error, however, can be markedly reduced by replication of technical procedures. It is also shown that a proportion selected of 0.1 at each tail will be appropriate for a wide range of experimental conditions. PMID:7896115

  9. Origin, dynamics, and implications of extracellular DNA pools in marine sediments.

    PubMed

    Torti, Andrea; Lever, Mark Alexander; Jørgensen, Bo Barker

    2015-12-01

    In marine sediments, DNA occurs both inside and outside living organisms. DNA not enclosed in living cells may account for the largest fraction of total DNA, and include molecules locked within dead cells, organic and inorganic aggregates, adsorbed onto mineral matrices, and viral DNA. This DNA comprises genetic material released in situ from sediment microbial communities, as well as DNA of pelagic and terrestrial origin deposited to the seafloor. DNA not enclosed in living cells undermines the assumption of a direct link between the overall DNA pool and the local, currently living microbial assemblages, in terms of both microbial cell abundance and diversity. At the same time, the extracellular DNA may provide an integrated view of the biodiversity and ecological processes occurring on land, in marine water columns, and sediments themselves, thereby acting as an archive of genetic information which can be used to reconstruct past changes in source environments. In this review, we identify and discuss DNA pools in marine sediments, with special focus on DNA not enclosed in living cells, its origin, dynamics, and ecological and methodological implications. Achievements in deciphering the genetic information held within each DNA pool are presented along with still-standing challenges and major gaps in current knowledge. PMID:26452301

  10. DNA--a molecule in search of additional functions: recipient of pool wave emissions? A hypothesis.

    PubMed

    Doerfler, Walter

    2010-09-01

    Almost the entire nucleotide sequence of human DNA is functionally unaccounted for, although large parts of the human genome are transcribed. The genes, as defined by current molecular biology, comprise about 1.5-2% of the DNA molecule. It is proposed that DNA encodes additional, hitherto unrecognized functions. In this discussion, the total information inside and outside the universe we live in is termed the pool or the sum total, known or unknown, of all laws, matter, energy, concepts and events. In a hypothetical model, a Gedankenexperiment, it is suggested that the total of all information emits pool waves of an unknown physical nature. They could be related to black energy or have completely different qualities. The designation pool waves should not imply any similarity to electromagnetism. Further, DNA is suggested to have the capability of interacting with the pool waves and thus permit humans - to some partly genetically determined and yet very limited extent - to perceive information from the pool. Pool emissions might be one of the forces that have been instrumental in and are still driving evolution from simple oligonucleotides to DNA with ever more complex recipient capacities. It will be a major challenge for researchers in the field to unravel these and less hypothetical undetected coding principles in DNA. It is uncertain whether the current trend to search the available DNA sequences with ever more refined computer technology on the basis of our present understanding of biology will detect unknown coding systems. For molecular medicine, research into the genetics of the most common human diseases could profit from the elucidation of presently still ephemeral codes in human DNA. Young scientists with a proven record of original research deserve support for the pursuit of unconventional ideas. This concept of granting priorities will be of the utmost importance in advancing the field beyond current concepts in molecular biology. PMID:20356684

  11. Combined roles of buoyancy and orientation in nucleate pool boiling

    NASA Technical Reports Server (NTRS)

    Merte, H., Jr.

    1988-01-01

    The paper presents results obtained during pool boiling from flat surfaces over body force levels of a/g = -1 to 0 to 20 for LN2 and R-113 under steady and transient conditions. Pyrex and metal surfaces are used. Particular attention is given to the case where the effective gravity is perpendicular to the heating surface. At the lower levels of heat flux with nucleate boiling, the heater surface superheat first increases and then decreases as the body force increases above a/g = 1. This is due to the greater contribution of nonboiling convection.

  12. Deoxyribonucleotide pool imbalance stimulates deletions in HeLa cell mitochondrial DNA.

    PubMed

    Song, Shiwei; Wheeler, Linda J; Mathews, Christopher K

    2003-11-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder associated with multiple mutations in mitochondrial DNA, both deletions and point mutations, and mutations in the nuclear gene for thymidine phosphorylase. Spinazzola et al. (Spinazzola, A., Marti, R., Nishino, I., Andreu, A., Naini, A., Tadesse, S., Pela, I., Zammarchi, E., Donati, M., Oliver, J., and Hirano, M. (2001) J. Biol. Chem. 277, 4128-4133) showed that MNGIE patients have elevated circulating thymidine levels and they hypothesized that this generates imbalanced mitochondrial deoxyribonucleoside triphosphate (dNTP) pools, which in turn are responsible for mitochondrial (mt) DNA mutagenesis. We tested this hypothesis by culturing HeLa cells in medium supplemented with 50 microM thymidine. After 8-month growth, mtDNA in the thymidine-treated culture, but not the control, showed multiple deletions, as detected both by Southern blotting and by long extension polymerase chain reaction. After 4-h growth in thymidine-supplemented medium, we found the mitochondrial dTTP and dGTP pools to expand significantly, the dCTP pool to drop significantly, and the dATP pool to drop slightly. In whole-cell extracts, dTTP and dGTP pools also expanded, but somewhat less than in mitochondria. The dCTP pool shrank by about 50%, and the dATP pool was essentially unchanged. These results are discussed in terms of the recent report by Nishigaki et al. (Nishigaki, Y., Marti, R., Copeland, W. C., and Hirano, M. (2003) J. Clin. Invest. 111, 1913-1921) that most mitochondrial point mutations in MNGIE patients involve T --> C transitions in sequences containing two As to the 5' side of a T residue. Our finding of dTTP and dGTP elevations and dATP depletion in mitochondrial dNTP pools are consistent with a mutagenic mechanism involving T-G mispairing followed by a next-nucleotide effect involving T insertion opposite A. PMID:13679382

  13. Evaluation of DNA pooling for the estimation of microsatellite allele frequencies: a case study using striped bass (Morone saxatilis).

    PubMed

    Skalski, Garrick T; Couch, Charlene R; Garber, Amber F; Weir, Bruce S; Sullivan, Craig V

    2006-06-01

    Using striped bass (Morone saxatilis) and six multiplexed microsatellite markers, we evaluated procedures for estimating allele frequencies by pooling DNA from multiple individuals, a method suggested as cost-effective relative to individual genotyping. Using moment-based estimators, we estimated allele frequencies in experimental DNA pools and found that the three primary laboratory steps, DNA quantitation and pooling, PCR amplification, and electrophoresis, accounted for 23, 48, and 29%, respectively, of the technical variance of estimates in pools containing DNA from 2-24 individuals. Exact allele-frequency estimates could be made for pools of sizes 2-8, depending on the locus, by using an integer-valued estimator. Larger pools of size 12 and 24 tended to yield biased estimates; however, replicates of these estimates detected allele frequency differences among pools with different allelic compositions. We also derive an unbiased estimator of Hardy-Weinberg disequilibrium coefficients that uses multiple DNA pools and analyze the cost-efficiency of DNA pooling. DNA pooling yields the most potential cost savings when a large number of loci are employed using a large number of individuals, a situation becoming increasingly common as microsatellite loci are developed in increasing numbers of taxa. PMID:16582444

  14. The deoxynucleotide triphosphohydrolase SAMHD1 is a major regulator of DNA precursor pools in mammalian cells

    PubMed Central

    Franzolin, Elisa; Pontarin, Giovanna; Rampazzo, Chiara; Miazzi, Cristina; Ferraro, Paola; Palumbo, Elisa; Reichard, Peter; Bianchi, Vera

    2013-01-01

    Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is a triphosphohydrolase converting deoxynucleoside triphosphates (dNTPs) to deoxynucleosides. The enzyme was recently identified as a component of the human innate immune system that restricts HIV-1 infection by removing dNTPs required for viral DNA synthesis. SAMHD1 has deep evolutionary roots and is ubiquitous in human organs. Here we identify a general function of SAMHD1 in the regulation of dNTP pools in cultured human cells. The protein was nuclear and variably expressed during the cell cycle, maximally during quiescence and minimally during S-phase. Treatment of lung or skin fibroblasts with specific siRNAs resulted in the disappearence of SAMHD1 accompanied by loss of the cell-cycle regulation of dNTP pool sizes and dNTP imbalance. Cells accumulated in G1 phase with oversized pools and stopped growing. Following removal of the siRNA, the pools were normalized and cell growth restarted, but only after SAMHD1 had reappeared. In quiescent cultures SAMHD1 down-regulation leads to a marked expansion of dNTP pools. In all cases the largest effect was on dGTP, the preferred substrate of SAMHD1. Ribonucleotide reductase, responsible for the de novo synthesis of dNTPs, is a cytosolic enzyme maximally induced in S-phase cells. Thus, in mammalian cells the cell cycle regulation of the two main enzymes controlling dNTP pool sizes is adjusted to the requirements of DNA replication. Synthesis by the reductase peaks during S-phase, and catabolism by SAMHD1 is maximal during G1 phase when large dNTP pools would prevent cells from preparing for a new round of DNA replication. PMID:23858451

  15. Mitochondrial DNA Replication Defects Disturb Cellular dNTP Pools and Remodel One-Carbon Metabolism.

    PubMed

    Nikkanen, Joni; Forsström, Saara; Euro, Liliya; Paetau, Ilse; Kohnz, Rebecca A; Wang, Liya; Chilov, Dmitri; Viinamäki, Jenni; Roivainen, Anne; Marjamäki, Päivi; Liljenbäck, Heidi; Ahola, Sofia; Buzkova, Jana; Terzioglu, Mügen; Khan, Nahid A; Pirnes-Karhu, Sini; Paetau, Anders; Lönnqvist, Tuula; Sajantila, Antti; Isohanni, Pirjo; Tyynismaa, Henna; Nomura, Daniel K; Battersby, Brendan J; Velagapudi, Vidya; Carroll, Christopher J; Suomalainen, Anu

    2016-04-12

    Mitochondrial dysfunction affects cellular energy metabolism, but less is known about the consequences for cytoplasmic biosynthetic reactions. We report that mtDNA replication disorders caused by TWINKLE mutations-mitochondrial myopathy (MM) and infantile onset spinocerebellar ataxia (IOSCA)-remodel cellular dNTP pools in mice. MM muscle shows tissue-specific induction of the mitochondrial folate cycle, purine metabolism, and imbalanced and increased dNTP pools, consistent with progressive mtDNA mutagenesis. IOSCA-TWINKLE is predicted to hydrolyze dNTPs, consistent with low dNTP pools and mtDNA depletion in the disease. MM muscle also modifies the cytoplasmic one-carbon cycle, transsulfuration, and methylation, as well as increases glucose uptake and its utilization for de novo serine and glutathione biosynthesis. Our evidence indicates that the mitochondrial replication machinery communicates with cytoplasmic dNTP pools and that upregulation of glutathione synthesis through glucose-driven de novo serine biosynthesis contributes to the metabolic stress response. These results are important for disorders with primary or secondary mtDNA instability and offer targets for metabolic therapy. PMID:26924217

  16. Sequences From First Settlers Reveal Rapid Evolution in Icelandic mtDNA Pool

    PubMed Central

    Helgason, Agnar; Lalueza-Fox, Carles; Ghosh, Shyamali; Sigurðardóttir, Sigrún; Sampietro, Maria Lourdes; Gigli, Elena; Baker, Adam; Bertranpetit, Jaume; Árnadóttir, Lilja; Þorsteinsdottir, Unnur; Stefánsson, Kári

    2009-01-01

    A major task in human genetics is to understand the nature of the evolutionary processes that have shaped the gene pools of contemporary populations. Ancient DNA studies have great potential to shed light on the evolution of populations because they provide the opportunity to sample from the same population at different points in time. Here, we show that a sample of mitochondrial DNA (mtDNA) control region sequences from 68 early medieval Icelandic skeletal remains is more closely related to sequences from contemporary inhabitants of Scotland, Ireland, and Scandinavia than to those from the modern Icelandic population. Due to a faster rate of genetic drift in the Icelandic mtDNA pool during the last 1,100 years, the sequences carried by the first settlers were better preserved in their ancestral gene pools than among their descendants in Iceland. These results demonstrate the inferential power gained in ancient DNA studies through the application of population genetics analyses to relatively large samples. PMID:19148284

  17. Unbalanced deoxynucleotide pools cause mitochondrial DNA instability in thymidine phosphorylase-deficient mice.

    PubMed

    López, Luis C; Akman, Hasan O; García-Cazorla, Angeles; Dorado, Beatriz; Martí, Ramón; Nishino, Ichizo; Tadesse, Saba; Pizzorno, Giuseppe; Shungu, Dikoma; Bonilla, Eduardo; Tanji, Kurenai; Hirano, Michio

    2009-02-15

    Replication and repair of DNA require equilibrated pools of deoxynucleoside triphosphate precursors. This concept has been proven by in vitro studies over many years, but in vivo models are required to demonstrate its relevance to multicellular organisms and to human diseases. Accordingly, we have generated thymidine phosphorylase (TP) and uridine phosphorylase (UP) double knockout (TP(-/-)UP(-/-)) mice, which show severe TP deficiency, increased thymidine and deoxyuridine in tissues and elevated mitochondrial deoxythymidine triphosphate. As consequences of the nucleotide pool imbalances, brains of mutant mice developed partial depletion of mtDNA, deficiencies of respiratory chain complexes and encephalopathy. These findings largely account for the pathogenesis of mitochondrial neurogastrointestinal encephalopathy (MNGIE), the first inherited human disorder of nucleoside metabolism associated with somatic DNA instability. PMID:19028666

  18. Pooling optimal combinations of energy thresholds in spectroscopic CT

    NASA Astrophysics Data System (ADS)

    Koenig, Thomas; Zuber, Marcus; Hamann, Elias; Runz, Armin; Fiederle, Michael; Baumbach, Tilo

    2014-03-01

    Photon counting detectors used in spectroscopic CT are often based on small pixels and therefore offer only limited space to include energy discriminators and their associated counters in each pixel cell. For this reason, it is important to make efficient use of the available energy discriminators in order to achieve an optimized material contrast at a radiation dose as low as possible. Unfortunately, the complexity of evaluating every possible combination of energy thresholds, given a fixed number of counters, rapidly increases with the resolution at which this search is performed, and makes brute-force approaches to this problem infeasible. In this work, we introduce methods from machine learning, in particular sparse regression, to perform a feature selection to determine optimal combinations of energy thresholds. We will demonstrate how methods enforcing row-sparsity on a linear regression's coefficient matrix can be applied to the multiple response problem in spectroscopic CT, i.e. the case in which a single set of energy thresholds is sought to simultaneously retrieve concentrations pertaining to a multitude of materials in an optimal way. These methods are applied to CT images experimentally obtained with a Medipix3RX detector operated in charge summing mode and with a CdTe sensor at a pixel pitch of 110μm. We show that the least absolute shrinkage and selection operator (lasso), generalized to the multiple response case, chooses four out of 20 possible threshold positions that allow discriminating PMMA, iodine and gadolinium in a contrast agent phantom at a higher accuracy than with equally spaced thresholds. Finally, we illustrate why it might be unwise to use a higher number of energy thresholds than absolutely necessary.

  19. Modulation of mutagenesis in eukaryotes by DNA replication fork dynamics and quality of nucleotide pools

    PubMed Central

    Waisertreiger, Irina S.-R.; Liston, Victoria G.; Menezes, Miriam R.; Kim, Hyun-Min; Lobachev, Kirill S.; Stepchenkova, Elena I.; Tahirov, Tahir H.; Rogozin, Igor B.; Pavlov, Youri. I.

    2014-01-01

    The rate of mutations in eukaryotes depends on a plethora of factors and is not immediately derived from the fidelity of DNA polymerases (Pols). Replication of chromosomes containing the anti-parallel strands of duplex DNA occurs through the copying of leading and lagging strand templates by a trio of Pols α, δ and ε, with the assistance of Pol ζ and Y-family Pols at difficult DNA template structures or sites of DNA damage. The parameters of the synthesis at a given location are dictated by the quality and quantity of nucleotides in the pools, replication fork architecture, transcription status, regulation of Pol switches, and structure of chromatin. The result of these transactions is a subject of survey and editing by DNA repair. PMID:23055184

  20. Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA

    PubMed Central

    Gemble, Simon; Ahuja, Akshay; Buhagiar-Labarchède, Géraldine; Onclercq-Delic, Rosine; Dairou, Julien; Biard, Denis S. F.; Lambert, Sarah; Lopes, Massimo; Amor-Guéret, Mounira

    2015-01-01

    Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at “difficult-to-replicate” sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3’-5’ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects. PMID:26181065

  1. Swimming Pools.

    ERIC Educational Resources Information Center

    Ministry of Housing and Local Government, London (England).

    Technical and engineering data are set forth on the design and construction of swimming pools. Consideration is given to site selection, pool construction, the comparative merits of combining open air and enclosed pools, and alternative uses of the pool. Guidelines are presented regarding--(1) pool size and use, (2) locker and changing rooms, (3)…

  2. Extra-binomial variation approach for analysis of pooled DNA sequencing data

    PubMed Central

    Wallace, Chris

    2012-01-01

    Motivation: The invention of next-generation sequencing technology has made it possible to study the rare variants that are more likely to pinpoint causal disease genes. To make such experiments financially viable, DNA samples from several subjects are often pooled before sequencing. This induces large between-pool variation which, together with other sources of experimental error, creates over-dispersed data. Statistical analysis of pooled sequencing data needs to appropriately model this additional variance to avoid inflating the false-positive rate. Results: We propose a new statistical method based on an extra-binomial model to address the over-dispersion and apply it to pooled case-control data. We demonstrate that our model provides a better fit to the data than either a standard binomial model or a traditional extra-binomial model proposed by Williams and can analyse both rare and common variants with lower or more variable pool depths compared to the other methods. Availability: Package ‘extraBinomial’ is on http://cran.r-project.org/ Contact: chris.wallace@cimr.cam.ac.uk Supplementary information: Supplementary data are available at Bioinformatics Online. PMID:22976083

  3. Mitochondrial DNA depletion and thymidine phosphate pool dynamics in a cellular model of mitochondrial neurogastrointestinal encephalomyopathy.

    PubMed

    Pontarin, Giovanna; Ferraro, Paola; Valentino, Maria L; Hirano, Michio; Reichard, Peter; Bianchi, Vera

    2006-08-11

    Mitochondrial (mt) neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease associated with depletion, deletions, and point mutations of mtDNA. Patients lack a functional thymidine phosphorylase and their plasma contains high concentrations of thymidine and deoxyuridine; elevation of the corresponding triphosphates probably impairs normal mtDNA replication and repair. To study metabolic events leading to MNGIE we used as model systems skin and lung fibroblasts cultured in the presence of thymidine and/or deoxyuridine at concentrations close to those in the plasma of the patients, a more than 100-fold excess relative to controls. The two deoxynucleosides increased the mt and cytosolic dTTP pools of skin fibroblasts almost 2-fold in cycling cells and 8-fold in quiescent cells. During up to a two-month incubation of quiescent fibroblasts with thymidine (but not with deoxyuridine), mtDNA decreased to approximately 50% without showing deletions or point mutations. When we removed thymidine, but maintained the quiescent state, mtDNA recovered rapidly. With thymidine in the medium, the dTTP pool of quiescent cells turned over rapidly at a rate depending on the concentration of thymidine, due to increased degradation and resynthesis of dTMP in a substrate (=futile) cycle between thymidine kinase and 5'-deoxyribonucleotidase. The cycle limited the expansion of the dTTP pool at the expense of ATP hydrolysis. We propose that the substrate cycle represents a regulatory mechanism to protect cells from harmful increases of dTTP. Thus MNGIE patients may increase their consumption of ATP to counteract an unlimited expansion of the dTTP pool caused by circulating thymidine. PMID:16774911

  4. A Molecular Bar-Coded DNA Repair Resource for Pooled Toxicogenomic Screens

    PubMed Central

    Rooney, John P.; Patil, Ashish; Zappala, Maria R.; Conklin, Douglas S.; Cunningham, Richard P.; Begley, Thomas J.

    2008-01-01

    DNA damage from exogenous and endogenous sources can promote mutations and cell death. Fortunately, cells contain DNA repair and damage signalling pathways to reduce the mutagenic and cytotoxic effects of DNA damage. The identification of specific DNA repair proteins and the coordination of DNA repair pathways after damage has been a central theme to the field of Genetic Toxicology and we have developed a tool for use in this area. We have produced 99 molecular bar-coded Escherichia coli gene-deletion mutants specific to DNA repair and damage signalling pathways, and each bar-coded mutant can be tracked in pooled format using bar-code specific microarrays. Our design adapted bar-codes developed for the Saccharomyces cerevisiae Gene Deletion Project, which allowed us to utilize an available microarray product for pooled gene-exposure studies. Microarray-based screens were used for en masse identification of individual mutants sensitive to methyl methanesulfonate (MMS). As expected, gene deletion mutants specific to direct, base excision, and recombinational DNA repair pathways were identified as MMS-sensitive in our pooled assay, thus validating our resource. We have demonstrated that molecular bar-codes designed for S. cerevisiae are transferable to E. coli, and that they can be used with pre-existing microarrays to perform competitive growth experiments. Further, when comparing microarray to traditional plate-based screens both over-lapping and distinct results were obtained, which is a novel technical finding, with discrepancies between the two approaches explained by differences in output measurements (DNA content verse cell mass). The microarray-based classification of Δtag and ΔdinG cells as depleted after MMS exposure, contrary to plate-based methods, led to the discovery that Δtag and ΔdinG cells show a filamentation phenotype after MMS exposure, thus accounting for the discrepancy. A novel biological finding is the observation that while ΔdinG cells

  5. Finding Markers That Make a Difference: DNA Pooling and SNP-Arrays Identify Population Informative Markers for Genetic Stock Identification

    PubMed Central

    Ozerov, Mikhail; Vasemägi, Anti; Wennevik, Vidar; Diaz-Fernandez, Rogelio; Kent, Matthew; Gilbey, John; Prusov, Sergey; Niemelä, Eero; Vähä, Juha-Pekka

    2013-01-01

    Genetic stock identification (GSI) using molecular markers is an important tool for management of migratory species. Here, we tested a cost-effective alternative to individual genotyping, known as allelotyping, for identification of highly informative SNPs for accurate genetic stock identification. We estimated allele frequencies of 2880 SNPs from DNA pools of 23 Atlantic salmon populations using Illumina SNP-chip. We evaluated the performance of four common strategies (global FST, pairwise FST, Delta and outlier approach) for selection of the most informative set of SNPs and tested their effectiveness for GSI compared to random sets of SNP and microsatellite markers. For the majority of cases, SNPs selected using the outlier approach performed best followed by pairwise FST and Delta methods. Overall, the selection procedure reduced the number of SNPs required for accurate GSI by up to 53% compared with randomly chosen SNPs. However, GSI accuracy was more affected by populations in the ascertainment group rather than the ranking method itself. We demonstrated for the first time the compatibility of different large-scale SNP datasets by compiling the largest population genetic dataset for Atlantic salmon to date. Finally, we showed an excellent performance of our top SNPs on an independent set of populations covering the main European distribution range of Atlantic salmon. Taken together, we demonstrate how combination of DNA pooling and SNP arrays can be applied for conservation and management of salmonids as well as other species. PMID:24358184

  6. Finding markers that make a difference: DNA pooling and SNP-arrays identify population informative markers for genetic stock identification.

    PubMed

    Ozerov, Mikhail; Vasemägi, Anti; Wennevik, Vidar; Diaz-Fernandez, Rogelio; Kent, Matthew; Gilbey, John; Prusov, Sergey; Niemelä, Eero; Vähä, Juha-Pekka

    2013-01-01

    Genetic stock identification (GSI) using molecular markers is an important tool for management of migratory species. Here, we tested a cost-effective alternative to individual genotyping, known as allelotyping, for identification of highly informative SNPs for accurate genetic stock identification. We estimated allele frequencies of 2880 SNPs from DNA pools of 23 Atlantic salmon populations using Illumina SNP-chip. We evaluated the performance of four common strategies (global F ST, pairwise F ST, Delta and outlier approach) for selection of the most informative set of SNPs and tested their effectiveness for GSI compared to random sets of SNP and microsatellite markers. For the majority of cases, SNPs selected using the outlier approach performed best followed by pairwise F ST and Delta methods. Overall, the selection procedure reduced the number of SNPs required for accurate GSI by up to 53% compared with randomly chosen SNPs. However, GSI accuracy was more affected by populations in the ascertainment group rather than the ranking method itself. We demonstrated for the first time the compatibility of different large-scale SNP datasets by compiling the largest population genetic dataset for Atlantic salmon to date. Finally, we showed an excellent performance of our top SNPs on an independent set of populations covering the main European distribution range of Atlantic salmon. Taken together, we demonstrate how combination of DNA pooling and SNP arrays can be applied for conservation and management of salmonids as well as other species. PMID:24358184

  7. The efficacy of detecting variants with small effects on the Affymetrix 6.0 platform using pooled DNA

    PubMed Central

    Chiang, Charleston W. K.; Gajdos, Zofia K. Z.; Butler, Johannah L.; Hackett, Rachel; Guiducci, Candace; Nguyen, Thutrang T.; Wilks, Rainford; Forrester, Terrence; Henderson, Katherine D.; Le Marchand, Loic; Henderson, Brian E.; Haiman, Christopher A.; Cooper, Richard S.; Lyon, Helen N.; Zhu, Xiaofeng; McKenzie, Colin A.; Palmer, Mark R.; Hirschhorn, Joel N.

    2012-01-01

    Genome-wide genotyping of a cohort using pools rather than individual samples has long been proposed as a cost-saving alternative for performing genome-wide association (GWA) studies. However, successful disease gene mapping using pooled genotyping has thus far been limited to detecting common variants with large effect sizes, which tend not to exist for many complex common diseases or traits. Therefore, for DNA pooling to be a viable strategy for conducting GWA studies, it is important to determine whether commonly used genome-wide SNP array platforms such as the Affymetrix 6.0 array can reliably detect common variants of small effect sizes using pooled DNA. Taking obesity and age at menarche as examples of human complex traits, we assessed the feasibility of genome-wide genotyping of pooled DNA as a single-stage design for phenotype association. By individually genotyping the top associations identified by pooling, we obtained a 14- to 16-fold enrichment of SNPs nominally associated with the phenotype, but we likely missed the top true associations. In addition, we assessed whether genotyping pooled DNA can serve as an inexpensive screen as the second stage of a multi-stage design with a large number of samples by comparing the most cost-effective 3-stage designs with 80% power to detect common variants with genotypic relative risk of 1.1, with and without pooling. Given the current state of the specific technology we employed and the associated genotyping costs, we showed through simulation that a design involving pooling would be 1.07 times more expensive than a design without pooling. Thus, while a significant amount of information exists within the data from pooled DNA, our analysis does not support genotyping pooled DNA as a means to efficiently identify common variants contributing small effects to phenotypes of interest. While our conclusions were based on the specific technology and study design we employed, the approach presented here will be useful for

  8. Detection of obesity QTLs on mouse chromosomes 1 and 7 by selective DNA pooling

    SciTech Connect

    Taylor, B.A.; Phillips, S.J.

    1996-06-15

    The inheritance of obesity has been analyzed in an intercross between the lean 129/Sv mouse strain and the obesity-prone EL/Suz mouse strain. The weights of three major fat pads were determined on 4-month-old mice, and the sum of these weights, divided by body weight, was used as an adiposity index. The strategy of selective DNA pooling was used as a primary screen to identify putative quantitative trait loci (QTLs) affecting adiposity index. DNA pools representing the leanest 15% and fattest 15% of the F2 progeny were compared for differential allelic enrichment using widely dispersed microsatellite variants. To evaluate putative QTLs, individual genotyping and interval mapping were employed to estimate QTL effects and assess statistical significance. One QTL affecting adiposity index, which accounted for 12.3% of phenotypic variance in gender-merged data, was mapped to the central region of Chromosome (Chr) 7. The QTL allele inherited from EL conferred increased adiposity. A second QTL that accounts for 6.3% of phenotypic variance was identified on Chr 1 near D1Mitt211. At both QTLs, the data are consistent with dominant inheritance of the allele contributing to obesity. The possible relationships between these QTLs and previously described obesity QYLs, major obesity mutations, and candidate genes are discussed. 42 refs., 3 figs., 3 tabs.

  9. A DNA pooling based system to detect Escherichia coli virulence factors in fecal and wastewater samples

    PubMed Central

    Luz María Chacón, J; Lizeth Taylor, C; Carmen Valiente, A; Irene Alvarado, P; Ximena Cortés, B

    2012-01-01

    The availability of a useful tool for simple and timely detection of the most important virulent varieties of Escherichia coli is indispensable. To this end, bacterial DNA pools which had previously been categorized were obtained from isolated colonies as well as selected in terms of utilized phenotype; the pools were assessed by two PCR Multiplex for the detection of virulent E. coli eaeA, bfpA, stx1, stx2, ipaH, ST, LT, and aatA genes, with the 16S gene used as DNA control. The system was validated with 66 fecal samples and 44 wastewater samples. At least one positive isolate was detected by a virulent gene among the 20 that were screened. The analysis of fecal samples from children younger than 6 years of age detected frequencies of 25% LT positive strains, 8.3% eae, 8.3% bfpA, 16.7% ipaH, as well as 12.5 % aatA and ST. On the other hand, wastewater samples revealed frequencies of 25.7% eaeA positive, 30.3% stx1, 15.1% LT and 19.7% aatA. This study is an initial step toward carrying out epidemiological field research that will reveal the presence of these bacterial varieties. PMID:24031959

  10. Cost-effective genome-wide estimation of allele frequencies from pooled DNA in Atlantic salmon (Salmo salar L.)

    PubMed Central

    2013-01-01

    Background New sequencing technologies have tremendously increased the number of known molecular markers (single nucleotide polymorphisms; SNPs) in a variety of species. Concurrently, improvements to genotyping technology have now made it possible to efficiently genotype large numbers of genome-wide distributed SNPs enabling genome wide association studies (GWAS). However, genotyping significant numbers of individuals with large number of SNPs remains prohibitively expensive for many research groups. A possible solution to this problem is to determine allele frequencies from pooled DNA samples, such ‘allelotyping’ has been presented as a cost-effective alternative to individual genotyping and has become popular in human GWAS. In this article we have tested the effectiveness of DNA pooling to obtain accurate allele frequency estimates for Atlantic salmon (Salmo salar L.) populations using an Illumina SNP-chip. Results In total, 56 Atlantic salmon DNA pools from 14 populations were analyzed on an Atlantic salmon SNP-chip containing probes for 5568 SNP markers, 3928 of which were bi-allelic. We developed an efficient quality control filter which enables exclusion of loci showing high error rate and minor allele frequency (MAF) close to zero. After applying multiple quality control filters we obtained allele frequency estimates for 3631 bi-allelic loci. We observed high concordance (r > 0.99) between allele frequency estimates derived from individual genotyping and DNA pools. Our results also indicate that even relatively small DNA pools (35 individuals) can provide accurate allele frequency estimates for a given sample. Conclusions Despite of higher level of variation associated with array replicates compared to pool construction, we suggest that both sources of variation should be taken into account. This study demonstrates that DNA pooling allows fast and high-throughput determination of allele frequencies in Atlantic salmon enabling cost

  11. International Lung Cancer Consortium: Pooled Analysis of Sequence Variants in DNA Repair and Cell Cycle Pathways

    PubMed Central

    Hung, Rayjean J.; Christiani, David C.; Risch, Angela; Popanda, Odilia; Haugen, Aage; Zienolddiny, Shan; Benhamou, Simone; Bouchardy, Christine; Lan, Qing; Spitz, Margaret R.; Wichmann, H.-Erich; LeMarchand, Loic; Vineis, Paolo; Matullo, Giuseppe; Kiyohara, Chikako; Zhang, Zuo-Feng; Pezeshki, Benhnaz; Harris, Curtis; Mechanic, Leah; Seow, Adeline; Ng, Daniel P.K.; Szeszenia-Dabrowska, Neonila; Zaridze, David; Lissowska, Jolanta; Rudnai, Peter; Fabianova, Eleonora; Mates, Dana; Foretova, Lenka; Janout, Vladimir; Bencko, Vladimir; Caporaso, Neil; Chen, Chu; Duell, Eric J.; Goodman, Gary; Field, John K.; Houlston, Richard S.; Hong, Yun-Chul; Landi, Maria Teresa; Lazarus, Philip; Muscat, Joshua; McLaughlin, John; Schwartz, Ann G.; Shen, Hongbing; Stucker, Isabelle; Tajima, Kazuo; Matsuo, Keitaro; Thun, Michael; Yang, Ping; Wiencke, John; Andrew, Angeline S.; Monnier, Stephanie; Boffetta, Paolo; Brennan, Paul

    2009-01-01

    Background The International Lung Cancer Consortium was established in 2004. To clarify the role of DNA repair genes in lung cancer susceptibility, we conducted a pooled analysis of genetic variants in DNA repair pathways, whose associations have been investigated by at least 3 individual studies. Methods Data from 14 studies were pooled for 18 sequence variants in 12 DNA repair genes, including APEX1, OGG1, XRCC1, XRCC2, XRCC3, ERCC1, XPD, XPF, XPG, XPA, MGMT, and TP53. The total number of subjects included in the analysis for each variant ranged from 2,073 to 13,955 subjects. Results Four of the variants were found to be weakly associated with lung cancer risk with borderline significance: these were XRCC3 T241M [heterozygote odds ratio (OR), 0.89; 95% confidence interval (95% CI), 0.79–0.99 and homozygote OR, 0.84; 95% CI, 0.71–1.00] based on 3,467 cases and 5,021 controls from 8 studies, XPD K751Q (heterozygote OR, 0.99; 95% CI, 0.89–1.10 and homozygote OR, 1.19; 95% CI, 1.02–1.39) based on 6,463 cases and 6,603 controls from 9 studies, and TP53 R72P (heterozygote OR, 1.14; 95% CI, 1.00–1.29 and homozygote OR, 1.20; 95% CI, 1.02–1.42) based on 3,610 cases and 5,293 controls from 6 studies. OGG1 S326C homozygote was suggested to be associated with lung cancer risk in Caucasians (homozygote OR, 1.34; 95% CI, 1.01–1.79) based on 2,569 cases and 4,178 controls from 4 studies but not in Asians. The other 14 variants did not exhibit main effects on lung cancer risk. Discussion In addition to data pooling, future priorities of International Lung Cancer Consortium include coordinated genotyping and multistage validation for ongoing genome-wide association studies. PMID:18990748

  12. Tracing European Founder Lineages in the Near Eastern mtDNA Pool

    PubMed Central

    Richards, Martin; Macaulay, Vincent; Hickey, Eileen; Vega, Emilce; Sykes, Bryan; Guida, Valentina; Rengo, Chiara; Sellitto, Daniele; Cruciani, Fulvio; Kivisild, Toomas; Villems, Richard; Thomas, Mark; Rychkov, Serge; Rychkov, Oksana; Rychkov, Yuri; Gölge, Mukaddes; Dimitrov, Dimitar; Hill, Emmeline; Bradley, Dan; Romano, Valentino; Calì, Francesco; Vona, Giuseppe; Demaine, Andrew; Papiha, Surinder; Triantaphyllidis, Costas; Stefanescu, Gheorghe; Hatina, Jiři; Belledi, Michele; Di Rienzo, Anna; Oppenheim, Ariella; Nørby, Søren; Al-Zaheri, Nadia; Santachiara-Benerecetti, Silvana; Scozzari, Rosaria; Torroni, Antonio; Bandelt, Hans-Jürgen

    2000-01-01

    Founder analysis is a method for analysis of nonrecombining DNA sequence data, with the aim of identification and dating of migrations into new territory. The method picks out founder sequence types in potential source populations and dates lineage clusters deriving from them in the settlement zone of interest. Here, using mtDNA, we apply the approach to the colonization of Europe, to estimate the proportion of modern lineages whose ancestors arrived during each major phase of settlement. To estimate the Palaeolithic and Neolithic contributions to European mtDNA diversity more accurately than was previously achievable, we have now extended the Near Eastern, European, and northern-Caucasus databases to 1,234, 2,804, and 208 samples, respectively. Both back-migration into the source population and recurrent mutation in the source and derived populations represent major obstacles to this approach. We have developed phylogenetic criteria to take account of both these factors, and we suggest a way to account for multiple dispersals of common sequence types. We conclude that (i) there has been substantial back-migration into the Near East, (ii) the majority of extant mtDNA lineages entered Europe in several waves during the Upper Palaeolithic, (iii) there was a founder effect or bottleneck associated with the Last Glacial Maximum, 20,000 years ago, from which derives the largest fraction of surviving lineages, and (iv) the immigrant Neolithic component is likely to comprise less than one-quarter of the mtDNA pool of modern Europeans. PMID:11032788

  13. Admixture Aberration Analysis: Application to Mapping in Admixed Population Using Pooled DNA

    NASA Astrophysics Data System (ADS)

    Bercovici, Sivan; Geiger, Dan

    Admixture mapping is a gene mapping approach used for the identification of genomic regions harboring disease susceptibility genes in the case of recently admixed populations such as African Americans. We present a novel method for admixture mapping, called admixture aberration analysis (AAA), that uses a DNA pool of affected admixed individuals. We demonstrate through simulations that AAA is a powerful and economical mapping method under a range of scenarios, capturing complex human diseases such as hypertension and end stage kidney disease. The method has a low false-positive rate and is robust to deviation from model assumptions. Finally, we apply AAA on 600 prostate cancer-affected African Americans, replicating a known risk locus. Simulation results indicate that the method can yield over 96% reduction in genotyping. Our method is implemented as a Java program called AAAmap and is freely available.

  14. Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples

    PubMed Central

    2012-01-01

    Background The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility. Results In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9% (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom® MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total). Conclusions The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post

  15. Effective High-Throughput Blood Pooling Strategy before DNA Extraction for Detection of Malaria in Low-Transmission Settings

    PubMed Central

    Nyunt, Myat Htut; Kyaw, Myat Phone; Thant, Kyaw Zin; Shein, Thinzer; Han, Soe Soe; Zaw, Ni Ni; Han, Jin-Hee; Lee, Seong-Kyun; Muh, Fauzi; Kim, Jung-Yeon; Cho, Shin-Hyeong; Lee, Sang-Eun; Yang, Eun-Jeong; Chang, Chulhun L.; Han, Eun-Taek

    2016-01-01

    In the era of (pre) elimination setting, the prevalence of malaria has been decreasing in most of the previously endemic areas. Therefore, effective cost- and time-saving validated pooling strategy is needed for detection of malaria in low transmission settings. In this study, optimal pooling numbers and lowest detection limit were assessed using known density samples prepared systematically, followed by genomic DNA extraction and nested PCR. Pooling strategy that composed of 10 samples in 1 pool, 20 µl in 1 sample, was optimal, and the parasite density as low as 2 p/µl for both falciparum and vivax infection was enough for detection of malaria. This pooling method showed effectiveness for handling of a huge number of samples in low transmission settings (<9% positive rate). The results indicated that pooling of the blood samples before DNA extraction followed by usual nested PCR is useful and effective for detection of malaria in screening of hidden cases in low-transmission settings. PMID:27417078

  16. Effective High-Throughput Blood Pooling Strategy before DNA Extraction for Detection of Malaria in Low-Transmission Settings.

    PubMed

    Nyunt, Myat Htut; Kyaw, Myat Phone; Thant, Kyaw Zin; Shein, Thinzer; Han, Soe Soe; Zaw, Ni Ni; Han, Jin-Hee; Lee, Seong-Kyun; Muh, Fauzi; Kim, Jung-Yeon; Cho, Shin-Hyeong; Lee, Sang-Eun; Yang, Eun-Jeong; Chang, Chulhun L; Han, Eun-Taek

    2016-06-01

    In the era of (pre) elimination setting, the prevalence of malaria has been decreasing in most of the previously endemic areas. Therefore, effective cost- and time-saving validated pooling strategy is needed for detection of malaria in low transmission settings. In this study, optimal pooling numbers and lowest detection limit were assessed using known density samples prepared systematically, followed by genomic DNA extraction and nested PCR. Pooling strategy that composed of 10 samples in 1 pool, 20 µl in 1 sample, was optimal, and the parasite density as low as 2 p/µl for both falciparum and vivax infection was enough for detection of malaria. This pooling method showed effectiveness for handling of a huge number of samples in low transmission settings (<9% positive rate). The results indicated that pooling of the blood samples before DNA extraction followed by usual nested PCR is useful and effective for detection of malaria in screening of hidden cases in low-transmission settings. PMID:27417078

  17. Combining of different data pools for calculating a reliable POD for real defects

    NASA Astrophysics Data System (ADS)

    Kanzler, Daniel; Müller, Christina; Pitkänen, Jorma

    2015-03-01

    Real defects are essential for the evaluation of the reliability of non destructive testing (NDT) methods, especially in relation to the integrity of components. But in most of the cases the amount of available real defects is not sufficient to evaluate the system. Model-assisted and transfer functions are one way to handle that challenge. This study is focused on a combination of different data pools to create a sufficient amount of data for the reliability estimation. A widespread approach for calculating the Probability of Detection (POD) was used on a radiographic testing (RT) method. The highest contrast to noise ratio (CNR) of each indication is usually selected as the signal in the "â vs. a" (signal-response) approach for RT. By combining real and artificial defects (flat bottom holes, side drill holes, flat bottom squares, notches, etc) in RT the highest signals are close to each other, but the process of creating and evaluating real defects is much more complex. The solution is seen in the combination of real and artificial data using a weighted least square approach. The weights for real or artificial data were based on the importance, the value and the different detection behavior of the different data. For comparison, the alternative combination through the Bayesian Updating was also applied. As verification, a data pool with a large amount of real data was available. In an advanced approach for evaluating the digital RT data, the size of the indication (perpendicular to the X-ray beam) was introduced as additional information. The signal now consists of the CNR and the area of the indication. The detectability is changing depending on the area of the indication, a fact that was ignored in the previous POD calculations for RT. This points out that a weighted least square approach to pool the data might no longer be adequate. The Bayesian Updating of the estimated parameters of the relationship between the signal field (the area of the indication) and

  18. Combining of different data pools for calculating a reliable POD for real defects

    SciTech Connect

    Kanzler, Daniel E-mail: christina.mueller@bam.de; Müller, Christina E-mail: christina.mueller@bam.de; Pitkänen, Jorma

    2015-03-31

    Real defects are essential for the evaluation of the reliability of non destructive testing (NDT) methods, especially in relation to the integrity of components. But in most of the cases the amount of available real defects is not sufficient to evaluate the system. Model-assisted and transfer functions are one way to handle that challenge. This study is focused on a combination of different data pools to create a sufficient amount of data for the reliability estimation. A widespread approach for calculating the Probability of Detection (POD) was used on a radiographic testing (RT) method. The highest contrast to noise ratio (CNR) of each indication is usually selected as the signal in the 'â vs. a' (signal-response) approach for RT. By combining real and artificial defects (flat bottom holes, side drill holes, flat bottom squares, notches, etc) in RT the highest signals are close to each other, but the process of creating and evaluating real defects is much more complex. The solution is seen in the combination of real and artificial data using a weighted least square approach. The weights for real or artificial data were based on the importance, the value and the different detection behavior of the different data. For comparison, the alternative combination through the Bayesian Updating was also applied. As verification, a data pool with a large amount of real data was available. In an advanced approach for evaluating the digital RT data, the size of the indication (perpendicular to the X-ray beam) was introduced as additional information. The signal now consists of the CNR and the area of the indication. The detectability is changing depending on the area of the indication, a fact that was ignored in the previous POD calculations for RT. This points out that a weighted least square approach to pool the data might no longer be adequate. The Bayesian Updating of the estimated parameters of the relationship between the signal field (the area of the indication) and

  19. Microarray-based estimation of SNP allele-frequency in pooled DNA using the Langmuir kinetic model

    PubMed Central

    Yin, Bin-Cheng; Li, Honghua; Ye, Bang-Ce

    2008-01-01

    Background High throughput genotyping of single nucleotide polymorphisms (SNPs) for genome-wide association requires technologies for generating millions of genotypes with relative ease but also at a reasonable cost and with high accuracy. In this work, we have developed a theoretical approach to estimate allele frequency in pooled DNA samples, based on the physical principles of DNA immobilization and hybridization on solid surface using the Langmuir kinetic model and quantitative analysis of the allelic signals. Results This method can successfully distinguish allele frequencies differing by 0.01 in the actual pool of clinical samples, and detect alleles with a frequency as low as 2%. The accuracy of measuring known allele frequencies is very high, with the strength of correlation between measured and actual frequencies having an r2 = 0.9992. These results demonstrated that this method could allow the accurate estimation of absolute allele frequencies in pooled samples of DNA in a feasible and inexpensive way. Conclusion We conclude that this novel strategy for quantitative analysis of the ratio of SNP allelic sequences in DNA pools is an inexpensive and feasible alternative for detecting polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans. PMID:19087310

  20. Disinfection of herbal spa pool using combined chlorine dioxide and sodium hypochlorite treatment.

    PubMed

    Hsu, Ching-Shan; Huang, Da-Ji

    2015-02-01

    The presence of pathogenic microorganisms in public spa pools poses a serious threat to human health. The problem is particularly acute in herbal spas, in which the herbs and microorganisms may interact and produce undesirable consequences. Accordingly, the present study investigated the effectiveness of a combined disinfectant containing chlorine dioxide and sodium hypochlorite in improving the water quality of a public herbal spa in Taiwan. Water samples were collected from the spa pool and laboratory tests were then performed to measure the variation over time of the microorganism content (total CFU and total coliforms) and residual disinfectant content given a single disinfection mode (SDM) with disinfectant concentrations of 5.2 × 10, 6.29 × 10, 7.4 × 10, and 11.4 × 10(-5) N, respectively. Utilizing the experience gained from the laboratory tests, a further series of on-site investigations was performed using three different disinfection modes, namely SDM, 3DM (once every 3 h disinfection mode), and 2DM (once every 2 h disinfection mode). The laboratory results showed that for all four disinfectant concentrations, the CFU concentration reduced for the first 6 h following SDM treatment, but then increased. Moreover, the ANOVA results showed that the sample treated with the highest disinfectant concentration (11.4 × 10(-5) N) exhibited the lowest rate of increase in the CFU concentration. In addition, the on-site test results showed that 3DM and 2DM treatments with disinfectant concentrations in excess of 9.3 × 10 and 5.5 × 10(-5) N, respectively, provided an effective reduction in the total CFU concentration. In conclusion, the experimental results presented in this study provide a useful source of reference for spa businesses seeking to improve the water quality of their spa pools. PMID:25632897

  1. Detection of deep vein thrombosis: combined flow and blood pool radionuclide venography vs contrast venography.

    PubMed

    Caner, B; Ozmen, M; Dincer, A; Kapucu, O; Bekdik, C

    1991-10-01

    This study was performed to validate the combined study of flow radionuclide venography (FRV) with subsequent 99mTc-red blood cell(RBC) blood pool radionuclide venography(BRV) for the detection of deep vein thrombosis (DVT). Findings in 32 patients with suspected DVT of lower extremities (n = 52) were compared with those of corresponding contrast venograms (CV) serving as a reference method. FRV was performed by using three separate doses of a large 99mTc04-bolus (10-12 cc) injection. The findings were as follows: concerning the detection of DVT in calf veins, agreement between FRV and CV, FRV+BRV and CV, and BRV and CV were 67%, 73% and 60%, respectively. For femoral veins, agreement between FRV and CV was 96%, while it was 87% between BRV and CV. When FRV and BRV of femoral veins were evaluated in combination, 100% agreement between radionuclide and radiologic method was observed. For iliac veins there was no disagreement in comparison of the methods either singly or in combination. In 7.6% of the extremities, collaterals not demonstrated by CV and BRV were visualized only by FRV. Although the radioactive agent was injected in a relatively large volume, all of the calf veins could not be filled even when they were completely patent. It was concluded that a combined study of FRV with BRV improved the diagnostic value of radionuclide venography for the detection of DVT in calf and femoral veins. PMID:1659257

  2. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types

    PubMed Central

    Lever, Mark A.; Torti, Andrea; Eickenbusch, Philip; Michaud, Alexander B.; Šantl-Temkiv, Tina; Jørgensen, Bo Barker

    2015-01-01

    A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world's oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals. PMID:26042110

  3. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types.

    PubMed

    Lever, Mark A; Torti, Andrea; Eickenbusch, Philip; Michaud, Alexander B; Šantl-Temkiv, Tina; Jørgensen, Bo Barker

    2015-01-01

    A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world's oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals. PMID:26042110

  4. Clines of nuclear DNA markers suggest a largely neolithic ancestry of the European gene pool.

    PubMed

    Chikhi, L; Destro-Bisol, G; Bertorelle, G; Pascali, V; Barbujani, G

    1998-07-21

    Comparisons between archaeological findings and allele frequencies at protein loci suggest that most genes of current Europeans descend from populations that have been expanding in Europe in the last 10, 000 years, in the Neolithic period. Recent mitochondrial data have been interpreted as indicating a much older, Paleolithic ancestry. In a spatial autocorrelation study at seven hypervariable loci in Europe (four microsatellites, two larger, tandem-repeat loci, and a sequence polymorphism) broad clinal patterns of DNA variation were recognized. The observed clines closely match those described at the protein level, in agreement with a possible Near Eastern origin for the ancestral population. Separation times between populations were estimated on the basis of a stepwise mutation model. Even assuming low mutation rates and long generation times, we found no evidence for population splits older than 10,000 years, with the predictable exception of Saami (Lapps). The simplest interpretation of these results is that the current nuclear gene pool largely reflects the westward and northward expansion of a Neolithic group. This conclusion is now supported by purely genetic evidence on the levels and patterns of microsatellite diversity, rather than by correlations of biological and nonbiological data. We argue that many mitochondrial lineages whose origin has been traced back to the Paleolithic period probably reached Europe at a later time. PMID:9671803

  5. DNA Profiling of Convicted Offender Samples for the Combined DNA Index System

    ERIC Educational Resources Information Center

    Millard, Julie T

    2011-01-01

    The cornerstone of forensic chemistry is that a perpetrator inevitably leaves trace evidence at a crime scene. One important type of evidence is DNA, which has been instrumental in both the implication and exoneration of thousands of suspects in a wide range of crimes. The Combined DNA Index System (CODIS), a network of DNA databases, provides…

  6. Reduced Representation Libraries from DNA Pools Analysed with Next Generation Semiconductor Based-Sequencing to Identify SNPs in Extreme and Divergent Pigs for Back Fat Thickness

    PubMed Central

    Bovo, Samuele; Bertolini, Francesca; Schiavo, Giuseppina; Mazzoni, Gianluca; Dall'Olio, Stefania; Fontanesi, Luca

    2015-01-01

    The aim of this study was to identify single nucleotide polymorphisms (SNPs) that could be associated with back fat thickness (BFT) in pigs. To achieve this goal, we evaluated the potential and limits of an experimental design that combined several methodologies. DNA samples from two groups of Italian Large White pigs with divergent estimating breeding value (EBV) for BFT were separately pooled and sequenced, after preparation of reduced representation libraries (RRLs), on the Ion Torrent technology. Taking advantage from SNAPE for SNPs calling in sequenced DNA pools, 39,165 SNPs were identified; 1/4 of them were novel variants not reported in dbSNP. Combining sequencing data with Illumina PorcineSNP60 BeadChip genotyping results on the same animals, 661 genomic positions overlapped with a good approximation of minor allele frequency estimation. A total of 54 SNPs showing enriched alleles in one or in the other RRLs might be potential markers associated with BFT. Some of these SNPs were close to genes involved in obesity related phenotypes. PMID:25821781

  7. Reduced Representation Libraries from DNA Pools Analysed with Next Generation Semiconductor Based-Sequencing to Identify SNPs in Extreme and Divergent Pigs for Back Fat Thickness.

    PubMed

    Bovo, Samuele; Bertolini, Francesca; Schiavo, Giuseppina; Mazzoni, Gianluca; Dall'Olio, Stefania; Fontanesi, Luca

    2015-01-01

    The aim of this study was to identify single nucleotide polymorphisms (SNPs) that could be associated with back fat thickness (BFT) in pigs. To achieve this goal, we evaluated the potential and limits of an experimental design that combined several methodologies. DNA samples from two groups of Italian Large White pigs with divergent estimating breeding value (EBV) for BFT were separately pooled and sequenced, after preparation of reduced representation libraries (RRLs), on the Ion Torrent technology. Taking advantage from SNAPE for SNPs calling in sequenced DNA pools, 39,165 SNPs were identified; 1/4 of them were novel variants not reported in dbSNP. Combining sequencing data with Illumina PorcineSNP60 BeadChip genotyping results on the same animals, 661 genomic positions overlapped with a good approximation of minor allele frequency estimation. A total of 54 SNPs showing enriched alleles in one or in the other RRLs might be potential markers associated with BFT. Some of these SNPs were close to genes involved in obesity related phenotypes. PMID:25821781

  8. A genome-wide study of preferential amplification/hybridization in microarray-based pooled DNA experiments

    PubMed Central

    Yang, H.-C.; Liang, Y.-J.; Huang, M.-C.; Li, L.-H.; Lin, C.-H.; Wu, J.-Y.; Chen, Y.-T.; Fann, C.S.J.

    2006-01-01

    Microarray-based pooled DNA methods overcome the cost bottleneck of simultaneously genotyping more than 100 000 markers for numerous study individuals. The success of such methods relies on the proper adjustment of preferential amplification/hybridization to ensure accurate and reliable allele frequency estimation. We performed a hybridization-based genome-wide single nucleotide polymorphisms (SNPs) genotyping analysis to dissect preferential amplification/hybridization. The majority of SNPs had less than 2-fold signal amplification or suppression, and the lognormal distributions adequately modeled preferential amplification/hybridization across the human genome. Comparative analyses suggested that the distributions of preferential amplification/hybridization differed among genotypes and the GC content. Patterns among different ethnic populations were similar; nevertheless, there were striking differences for a small proportion of SNPs, and a slight ethnic heterogeneity was observed. To fulfill appropriate and gratuitous adjustments, databases of preferential amplification/hybridization for African Americans, Caucasians and Asians were constructed based on the Affymetrix GeneChip Human Mapping 100 K Set. The robustness of allele frequency estimation using this database was validated by a pooled DNA experiment. This study provides a genome-wide investigation of preferential amplification/hybridization and suggests guidance for the reliable use of the database. Our results constitute an objective foundation for theoretical development of preferential amplification/hybridization and provide important information for future pooled DNA analyses. PMID:16931491

  9. SNP discovery using Paired-End RAD-tag sequencing on pooled genomic DNA of Sisymbrium austriacum (Brassicaceae).

    PubMed

    Vandepitte, K; Honnay, O; Mergeay, J; Breyne, P; Roldán-Ruiz, I; De Meyer, T

    2013-03-01

    Single nucleotide polymorphisms SNPs are rapidly replacing anonymous markers in population genomic studies, but their use in non model organisms is hampered by the scarcity of cost-effective approaches to uncover genome-wide variation in a comprehensive subset of individuals. The screening of one or only a few individuals induces ascertainment bias. To discover SNPs for a population genomic study of the Pyrenean rocket (Sisymbrium austriacum subsp. chrysanthum), we undertook a pooled RAD-PE (Restriction site Associated DNA Paired-End sequencing) approach. RAD tags were generated from the PstI-digested pooled genomic DNA of 12 individuals sampled across the species distribution range and paired-end sequenced using Illumina technology to produce ~24.5 Mb of sequences, covering ~7% of the specie's genome. Sequences were assembled into ~76 000 contigs with a mean length of 323 bp (N(50)  = 357 bp, sequencing depth = 24x). In all, >15 000 SNPs were called, of which 47% were annotated in putative genic regions based on homology with the Arabidopsis thaliana genome. Gene ontology (GO) slim categorization demonstrated that the identified SNPs covered extant genic variation well. The validation of 300 SNPs on a larger set of individuals using a KASPar assay underpinned the utility of pooled RAD-PE as an inexpensive genome-wide SNP discovery technique (success rate: 87%). In addition to SNPs, we discovered >600 putative SSR markers. PMID:23231662

  10. Sequence evaluation of four pooled-tissue normalized bovine cDNA libraries and construction of a gene index for cattle.

    PubMed

    Smith, T P; Grosse, W M; Freking, B A; Roberts, A J; Stone, R T; Casas, E; Wray, J E; White, J; Cho, J; Fahrenkrug, S C; Bennett, G L; Heaton, M P; Laegreid, W W; Rohrer, G A; Chitko-McKown, C G; Pertea, G; Holt, I; Karamycheva, S; Liang, F; Quackenbush, J; Keele, J W

    2001-04-01

    An essential component of functional genomics studies is the sequence of DNA expressed in tissues of interest. To provide a resource of bovine-specific expressed sequence data and facilitate this powerful approach in cattle research, four normalized cDNA libraries were produced and arrayed for high-throughput sequencing. The libraries were made with RNA pooled from multiple tissues to increase efficiency of normalization and maximize the number of independent genes for which sequence data were obtained. Target tissues included those with highest likelihood to have impact on production parameters of animal health, growth, reproductive efficiency, and carcass merit. Success of normalization and inter- and intralibrary redundancy were assessed by collecting 6000-23,000 sequences from each of the libraries (68,520 total sequences deposited in GenBank). Sequence comparison and assembly of these sequences was performed in combination with 56,500 other bovine EST sequences present in the GenBank dbEST database to construct a cattle Gene Index (available from The Institute for Genomic Research at http://www.tigr.org/tdb/tgi.shtml). The 124,381 bovine ESTs present in GenBank at the time of the analysis form 16,740 assemblies that are listed and annotated on the Web site. Analysis of individual library sequence data indicates that the pooled-tissue approach was highly effective in preparing libraries for efficient deep sequencing. PMID:11282978

  11. Cost-Effective Pooling of DNA from Nasopharyngeal Swab Samples for Large-Scale Detection of Bacteria by Real-Time PCR

    PubMed Central

    Edouard, Sophie; Prudent, Elsa; Gautret, Philippe; Memish, Ziad A.

    2014-01-01

    We investigated the potential of pooling DNA from nasopharyngeal specimens to reduce the cost of real-time PCR (RT-PCR) for bacterial detection. Lyophilization is required to reconcentrate DNA. This strategy yields a high specificity (86%) and a high sensitivity (96%). We estimate that compared to individual testing, 37% fewer RT-PCR tests are needed. PMID:25552360

  12. Fish scales and SNP chips: SNP genotyping and allele frequency estimation in individual and pooled DNA from historical samples of Atlantic salmon (Salmo salar)

    PubMed Central

    2013-01-01

    Background DNA extracted from historical samples is an important resource for understanding genetic consequences of anthropogenic influences and long-term environmental change. However, such samples generally yield DNA of a lower amount and quality, and the extent to which DNA degradation affects SNP genotyping success and allele frequency estimation is not well understood. We conducted high density SNP genotyping and allele frequency estimation in both individual DNA samples and pooled DNA samples extracted from dried Atlantic salmon (Salmo salar) scales stored at room temperature for up to 35 years, and assessed genotyping success, repeatability and accuracy of allele frequency estimation using a high density SNP genotyping array. Results In individual DNA samples, genotyping success and repeatability was very high (> 0.973 and > 0.998, respectively) in samples stored for up to 35 years; both increased with the proportion of DNA of fragment size > 1000 bp. In pooled DNA samples, allele frequency estimation was highly repeatable (Repeatability = 0.986) and highly correlated with empirical allele frequency measures (Mean Adjusted R2 = 0.991); allele frequency could be accurately estimated in > 95% of pooled DNA samples with a reference group of at least 30 individuals. SNPs located in polyploid regions of the genome were more sensitive to DNA degradation: older samples had lower genotyping success at these loci, and a larger reference panel of individuals was required to accurately estimate allele frequencies. Conclusions SNP genotyping was highly successful in degraded DNA samples, paving the way for the use of degraded samples in SNP genotyping projects. DNA pooling provides the potential for large scale population genetic studies with fewer assays, provided enough reference individuals are also genotyped and DNA quality is properly assessed beforehand. We provide recommendations for future studies intending to conduct high-throughput SNP

  13. Uniparental Genetic Heritage of Belarusians: Encounter of Rare Middle Eastern Matrilineages with a Central European Mitochondrial DNA Pool

    PubMed Central

    Kushniarevich, Alena; Sivitskaya, Larysa; Danilenko, Nina; Novogrodskii, Tadeush; Tsybovsky, Iosif; Kiseleva, Anna; Kotova, Svetlana; Chaubey, Gyaneshwer; Metspalu, Ene; Sahakyan, Hovhannes; Bahmanimehr, Ardeshir; Reidla, Maere; Rootsi, Siiri; Parik, Jüri; Reisberg, Tuuli; Achilli, Alessandro; Hooshiar Kashani, Baharak; Gandini, Francesca; Olivieri, Anna; Behar, Doron M.; Torroni, Antonio; Davydenko, Oleg; Villems, Richard

    2013-01-01

    Ethnic Belarusians make up more than 80% of the nine and half million people inhabiting the Republic of Belarus. Belarusians together with Ukrainians and Russians represent the East Slavic linguistic group, largest both in numbers and territory, inhabiting East Europe alongside Baltic-, Finno-Permic- and Turkic-speaking people. Till date, only a limited number of low resolution genetic studies have been performed on this population. Therefore, with the phylogeographic analysis of 565 Y-chromosomes and 267 mitochondrial DNAs from six well covered geographic sub-regions of Belarus we strove to complement the existing genetic profile of eastern Europeans. Our results reveal that around 80% of the paternal Belarusian gene pool is composed of R1a, I2a and N1c Y-chromosome haplogroups – a profile which is very similar to the two other eastern European populations – Ukrainians and Russians. The maternal Belarusian gene pool encompasses a full range of West Eurasian haplogroups and agrees well with the genetic structure of central-east European populations. Our data attest that latitudinal gradients characterize the variation of the uniparentally transmitted gene pools of modern Belarusians. In particular, the Y-chromosome reflects movements of people in central-east Europe, starting probably as early as the beginning of the Holocene. Furthermore, the matrilineal legacy of Belarusians retains two rare mitochondrial DNA haplogroups, N1a3 and N3, whose phylogeographies were explored in detail after de novo sequencing of 20 and 13 complete mitogenomes, respectively, from all over Eurasia. Our phylogeographic analyses reveal that two mitochondrial DNA lineages, N3 and N1a3, both of Middle Eastern origin, might mark distinct events of matrilineal gene flow to Europe: during the mid-Holocene period and around the Pleistocene-Holocene transition, respectively. PMID:23785503

  14. Exome sequencing in pooled DNA samples to identify maternal pre-eclampsia risk variants

    PubMed Central

    Kaartokallio, Tea; Wang, Jingwen; Heinonen, Seppo; Kajantie, Eero; Kivinen, Katja; Pouta, Anneli; Gerdhem, Paul; Jiao, Hong; Kere, Juha; Laivuori, Hannele

    2016-01-01

    Pre-eclampsia is a common pregnancy disorder that is a major cause for maternal and perinatal mortality and morbidity. Variants predisposing to pre-eclampsia might be under negative evolutionary selection that is likely to keep their population frequencies low. We exome sequenced samples from a hundred Finnish pre-eclamptic women in pools of ten to screen for low-frequency, large-effect risk variants for pre-eclampsia. After filtering and additional genotyping steps, we selected 28 low-frequency missense, nonsense and splice site variants that were enriched in the pre-eclampsia pools compared to reference data, and genotyped the variants in 1353 pre-eclamptic and 699 non-pre-eclamptic women to test the association of them with pre-eclampsia and quantitative traits relevant for the disease. Genotypes from the SISu project (n = 6118 exome sequenced Finnish samples) were included in the binary trait association analysis as a population reference to increase statistical power. In these analyses, none of the variants tested reached genome-wide significance. In conclusion, the genetic risk for pre-eclampsia is likely complex even in a population isolate like Finland, and larger sample sizes will be necessary to detect risk variants. PMID:27384325

  15. Exome sequencing in pooled DNA samples to identify maternal pre-eclampsia risk variants.

    PubMed

    Kaartokallio, Tea; Wang, Jingwen; Heinonen, Seppo; Kajantie, Eero; Kivinen, Katja; Pouta, Anneli; Gerdhem, Paul; Jiao, Hong; Kere, Juha; Laivuori, Hannele

    2016-01-01

    Pre-eclampsia is a common pregnancy disorder that is a major cause for maternal and perinatal mortality and morbidity. Variants predisposing to pre-eclampsia might be under negative evolutionary selection that is likely to keep their population frequencies low. We exome sequenced samples from a hundred Finnish pre-eclamptic women in pools of ten to screen for low-frequency, large-effect risk variants for pre-eclampsia. After filtering and additional genotyping steps, we selected 28 low-frequency missense, nonsense and splice site variants that were enriched in the pre-eclampsia pools compared to reference data, and genotyped the variants in 1353 pre-eclamptic and 699 non-pre-eclamptic women to test the association of them with pre-eclampsia and quantitative traits relevant for the disease. Genotypes from the SISu project (n = 6118 exome sequenced Finnish samples) were included in the binary trait association analysis as a population reference to increase statistical power. In these analyses, none of the variants tested reached genome-wide significance. In conclusion, the genetic risk for pre-eclampsia is likely complex even in a population isolate like Finland, and larger sample sizes will be necessary to detect risk variants. PMID:27384325

  16. Telomere length in white blood cell DNA and lung cancer: a pooled analysis of three prospective cohorts.

    PubMed

    Seow, Wei Jie; Cawthon, Richard M; Purdue, Mark P; Hu, Wei; Gao, Yu-Tang; Huang, Wen-Yi; Weinstein, Stephanie J; Ji, Bu-Tian; Virtamo, Jarmo; Hosgood, H Dean; Bassig, Bryan A; Shu, Xiao-Ou; Cai, Qiuyin; Xiang, Yong-Bing; Min, Shen; Chow, Wong-Ho; Berndt, Sonja I; Kim, Christopher; Lim, Unhee; Albanes, Demetrius; Caporaso, Neil E; Chanock, Stephen; Zheng, Wei; Rothman, Nathaniel; Lan, Qing

    2014-08-01

    We investigated the relationship between telomere length and lung cancer in a pooled analysis from three prospective cohort studies: the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial, conducted among men and women in the United States, and previously published data from the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Trial conducted among male smokers in Finland, and the Shanghai Women's Health Study (SWHS), which is comprised primarily of never-smokers. The pooled population included 847 cases and 847 controls matched by study, age, and sex. Leukocyte telomere length was measured by a monochrome multiplex qPCR assay. We used conditional logistic regression models to calculate ORs and their 95% confidence intervals (CI) for the association between telomere length and lung cancer risk, adjusted for age and pack-years of smoking. Longer telomere length was associated with increased lung cancer risk in the pooled analysis [OR (95% CI) by quartile: 1.00; 1.24 (0.90-1.71); 1.27 (0.91-1.78); and 1.86 (1.33-2.62); P trend = 0.000022]. Findings were consistent across the three cohorts and strongest for subjects with very long telomere length, i.e., lung cancer risks for telomere length [OR (95% CI)] in the upper half of the fourth quartile were 2.41 (1.28-4.52), 2.16 (1.11-4.23), and 3.02(1.39-6.58) for the PLCO trial, the ATBC trial, and the SWHS, respectively. In addition, the association persisted among cases diagnosed more than 6 years after blood collection and was particularly evident for female adenocarcinoma cases. Telomere length in white blood cell DNA may be a biomarker of future increased risk of lung cancer in diverse populations. PMID:24853549

  17. DNA methyltransferase detection based on digestion triggering the combination of poly adenine DNA with gold nanoparticles.

    PubMed

    Liu, Pei; Wang, Dandan; Zhou, Yunlei; Wang, Haiyan; Yin, Huanshun; Ai, Shiyun

    2016-06-15

    DNA methyltransferase (MTase) has received a large amount of attention due to its catalyzation of DNA methylation in both eukaryotes and prokaryotes, which has a close relationship to cancer and bacterial diseases. Herein, a novel electrochemical strategy based on Dpn I digestion triggering the combination of poly adenine (polyA) DNA with a gold nanoparticles functioned glassy carbon electrode (AuNPs/GCE), is developed for the simple and efficient detection of DNA MTase and inhibitor screening. Only one methylene blue (MB)-labeled DNA hairpin probe and two enzymes are involved in this designed method. In the presence of Dam MTase, the hairpin probe can be methylated and then cleaved by the restriction endonuclease. Thus, a MB-labeled polyA signal-stranded DNA product is introduced to the surface of AuNPs/GCE through the effect between polyA and AuNPs, resulting in an obvious electrochemical signal. On the contrary, in the absence of Dam MTase, the DNA probe cannot be cleaved and a relatively small electrochemical response can be observed. As a result, the as-proposed biosensor offered an efficient way for Dam MTase activity monitoring with a low detection of 0.27U/mL, a wide linear range and good stability. Additionally, this assay holds great potential for further application in real biological matrices and inhibitors screening, which is expected to be useful in disease diagnosis and drug discovery. PMID:26807517

  18. Quantitative trait locus mapping in chickens by selective DNA pooling with dinucleotide microsatellite markers by using purified DNA and fresh or frozen red blood cells as applied to marker-assisted selection.

    PubMed

    Lipkin, E; Fulton, J; Cheng, H; Yonash, N; Soller, M

    2002-03-01

    Many large, half-sib sire families are an integral component of chicken genetic improvement programs. These family structures include a sufficient number of individuals for mapping quantitative trait loci (QTL) at high statistical power. However, realizing this statistical power through individual or selective genotyping is yet too costly to be feasible under current genotyping methodologies. Genotyping costs can be greatly reduced through selective DNA pooling, involving densitometric estimates of marker allele frequencies in pooled DNA samples. When using dinucleotide microsatellite markers, however, such estimates are often confounded by overlapping "shadow" bands and can be confounded further by differential amplification of alleles. In the present study a shadow correction procedure provided accurate densitometric estimates of allele frequency for dinucleotide microsatellite markers in pools made from chicken purified DNA samples, fresh blood samples, and frozen-thawed blood samples. In a retrospective study, selective DNA pooling with thawed blood samples successfully identified two QTL previously shown by selective genotyping to affect resistance in chickens to Marek's disease. It is proposed that use of selective DNA pooling can provide relatively low-cost mapping and use in marker-assisted selection of QTL that affect production traits in chickens. PMID:11902402

  19. Detection of Onchocerca volvulus in Latin American black flies for pool screening PCR using high-throughput automated DNA isolation for transmission surveillance.

    PubMed

    Rodríguez-Pérez, Mario A; Gopal, Hemavathi; Adeleke, Monsuru Adebayo; De Luna-Santillana, Erick Jesús; Gurrola-Reyes, J Natividad; Guo, Xianwu

    2013-11-01

    The posttreatment entomological surveillance (ES) of onchocerciasis in Latin America requires quite large numbers of flies to be examined for parasite infection to prove that the control strategies have worked and that the infection is on the path of elimination. Here, we report a high-throughput automated DNA isolation of Onchocerca volvulus for PCR using a major Latin American black fly vector of onchocerciasis. The sensitivity and relative effectiveness of silica-coated paramagnetic beads was evaluated in comparison with phenol chloroform (PC) method which is known as the gold standard of DNA extraction for ES in Latin America. The automated method was optimized in the laboratory and validated in the field to detect parasite DNA in Simulium ochraceum sensu lato flies in comparison with PC. The optimization of the automated method showed that it is sensitive to detect O. volvulus with a pool size of 100 flies as compared with PC which utilizes 50 flies pool size. The validation of the automated method in comparison with PC in an endemic community showed that 5/67 and 3/134 heads pools were positive for the two methods, respectively. There was no statistical variation (P < 0.05) in the estimation of transmission indices generated by automated method when compared with PC method. The fact that the automated method is sensitive to pool size up to 100 confers advantage over PC method and can, therefore, be employed in large-scale ES of onchocerciasis transmission in endemic areas of Latin America. PMID:24030195

  20. Quantify single nucleotide polymorphism (SNP) ratio in pooled DNA based on normalized fluorescence real-time PCR

    PubMed Central

    Yu, Airong; Geng, Haifeng; Zhou, Xuerui

    2006-01-01

    Background Conventional real-time PCR to quantify the allele ratio in pooled DNA mainly depends on PCR amplification efficiency determination and Ct value, which is defined as the PCR cycle number at which the fluorescence emission exceeds the fixed threshold. Because of the nature of exponential calculation, slight errors are multiplied and the variations of the results seem too large. We have developed a new PCR data point analysis strategy for allele ratio quantification based on normalized fluorescence ratio. Results In our method, initial reaction background fluorescence was determined based upon fitting of raw fluorescence data to four-parametric sigmoid function. After that, each fluorescence data point was first subtracted by respective background fluorescence and then each subtracted fluorescence data point was divided by the specific background fluorescence to get normalized fluorescence. By relating the normalized fluorescence ratio to the premixed known allele ratio of two alleles in standard samples, standard linear regression equation was generated, from which unknown specimens allele ratios were extrapolated using the measured normalized fluorescence ratio. In this article, we have compared the results of the proposed method with those of baseline subtracted fluorescence ratio method and conventional Ct method. Conclusion Results demonstrated that the proposed method could improve the reliability, precision, and repeatability for quantifying allele ratios. At the same time, it has the potential of fully automatic allelic ratio quantification. PMID:16764712

  1. Mutation analysis with random DNA identifiers (MARDI) catalogs Pig-a mutations in heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats.

    PubMed

    Revollo, Javier R; Crabtree, Nathaniel M; Pearce, Mason G; Pacheco-Martinez, M Monserrat; Dobrovolsky, Vasily N

    2016-03-01

    Identification of mutations induced by xenotoxins is a common task in the field of genetic toxicology. Mutations are often detected by clonally expanding potential mutant cells and genotyping each viable clone by Sanger sequencing. Such a "clone-by-clone" approach requires significant time and effort, and sometimes is even impossible to implement. Alternative techniques for efficient mutation identification would greatly benefit both basic and regulatory genetic toxicology research. Here, we report the development of Mutation Analysis with Random DNA Identifiers (MARDI), a novel high-fidelity Next Generation Sequencing (NGS) approach that circumvents clonal expansion and directly catalogs mutations in pools of mutant cells. MARDI uses oligonucleotides carrying Random DNA Identifiers (RDIs) to tag progenitor DNA molecules before PCR amplification, enabling clustering of descendant DNA molecules and eliminating NGS- and PCR-induced sequencing artifacts. When applied to the Pig-a cDNA analysis of heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats, MARDI detected nearly all Pig-a mutations that were previously identified by conventional clone-by-clone analysis and discovered many additional ones consistent with DMBA exposure: mostly A to T transversions, with the mutated A located on the non-transcribed DNA strand. PMID:26683280

  2. Simple combined model for nonlinear excitations in DNA.

    PubMed

    Hien, D L; Nhan, N T; Ngo, V Thanh; Viet, N A

    2007-08-01

    We propose a simple model for DNA denaturation bases on the pendulum model of Englander [Proc. Natl. Acad. Sci. U.S.A. 77, 7222 (1980)] and the microscopic model of Peyrard and Bishop [Phys. Rev. Lett. 62, 2755 (1989)], so-called "combined model." The main parameters of our model are the coupling constant k along each strand, the mean stretching y* of the hydrogen bonds, the ratio of the damping constant and driven force gamma/F. We show that both the length L of unpaired bases and the velocity v of kinks depend on not only the coupling constant k but also the temperature T. Our results are in good agreement with previous works. PMID:17930079

  3. Genome-wide Association Study of Subtype-Specific Epithelial Ovarian Cancer Risk Alleles Using Pooled DNA

    PubMed Central

    Earp, Madalene A.; Kelemen, Linda E.; Magliocco, Anthony M.; Swenerton, Kenneth D.; Chenevix–Trench, Georgia; Lu, Yi; Hein, Alexander; Ekici, Arif B.; Beckmann, Matthias W.; Fasching, Peter A.; Lambrechts, Diether; Despierre, Evelyn; Vergote, Ignace; Lambrechts, Sandrina; Doherty, Jennifer A.; Rossing, Mary Anne; Chang-Claude, Jenny; Rudolph, Anja; Friel, Grace; Moysich, Kirsten B.; Odunsi, Kunle; Sucheston-Campbell, Lara; Lurie, Galina; Goodman, Marc T.; Carney, Michael E.; Thompson, Pamela J.; Runnebaum, Ingo B.; Dürst, Matthias; Hillemanns, Peter; Dörk, Thilo; Antonenkova, Natalia; Bogdanova, Natalia; Leminen, Arto; Nevanlinna, Heli; Pelttari, Liisa M.; Butzow, Ralf; Bunker, Clareann H.; Modugno, Francesmary; Edwards, Robert P.; Ness, Roberta B.; du Bois, Andreas; Heitz, Florian; Schwaab, Ira; Harter, Philipp; Karlan, Beth Y.; Walsh, Christine; Lester, Jenny; Jensen, Allan; Kjær, Susanne K.; Høgdall, Claus K.; Høgdall, Estrid; Lundvall, Lene; Sellers, Thomas A.; Fridley, Brooke L.; Goode, Ellen L.; Cunningham, Julie M.; Vierkant, Robert A.; Giles, Graham G.; Baglietto, Laura; Severi, Gianluca; Southey, Melissa C.; Liang, Dong; Wu, Xifeng; Lu, Karen; Hildebrandt, Michelle A.T.; Levine, Douglas A.; Bisogna, Maria; Schildkraut, Joellen M.; Iversen, Edwin S.; Weber, Rachel Palmieri; Berchuck, Andrew; Cramer, Daniel W.; Terry, Kathryn L.; Poole, Elizabeth M.; Tworoger, Shelley S.; Bandera, Elisa V.; Chandran, Urmila; Orlow, Irene; Olson, Sara H.; Wik, Elisabeth; Salvesen, Helga B.; Bjorge, Line; Halle, Mari K.; van Altena, Anne M.; Aben, Katja K.H.; Kiemeney, Lambertus A.; Massuger, Leon F.A.G.; Pejovic, Tanja; Bean, Yukie T.; Cybulski, Cezary; Gronwald, Jacek; Lubinski, Jan; Wentzensen, Nicolas; Brinton, Louise A.; Lissowska, Jolanta; Garcia–Closas, Montserrat; Dicks, Ed; Dennis, Joe; Easton, Douglas F.; Song, Honglin; Tyrer, Jonathan P.; Pharoah, Paul D. P.; Eccles, Diana; Campbell, Ian G.; Whittemore, Alice S.; McGuire, Valerie; Sieh, Weiva; Rothstein, Joseph H.; Flanagan, James M.; Paul, James; Brown, Robert; Phelan, Catherine M.; Risch, Harvey A.; McLaughlin, John R.; Narod, Steven A.; Ziogas, Argyrios; Anton-Culver, Hoda; Gentry-Maharaj, Aleksandra; Menon, Usha; Gayther, Simon A.; Ramus, Susan J.; Wu, Anna H.; Pearce, Celeste L.; Pike, Malcolm C.; Dansonka-Mieszkowska, Agnieszka; Rzepecka, Iwona K; Szafron, Lukasz M; Kupryjanczyk, Jolanta; Cook, Linda S.; Le, Nhu D.; Brooks–Wilson, Angela

    2014-01-01

    Epithelial ovarian cancer (EOC) is a heterogeneous cancer with both genetic and environmental risk factors. Variants influencing the risk of developing the less-common EOC subtypes have not been fully investigated. We performed a genome-wide association study (GWAS) of EOC according to subtype by pooling genomic DNA from 545 cases and 398 controls of European descent, and testing for allelic associations. We evaluated for replication 188 variants from the GWAS (56 variants for mucinous, 55 for endometrioid and clear cell, 53 for low malignant potential (LMP) serous, and 24 for invasive serous EOC), selected using pre-defined criteria. Genotypes from 13,188 cases and 23,164 controls of European descent were used to perform unconditional logistic regression under the log-additive genetic model; odds ratios (OR) and 95% confidence intervals are reported. Nine variants tagging 6 loci were associated with subtype-specific EOC risk at P<0.05, and had an OR that agreed in direction of effect with the GWAS results. Several of these variants are in or near genes with a biological rationale for conferring EOC risk, including ZFP36L1 and RAD51B for mucinous EOC (rs17106154, OR=1.17, P=0.029, n=1,483 cases), GRB10 for endometrioid and clear cell EOC (rs2190503, P=0.014, n=2,903 cases), and C22orf26/BPIL2 for LMP serous EOC (rs9609538, OR=0.86, P=0.0043, n=892 cases). In analyses that included the 75 GWAS samples, the association between rs9609538 (OR=0.84, P=0.0007) and LMP serous EOC risk remained statistically significant at P<0.0012 adjusted for multiple testing. Replication in additional samples will be important to verify these results for the less-common EOC subtypes. PMID:24190013

  4. A combined DNA vaccine provides protective immunity against Mycobacterium bovis and Brucella abortus in cattle.

    PubMed

    Hu, Xi-Dan; Yu, Da-Hai; Chen, Su-Ting; Li, Shu-Xia; Cai, Hong

    2009-04-01

    We evaluated the immunogenicity and protective efficacy of a combined DNA vaccine containing six genes encoding immunodominant antigens from Mycobacterium bovis and Brucella abortus. The number of lymph node and spleen cultures positive for M. bovis and B. abortus from calves immunized with the combined DNA vaccine was significantly reduced (p < 0.01) compared with unvaccinated calves after challenge with virulent M. bovis and B. abortus 544. The combined DNA vaccine group displayed stronger antigen-specific interferon-gamma (IFN-gamma) responses and antigen-specific IFN-gamma ELISPOT activities 2 months after final immunization and after challenge. Antigen-specific CD4(+) and CD8(+) T cell responses in the combined DNA vaccine group were higher than either the Bacillus Calmette-Guerin (BCG)-positive or S19-positive control group. Likewise, more calves in the DNA vaccine group exhibited antigen-specific IgG titers and had higher IgG titers than those in the BCG- or S19-immunized groups 2 months after the final immunization. Moreover, two antigens in the combined DNA vaccine induced significant antigen-specific IFN-gamma responses 6 months after challenge (p < 0.05). Bacterial counts and pathological analyses of the challenged animals indicated that the combined DNA vaccine provided significantly better protection than the BCG vaccine against M. bovis, and the protection level induced by the combined DNA vaccine was comparable to S19 against B. abortus. This is the first report to demonstrate that a single combined DNA vaccine protects cattle against two infectious diseases. PMID:19364278

  5. Combinative exposure effect of radio frequency signals from CDMA mobile phones and aphidicolin on DNA integrity.

    PubMed

    Tiwari, R; Lakshmi, N K; Surender, V; Rajesh, A D V; Bhargava, S C; Ahuja, Y R

    2008-01-01

    The aim of present study is to assess DNA integrity on the effect of exposure to a radio frequency (RF) signal from Code Division Multiple Access (CDMA) mobile phones. Whole blood samples from six healthy male individuals were exposed for RF signals from a CDMA mobile phone for 1 h. Alkaline comet assay was performed to assess the DNA damage. The combinative exposure effect of the RF signals and APC at two concentrations on DNA integrity was studied. DNA repair efficiency of the samples was also studied after 2 h of exposure. The RF signals and APC (0.2 microg/ml) alone or in synergism did not have any significant DNA damage as compared to sham exposed. However, univariate analysis showed that DNA damage was significantly different among combinative exposure of RF signals and APC at 0.2 microg/ml (p < 0.05) and at 2 microg/ml (p < 0.02). APC at 2 microg/ml concentration also showed significant damage levels (p < 0.05) when compared to sham exposed. DNA repair efficiency also varied in a significant way in combinative exposure sets (p < 0.05). From these results, it appears that the repair inhibitor APC enhances DNA breaks at 2 microg/ml concentration and that the damage is possibly repairable. Thus, it can be inferred that the in vitro exposure to RF signals induces reversible DNA damage in synergism with APC. PMID:19037791

  6. Pool Purification

    NASA Technical Reports Server (NTRS)

    1988-01-01

    Caribbean Clear, Inc. used NASA's silver ion technology as a basis for its automatic pool purifier. System offers alternative approach to conventional purification chemicals. Caribbean Clear's principal markets are swimming pool owners who want to eliminate chlorine and bromine. Purifiers in Caribbean Clear System are same silver ions used in Apollo System to kill bacteria, plus copper ions to kill algae. They produce spa or pool water that exceeds EPA Standards for drinking water.

  7. Enhancement of anti-proliferative activities of Metformin, when combined with Celecoxib, without increasing DNA damage.

    PubMed

    Ullah, Asad; Ashraf, Muhammad; Javeed, Aqeel; Anjum, Aftab Ahmad; Attiq, Ali; Ali, Sarwat

    2016-07-01

    Pathophysiological changes in diabetes like hyperglycemia, oxidative stress, insulin resistance and compensatory hyperinsulinemia predispose cells to malignant transformation and damage DNA repair mechanism. This study was designed to explore the potential synergistic toxic effects of anti-diabetic drug (Metformin), and an analgesic drug (Celecoxib) at cellular level. MTT assay run on Vero cell line revealed that the combinations of Metformin and Celecoxib augment the anti-proliferative effects, whereas Single cell gel electrophoresis spotlighted that Metformin produce non-significant DNA damage with the threshold concentration of 400μg/ml in peripheral blood mononuclear cells (lymphocytes and monocytes), while Celecoxib produced significant (P<0.05) DNA damage (class III comets) above the concentration of 75μg/ml, however the DNA damage or DNA tail protrusions by combinations of both drugs were less than what was observed with Celecoxib alone. Metformin or Celecoxib did not appear mutagenic against any mutant strains (TA 100 and TA 98) but their combination exhibited slight mutagenicity at much higher concentration. The results obtained at concentrations higher than the therapeutic level of drugs and reflect that Metformin in combination with Celecoxib synergistically inhibits the cell proliferation in a concentration dependent pattern. Since, this increase in cytotoxicity did not confer an increase in DNA damage; this combination could be adopted to inhibit the growth of malignant cell without producing any genotoxic or mutagenic effects at cellular level. PMID:27327526

  8. Ultrasensitive electrochemical biosensing for DNA using quantum dots combined with restriction endonuclease.

    PubMed

    Zhang, Can; Lou, Jing; Tu, Wenwen; Bao, Jianchun; Dai, Zhihui

    2015-01-21

    A universal and sensitive electrochemical biosensing platform for the detection and identification of DNA using CdSe quantum dots (CdSe QDs) as signal markers was designed. The detection mechanism was based on the specific recognition of MspI endonuclease combined with the signal amplification of gold nanoparticles (AuNPs). MspI endonuclease could recognize its specific sequence in the double-strand DNA (dsDNA) and cleave the dsDNA fragments linked with CdSe QDs from the electrode. The remaining attached CdSe QDs can be easily read out by square-wave voltammetry using an electrodeposited bismuth (Bi) film-modified glass carbon electrode. The concentrations of target DNA could be simultaneously detected by the signal of metal markers. Using mycobacterium tuberculosis (Mtb) DNA as a model, under the optimal conditions, the proposed biosensor could detect Mtb DNA down to 8.7 × 10(-15) M with a linear range of 5 orders of magnitude (from 1.0 × 10(-14) to 1.0 × 10(-9) M) and discriminate mismatched DNA with high selectivity. This strategy presented a universal and convenient biosensing platform for DNA assay, and its satisfactory performances make it a potential candidate for the early diagnosis of gene-related diseases. PMID:25408952

  9. Deep Sequencing of the Nicastrin Gene in Pooled DNA, the Identification of Genetic Variants That Affect Risk of Alzheimer's Disease

    PubMed Central

    Lupton, Michelle K.; Proitsi, Petroula; Danillidou, Makrina; Tsolaki, Magda; Hamilton, Gillian; Wroe, Richard; Pritchard, Megan; Lord, Kathryn; Martin, Belinda M.; Kloszewska, Iwona; Soininen, Hilkka; Mecocci, Patrizia; Vellas, Bruno; Harold, Denise; Hollingworth, Paul; Lovestone, Simon; Powell, John F.

    2011-01-01

    Nicastrin is an obligatory component of the γ-secretase; the enzyme complex that leads to the production of Aβ fragments critically central to the pathogenesis of Alzheimer's disease (AD). Analyses of the effects of common variation in this gene on risk for late onset AD have been inconclusive. We investigated the effect of rare variation in the coding regions of the Nicastrin gene in a cohort of AD patients and matched controls using an innovative pooling approach and next generation sequencing. Five SNPs were identified and validated by individual genotyping from 311 cases and 360 controls. Association analysis identified a non-synonymous rare SNP (N417Y) with a statistically higher frequency in cases compared to controls in the Greek population (OR 3.994, CI 1.105–14.439, p = 0.035). This finding warrants further investigation in a larger cohort and adds weight to the hypothesis that rare variation explains some of genetic heritability still to be identified in Alzheimer's disease. PMID:21364883

  10. Constructing the parental linkage phase and the genetic map over distances <1 cM using pooled haploid DNA.

    PubMed

    Gasbarra, Dario; Sillanpää, Mikko J

    2006-02-01

    A new statistical approach for construction of the genetic linkage map and estimation of the parental linkage phase based on allele frequency data from pooled gametic (sperm or egg) samples is introduced. This method can be applied for estimation of recombination fractions (over distances <1 cM) and ordering of large numbers (even hundreds) of closely linked markers. This method should be extremely useful in species with a long generation interval and a large genome size such as in dairy cattle or in forest trees; the conifer species have haploid tissues available in megagametophytes. According to Mendelian expectation, two parental alleles should occur in gametes in 1:1 proportions, if segregation distortion does not occur. However, due to mere sampling variation, the observed proportions may deviate from their expected value in practice. These deviations and their dependence along the chromosome can provide information on the parental linkage phase and on the genetic linkage map. Usefulness of the method is illustrated with simulations. The role of segregation distortion as a source of these deviations is also discussed. The software implementing this method is freely available for research purposes from the authors. PMID:16301209

  11. Single DNA molecule grafting and manipulation using a combined atomic force microscope and an optical tweezer

    NASA Astrophysics Data System (ADS)

    Shivashankar, G. V.; Libchaber, A.

    1997-12-01

    In this letter, we report on spatially selecting and grafting a DNA-tethered bead to an atomic force microscope (AFM) cantilever, using an optical tweezer. To quantify this technique, we measure force versus extension of a single DNA molecule using AFM. For such studies, we have developed a micromanipulation approach by combining an AFM, an optical tweezer, and visualization setup. The ability to select a single DNA polymer and specifically graft it to a localized position on a substrate opens up new possibilities in biosensors and bioelectronic devices.

  12. Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability

    PubMed Central

    Cilli, Piera; Ventura, Ilenia; Minoprio, Anna; Meccia, Ettore; Martire, Alberto; Wilson, Samuel H.; Bignami, Margherita; Mazzei, Filomena

    2016-01-01

    DNA trinucleotide repeat (TNR) expansion underlies several neurodegenerative disorders including Huntington's disease (HD). Accumulation of oxidized DNA bases and their inefficient processing by base excision repair (BER) are among the factors suggested to contribute to TNR expansion. In this study, we have examined whether oxidation of the purine dNTPs in the dNTP pool provides a source of DNA damage that promotes TNR expansion. We demonstrate that during BER of 8-oxoguanine (8-oxodG) in TNR sequences, DNA polymerase β (POL β) can incorporate 8-oxodGMP with the formation of 8-oxodG:C and 8-oxodG:A mispairs. Their processing by the OGG1 and MUTYH DNA glycosylases generates closely spaced incisions on opposite DNA strands that are permissive for TNR expansion. Evidence in HD model R6/2 mice indicates that these DNA glycosylases are present in brain areas affected by neurodegeneration. Consistent with prevailing oxidative stress, the same brain areas contained increased DNA 8-oxodG levels and expression of the p53-inducible ribonucleotide reductase. Our in vitro and in vivo data support a model where an oxidized dNTPs pool together with aberrant BER processing contribute to TNR expansion in non-replicating cells. PMID:26980281

  13. Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability.

    PubMed

    Cilli, Piera; Ventura, Ilenia; Minoprio, Anna; Meccia, Ettore; Martire, Alberto; Wilson, Samuel H; Bignami, Margherita; Mazzei, Filomena

    2016-06-20

    DNA trinucleotide repeat (TNR) expansion underlies several neurodegenerative disorders including Huntington's disease (HD). Accumulation of oxidized DNA bases and their inefficient processing by base excision repair (BER) are among the factors suggested to contribute to TNR expansion. In this study, we have examined whether oxidation of the purine dNTPs in the dNTP pool provides a source of DNA damage that promotes TNR expansion. We demonstrate that during BER of 8-oxoguanine (8-oxodG) in TNR sequences, DNA polymerase β (POL β) can incorporate 8-oxodGMP with the formation of 8-oxodG:C and 8-oxodG:A mispairs. Their processing by the OGG1 and MUTYH DNA glycosylases generates closely spaced incisions on opposite DNA strands that are permissive for TNR expansion. Evidence in HD model R6/2 mice indicates that these DNA glycosylases are present in brain areas affected by neurodegeneration. Consistent with prevailing oxidative stress, the same brain areas contained increased DNA 8-oxodG levels and expression of the p53-inducible ribonucleotide reductase. Our in vitro and in vivo data support a model where an oxidized dNTPs pool together with aberrant BER processing contribute to TNR expansion in non-replicating cells. PMID:26980281

  14. Evaluation of large-scale genetic structure in complex demographic and historical scenarios: the mitochondrial DNA and Y-chromosome pools of the Iberian Atlantic façade.

    PubMed

    Pardiñas, Antonio F; Roca, Agustín; García-Vazquez, Eva; López, Belén

    2014-04-01

    Genetic structural patterns of human populations are usually a combination of long-term evolutionary forces and short-term social, cultural, and demographic processes. Recently, using mitochondrial DNA and Y-chromosome loci, various studies in northern Spain have found evidence that the geographical distribution of Iron Age tribal peoples might have influenced current patterns of genetic structuring in several autochthonous populations. Using the wealth of data that are currently available from the whole territory of the Iberian Peninsula, we have evaluated its genetic structuring in the spatial scale of the Atlantic façade. Hierarchical tree modeling procedures, combined with a classic analysis of molecular variance (AMOVA), were used to model known sociocultural divisions from the third century BCE to the eighth century CE, contrasting them with uniparental marker data. Our results show that, while mountainous and abrupt areas of the Iberian North bear the signals of long-term isolation in their maternal and paternal gene pools, the makeup of the Atlantic façade as a whole can be related to tribal population groups that predate the Roman conquest of the Peninsula. The maintenance through time of such a structure can be related to the numerous geographic barriers of the Iberian mainland, which have historically conditioned its settlement patterns and the occurrence of genetic drift processes. PMID:24375152

  15. Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ

    SciTech Connect

    Barends, Thomas R. M.; Brosi, Richard W. W.; Steinmetz, Andrea; Scherer, Anna; Hartmann, Elisabeth; Eschenbach, Jessica; Lorenz, Thorsten; Seidel, Ralf; Shoeman, Robert L.; Zimmermann, Sabine; Bittl, Robert; Schlichting, Ilme; Reinstein, Jochen

    2013-08-01

    The crystal structure of the N-terminal part of T. thermophilus DnaJ unexpectedly showed an ordered GF domain and guided the design of a construct enabling the first structure determination of a complete DnaJ cochaperone molecule. By combining the crystal structures with spin-labelling EPR and cross-linking in solution, a dynamic view of this flexible molecule was developed. Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.

  16. Low-density lipoprotein peptide-combined DNA nanocomplex as an efficient anticancer drug delivery vehicle.

    PubMed

    Zhang, Nan; Tao, Jun; Hua, Haiying; Sun, Pengchao; Zhao, Yongxing

    2015-08-01

    DNA is a type of potential biomaterials for drug delivery due to its nanoscale geometry, loading capacity of therapeutics, biocompatibility, and biodegradability. Unfortunately, DNA is easily degraded by DNases in the body circulation and has low intracellular uptake. In the present study, we selected three cationic polymers polyethylenimine (PEI), hexadecyl trimethyl ammonium bromide (CTAB), and low-density lipoprotein (LDL) receptor targeted peptide (RLT), to modify DNA and improve the issues. A potent anti-tumor anthracycline-doxorubicin (DOX) was intercalated into DNA non-covalently and the DOX/DNA was then combined with PEI, CTAB, and RLT, respectively. Compact nanocomplexes were formed by electrostatic interaction and could potentially protect DNA from DNases. More importantly, RLT had the potential to enhance intracellular uptake by LDL receptor mediated endocytosis. In a series of in vitro experiments, RLT complexed DNA enhanced intracellular delivery of DOX, increased tumor cell death and intracellular ROS production, and reduced intracellular elimination of DOX. All results suggested that the easily prepared and targeted RLT/DNA nanocomplexes had great potential to be developed into a formulation for doxorubicin with enhanced anti-tumor activity. PMID:25960329

  17. Genome-wide SNP scan of pooled DNA reveals nonsense mutation in FGF20 in the scaleless line of featherless chickens

    PubMed Central

    2012-01-01

    Background Scaleless (sc/sc) chickens carry a single recessive mutation that causes a lack of almost all body feathers, as well as foot scales and spurs, due to a failure of skin patterning during embryogenesis. This spontaneous mutant line, first described in the 1950s, has been used extensively to explore the tissue interactions involved in ectodermal appendage formation in embryonic skin. Moreover, the trait is potentially useful in tropical agriculture due to the ability of featherless chickens to tolerate heat, which is at present a major constraint to efficient poultry meat production in hot climates. In the interests of enhancing our understanding of feather placode development, and to provide the poultry industry with a strategy to breed heat-tolerant meat-type chickens (broilers), we mapped and identified the sc mutation. Results Through a cost-effective and labour-efficient SNP array mapping approach using DNA from sc/sc and sc/+ blood sample pools, we map the sc trait to chromosome 4 and show that a nonsense mutation in FGF20 is completely associated with the sc/sc phenotype. This mutation, common to all sc/sc individuals and absent from wild type, is predicted to lead to loss of a highly conserved region of the FGF20 protein important for FGF signalling. In situ hybridisation and quantitative RT-PCR studies reveal that FGF20 is epidermally expressed during the early stages of feather placode patterning. In addition, we describe a dCAPS genotyping assay based on the mutation, developed to facilitate discrimination between wild type and sc alleles. Conclusions This work represents the first loss of function genetic evidence supporting a role for FGF ligand signalling in feather development, and suggests FGF20 as a novel central player in the development of vertebrate skin appendages, including hair follicles and exocrine glands. In addition, this is to our knowledge the first report describing the use of the chicken SNP array to map genes based on

  18. STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA (presentation video)

    NASA Astrophysics Data System (ADS)

    Heller, Iddo; Sitters, Gerrit; Broekmans, Onno D.; Farge, Géraldine; Menges, Carolin; Wende, Wolfgang; Hell, Stefan W.; Peterman, Erwin J.; Wuite, Gijs J.

    2014-09-01

    Dense coverage of DNA by proteins is a ubiquitous feature of cellular processes such as DNA organization, replication and repair. We present a single-molecule approach capable of visualizing individual DNA-binding proteins on densely covered DNA and in the presence of high protein concentrations. Our approach combines optical tweezers with multicolor confocal and stimulated emission depletion (STED) fluorescence microscopy. Proteins on DNA are visualized at a resolution of 50 nm, a sixfold resolution improvement over that of confocal microscopy. High temporal resolution (<50 ms) is ensured by fast one-dimensional beam scanning. Individual trajectories of proteins translocating on DNA can thus be distinguished and tracked with high precision. We demonstrate our multimodal approach by visualizing the assembly of dense nucleoprotein filaments with unprecedented spatial resolution in real time. Experimental access to the force-dependent kinetics and motility of DNA-associating proteins at biologically relevant protein densities is essential for linking idealized in vitro experiments with the in vivo situation.

  19. A whole genome scan for quantitative trait loci affecting milk protein percentage in Israeli-Holstein cattle, by means of selective milk DNA pooling in a daughter design, using an adjusted false discovery rate criterion.

    PubMed Central

    Mosig, M O; Lipkin, E; Khutoreskaya, G; Tchourzyna, E; Soller, M; Friedmann, A

    2001-01-01

    Selective DNA pooling was employed in a daughter design to screen all bovine autosomes for quantitative trait loci (QTL) affecting estimated breeding value for milk protein percentage (EBVP%). Milk pools prepared from high and low daughters of each of seven sires were genotyped for 138 dinucleotide microsatellites. Shadow-corrected estimates of sire allele frequencies were compared between high and low pools. An adjusted false discovery rate (FDR) method was employed to calculate experimentwise significance levels and empirical power. Significant associations with milk protein percentage were found for 61 of the markers (adjusted FDR = 0.10; estimated power, 0.68). The significant markers appear to be linked to 19--28 QTL. Mean allele substitution effects of the putative QTL averaged 0.016 (0.009--0.028) in units of the within-sire family standard deviation of EBVP% and summed to 0.460 EBVP%. Overall QTL heterozygosity was 0.40. The identified QTL appear to account for all of the variation in EBVP% in the population. Through use of selective DNA pooling, 4400 pool data points provided the statistical power of 600,000 individual data points. PMID:11290723

  20. Synergistic antitumor efficacy of combined DNA vaccines targeting tumor cells and angiogenesis.

    PubMed

    Yin, Xiaotao; Wang, Wei; Zhu, Xiaoming; Wang, Yu; Wu, Shuai; Wang, Zicheng; Wang, Lin; Du, Zhiyan; Gao, Jiangping; Yu, Jiyun

    2015-09-18

    To further enhance the antitumor efficacy of DNA vaccine, we proposed a synergistic strategy that targeted tumor cells and angiogenesis simultaneously. In this study, a Semliki Forest Virus (SFV) replicon DNA vaccine expressing 1-4 domains of murine VEGFR2 and IL12 was constructed, and was named pSVK-VEGFR2-GFc-IL12 (CAVE). The expression of VEGFR2 antigen and IL12 adjuvant molecule in 293T cells in vitro were verified by western blot and enzyme-linked immune sorbent assay (ELISA). Then CAVE was co-immunized with CAVA, a SFV replicon DNA vaccine targeting survivin and β-hCG antigens constructed previously. The antitumor efficacy of our combined replicon vaccines was evaluated in mice model and the possible mechanism was further investigated. The combined vaccines could elicit efficient humoral and cellular immune responses against survivin, β-hCG and VEGFR2 simultaneously. Compared with CAVE or CAVA vaccine alone, the combined vaccines inhibited the tumor growth and improved the survival rate in B16 melanoma mice model more effectively. Furthermore, the intratumoral microvessel density was lowest in combined vaccines group than CAVE or CAVA alone group. Therefore, this synergistic strategy of DNA vaccines for tumor treatment results in an increased antitumor efficacy, and may be more suitable for translation to future research and clinic. PMID:26253468

  1. Combined antibody and DNA detection for early diagnosis of leptospirosis after a disaster.

    PubMed

    Iwasaki, Hiroko; Chagan-Yasutan, Haorile; Leano, Prisca Susan A; Koizumi, Nobuo; Nakajima, Chie; Taurustiati, Delsi; Hanan, Firmanto; Lacuesta, Talitha Lea; Ashino, Yugo; Suzuki, Yasuhiko; Gloriani, Nina G; Telan, Elizabeth Freda O; Hattori, Toshio

    2016-04-01

    Early diagnosis based on laboratory confirmation is essential for managing leptospirosis. This study investigated the effectiveness of a novel method of detecting leptospirosis that combines measurement of anti-Leptospira antibodies by the microscopic agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA), and immunochromatographic test (ICT) and leptospiral DNA by loop-mediated isothermal amplification (LAMP) and real-time PCR in plasma and 2 types of urine pellets. Of 113 suspected cases, 68.1%, 76.1%, and 60.2% were positive by MAT, ELISA, and ICT, respectively. Real-time PCR using DNA purified from urine pellets collected by low-speed centrifugation yielded positive signals for patients in late acute as well as early phase who were positive by LAMP using plasma DNA or urine pellets. Among antibody-negative patients, 9.5% were positive by DNA detection. These findings indicate that the leptospirosis detection rate is increased by combining antibody and DNA detection, providing a new tool for timely diagnosis of infection. PMID:26860351

  2. The Updated Phylogenies of the Phasianidae Based on Combined Data of Nuclear and Mitochondrial DNA

    PubMed Central

    Shen, Yong-Yi; Dai, Kun; Cao, Xue; Murphy, Robert W.; Shen, Xue-Juan; Zhang, Ya-Ping

    2014-01-01

    The phylogenetic relationships of species in the Phasianidae, Order Galliformes, are the object of intensive study. However, convergent morphological evolution and rapid species radiation result in much ambiguity in the group. Further, matrilineal (mtDNA) genealogies conflict with trees based on nuclear DNA retrotransposable elements. Herein, we analyze 39 nearly complete mitochondrial genomes (three new) and up to seven nuclear DNA segments. We combine these multiple unlinked, more informative genetic markers to infer historical relationships of the major groups of phasianids. The nuclear DNA tree is largely congruent with the tree derived from mt genomes. However, branching orders of mt/nuclear trees largely conflict with those based on retrotransposons. For example, Gallus/Bambusicola/Francolinus forms the sister-group of Coturnix/Alectoris in the nuclear/mtDNA trees, yet the tree based on retrotransposable elements roots the former at the base of the tree and not with the latter. Further, while peafowls cluster with Gallus/Coturnix in the mt tree, they root at the base of the phasianids following Gallus in the tree based on retrotransposable elements. The conflicting branch orders in nuclear/mtDNA and retrotransposons-based trees in our study reveal the complex topology of the Phasianidae. PMID:24748132

  3. Functional Flow Patterns and Static Blood Pooling in Tumors Revealed by Combined Contrast-Enhanced Ultrasound and Photoacoustic Imaging.

    PubMed

    Bar-Zion, Avinoam; Yin, Melissa; Adam, Dan; Foster, F Stuart

    2016-08-01

    Alterations in tumor perfusion and microenvironment have been shown to be associated with aggressive cancer phenotypes, raising the need for noninvasive methods of tracking these changes. Dynamic contrast-enhanced ultrasound (DCEUS) and photoacoustic (PA) imaging serve as promising candidates-one has the ability to measure tissue perfusion, whereas the other can be used to monitor tissue oxygenation and hemoglobin concentration. In this study, we investigated the relationship between the different functional parameters measured with DCEUS and PA imaging, using two morphologically different hind-limb tumor models and drug-induced alterations in an orthotopic breast tumor model. Imaging results showed some correlation between perfusion and oxygen saturation maps and the ability to sensitively monitor antivascular treatment. In addition, DCEUS measurements revealed different vascular densities in the core of specific tumors compared with their rims. Noncorrelated perfusion and hemoglobin concentration measurements facilitated discrimination between blood lakes and necrotic areas. Taken together, our results illustrate the utility of a combined contrast-enhanced ultrasound method with photoacoustic imaging to visualize blood flow patterns in tumors. Cancer Res; 76(15); 4320-31. ©2016 AACR. PMID:27325651

  4. Effect of Suberoylanilide Hydroxamic Acid (SAHA) Administration on the Residual Virus Pool in a Model of Combination Antiretroviral Therapy-Mediated Suppression in SIVmac239-Infected Indian Rhesus Macaques

    PubMed Central

    Del Prete, Gregory Q.; Shoemaker, Rebecca; Oswald, Kelli; Lara, Abigail; Trubey, Charles M.; Fast, Randy; Schneider, Douglas K.; Kiser, Rebecca; Coalter, Vicky; Wiles, Adam; Wiles, Rodney; Freemire, Brandi; Keele, Brandon F.; Estes, Jacob D.; Quiñones, Octavio A.; Smedley, Jeremy; Macallister, Rhonda; Sanchez, Rosa I.; Wai, John S.; Tan, Christopher M.; Alvord, W. Gregory; Hazuda, Daria J.; Piatak, Michael

    2014-01-01

    Nonhuman primate models are needed for evaluations of proposed strategies targeting residual virus that persists in HIV-1-infected individuals receiving suppressive combination antiretroviral therapy (cART). However, relevant nonhuman primate (NHP) models of cART-mediated suppression have proven challenging to develop. We used a novel three-class, six-drug cART regimen to achieve durable 4.0- to 5.5-log reductions in plasma viremia levels and declines in cell-associated viral RNA and DNA in blood and tissues of simian immunodeficiency virus SIVmac239-infected Indian-origin rhesus macaques, then evaluated the impact of treatment with the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA; Vorinostat) on the residual virus pool. Ex vivo SAHA treatment of CD4+ T cells obtained from cART-suppressed animals increased histone acetylation and viral RNA levels in culture supernatants. cART-suppressed animals each received 84 total doses of oral SAHA. We observed SAHA dose-dependent increases in acetylated histones with evidence for sustained modulation as well as refractoriness following prolonged administration. In vivo virologic activity was demonstrated based on the ratio of viral RNA to viral DNA in peripheral blood mononuclear cells, a presumptive measure of viral transcription, which significantly increased in SAHA-treated animals. However, residual virus was readily detected at the end of treatment, suggesting that SAHA alone may be insufficient for viral eradication in the setting of suppressive cART. The effects observed were similar to emerging data for repeat-dose SAHA treatment of HIV-infected individuals on cART, demonstrating the feasibility, utility, and relevance of NHP models of cART-mediated suppression for in vivo assessments of AIDS virus functional cure/eradication approaches. PMID:25182644

  5. [Release of Extracellular DNA after Administration of Radioprotective Combination of α-Tocopherol and Ascorbic Acid].

    PubMed

    Vasilyeval, I N; Bespalov, V G

    2015-01-01

    Radioprotective and apoptotic activities of α-tocopherol acetate (vitamin E) and ascorbic acid (vitamin C) have been studied in 180 Wistar male rats. Rats were administered a single oral dose with vitamin E, vitamin C or their combination at prophylactic doses before or after the single whole body exposure to irradiation at the doses of 2 or 8 Gy. The radioprotective effect was evaluated by the frequency of chromosomal aberrations at metaphase plates of the bone marrow cells, apoptotic--by the level of circulating low-molecular-weight DNA (ImwDNA) in the blood plasma of irradiated rats. Administration of the combination of vitamins E and C before and after the irradiation at the dose of 2 Gy reduced the number of the cells with chromosomal aberrations thus providing the radioprotective effect, but separately administration of these vitamins did not show the significant radioprotective activity. Administration of the combination of vitamins E and C before irradiation with 8 Gy increased the lmwDNA in blood thus providing the apoptotic effect. So, synergy of radioprotective activities has been revealed in vitamins E and C action at prophylactic doses. Radioprotective effect of the combination of vitamins E and C can be associated with the apoptotic activity and can be explained by elimination of the least viable irradiated cells from the cell population. PMID:26863779

  6. A DNA-based system for selecting and displaying the combined result of two input variables.

    PubMed

    Liu, Huajie; Wang, Jianbang; Song, Shiping; Fan, Chunhai; Gothelf, Kurt V

    2015-01-01

    Oligonucleotide-based technologies for biosensing or bio-regulation produce huge amounts of rich high-dimensional information. There is a consequent need for flexible means to combine diverse pieces of such information to form useful derivative outputs, and to display those immediately. Here we demonstrate this capability in a DNA-based system that takes two input numbers, represented in DNA strands, and returns the result of their multiplication, writing this as a number in a display. Unlike a conventional calculator, this system operates by selecting the result from a library of solutions rather than through logic operations. The multiplicative example demonstrated here illustrates a much more general capability--to generate a unique output for any distinct pair of DNA inputs. The system thereby functions as a lookup table and could be a key component in future, more powerful data-processing systems for diagnostics and sensing. PMID:26646059

  7. A DNA-based system for selecting and displaying the combined result of two input variables

    PubMed Central

    Liu, Huajie; Wang, Jianbang; Song, Shiping; Fan, Chunhai; Gothelf, Kurt V.

    2015-01-01

    Oligonucleotide-based technologies for biosensing or bio-regulation produce huge amounts of rich high-dimensional information. There is a consequent need for flexible means to combine diverse pieces of such information to form useful derivative outputs, and to display those immediately. Here we demonstrate this capability in a DNA-based system that takes two input numbers, represented in DNA strands, and returns the result of their multiplication, writing this as a number in a display. Unlike a conventional calculator, this system operates by selecting the result from a library of solutions rather than through logic operations. The multiplicative example demonstrated here illustrates a much more general capability—to generate a unique output for any distinct pair of DNA inputs. The system thereby functions as a lookup table and could be a key component in future, more powerful data-processing systems for diagnostics and sensing. PMID:26646059

  8. Dual Targeting Biomimetic Liposomes for Paclitaxel/DNA Combination Cancer Treatment

    PubMed Central

    Liu, Guo-Xia; Fang, Gui-Qing; Xu, Wei

    2014-01-01

    Combinations of chemotherapeutic drugs with nucleic acid has shown great promise in cancer therapy. In the present study, paclitaxel (PTX) and DNA were co-loaded in the hyaluronic acid (HA) and folate (FA)-modified liposomes (HA/FA/PPD), to obtain the dual targeting biomimetic nanovector. The prepared HA/FA/PPD exhibited nanosized structure and narrow size distributions (247.4 ± 4.2 nm) with appropriate negative charge of −25.40 ± 2.7 mV. HA/FA/PD (PTX free HA/FA/PPD) showed almost no toxicity on murine malignant melanoma cell line (B16) and human hepatocellular carcinoma cell line (HepG2) (higher than 80% cell viability), demonstrating the safety of the blank nanovector. In comparison with the FA-modified PTX/DNA co-loaded liposomes (FA/PPD), HA/FA/PPD showed significant superiority in protecting the nanoparticles from aggregation in the presence of plasma and degradation by DNase I. Moreover, HA/FA/PPD could also significantly improve the transfection efficiency and cellular internalization rates on B16 cells comparing to that of FA/PPD (p < 0.05) and PPD (p < 0.01), demonstrating the great advantages of dual targeting properties. Furthermore, fluorescence microscope and flow cytometry results showed that PTX and DNA could be effectively co-delivered into the same tumor cell via HA/FA/PPD, contributing to PTX/DNA combination cancer treatment. In conclusion, the obtained HA/FA/PPD in the study could effectively target tumor cells, enhance transfection efficiency and subsequently achieve the co-delivery of PTX and DNA, displaying great potential for optimal combination therapy. PMID:25177862

  9. Nanoparticles and DNA - a powerful and growing functional combination in bionanotechnology.

    PubMed

    Samanta, Anirban; Medintz, Igor L

    2016-04-28

    Functionally integrating DNA and other nucleic acids with nanoparticles in all their different physicochemical forms has produced a rich variety of composite nanomaterials which, in many cases, display unique or augmented properties due to the synergistic activity of both components. These capabilities, in turn, are attracting greater attention from various research communities in search of new nanoscale tools for diverse applications that include (bio)sensing, labeling, targeted imaging, cellular delivery, diagnostics, therapeutics, theranostics, bioelectronics, and biocomputing to name just a few amongst many others. Here, we review this vibrant and growing research area from the perspective of the materials themselves and their unique capabilities. Inorganic nanocrystals such as quantum dots or those made from gold or other (noble) metals along with metal oxides and carbon allotropes are desired as participants in these hybrid materials since they can provide distinctive optical, physical, magnetic, and electrochemical properties. Beyond this, synthetic polymer-based and proteinaceous or viral nanoparticulate materials are also useful in the same role since they can provide a predefined and biocompatible cargo-carrying and targeting capability. The DNA component typically provides sequence-based addressability for probes along with, more recently, unique architectural properties that directly originate from the burgeoning structural DNA field. Additionally, DNA aptamers can also provide specific recognition capabilities against many diverse non-nucleic acid targets across a range of size scales from ions to full protein and cells. In addition to appending DNA to inorganic or polymeric nanoparticles, purely DNA-based nanoparticles have recently surfaced as an excellent assembly platform and have started finding application in areas like sensing, imaging and immunotherapy. We focus on selected and representative nanoparticle-DNA materials and highlight their

  10. Nanoparticles and DNA - a powerful and growing functional combination in bionanotechnology

    NASA Astrophysics Data System (ADS)

    Samanta, Anirban; Medintz, Igor L.

    2016-04-01

    Functionally integrating DNA and other nucleic acids with nanoparticles in all their different physicochemical forms has produced a rich variety of composite nanomaterials which, in many cases, display unique or augmented properties due to the synergistic activity of both components. These capabilities, in turn, are attracting greater attention from various research communities in search of new nanoscale tools for diverse applications that include (bio)sensing, labeling, targeted imaging, cellular delivery, diagnostics, therapeutics, theranostics, bioelectronics, and biocomputing to name just a few amongst many others. Here, we review this vibrant and growing research area from the perspective of the materials themselves and their unique capabilities. Inorganic nanocrystals such as quantum dots or those made from gold or other (noble) metals along with metal oxides and carbon allotropes are desired as participants in these hybrid materials since they can provide distinctive optical, physical, magnetic, and electrochemical properties. Beyond this, synthetic polymer-based and proteinaceous or viral nanoparticulate materials are also useful in the same role since they can provide a predefined and biocompatible cargo-carrying and targeting capability. The DNA component typically provides sequence-based addressability for probes along with, more recently, unique architectural properties that directly originate from the burgeoning structural DNA field. Additionally, DNA aptamers can also provide specific recognition capabilities against many diverse non-nucleic acid targets across a range of size scales from ions to full protein and cells. In addition to appending DNA to inorganic or polymeric nanoparticles, purely DNA-based nanoparticles have recently surfaced as an excellent assembly platform and have started finding application in areas like sensing, imaging and immunotherapy. We focus on selected and representative nanoparticle-DNA materials and highlight their

  11. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    PubMed Central

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-01-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/. PMID:26482832

  12. Prenatal diagnosis of X-linked adrenoleukodystrophy combining biochemical, immunocytochemical and DNA analyses.

    PubMed

    Maier, E M; Roscher, A A; Kammerer, S; Mehnert, K; Conzelmann, E; Holzinger, A

    1999-04-01

    Amniocentesis was performed at 17 weeks' gestation on a 39-year-old woman at risk of being a carrier for X-linked adrenoleukodystrophy (X-ALD). Her first son had been affected with childhood cerebral X-ALD and had died at the age of nine years. DNA analysis had not been performed nor was any material available. The amniotic fluid cells (AFC) karyotype was found to be male and initial determination of very long chain fatty acids (VLCFA) in cultured amniocytes revealed borderline values. As an alternative strategy the complete coding region of the ALD gene was amplified and sequenced using DNA isolated from both AFC and maternal leukocytes as templates. Sequencing of the mother's DNA revealed the heterozygous pattern of a 2 bp deletion in exon 5, the most frequent individual mutation leading to X-ALD. It has previously been described to result in a complete loss of protein. This deletion was excluded in the fetus. Accordingly, ALDP was readily detected in AFC by immunofluorescence. We conclude that under circumstances of incomplete data about the index case the combination of methods, namely DNA analysis of the heterozygous mother, and biochemical, immunocytochemical and DNA analyses in fetal cells can secure a reliable prenatal diagnosis of X-ALD. PMID:10327143

  13. Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

    PubMed Central

    Monico, Carina; Belcastro, Gionata; Vanzi, Francesco; Pavone, Francesco S.; Capitanio, Marco

    2014-01-01

    The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable. PMID:25226304

  14. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    NASA Astrophysics Data System (ADS)

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-10-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/.

  15. A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

    PubMed Central

    Asanov, Alexander; Zepeda, Angélica; Vaca, Luis

    2012-01-01

    We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF) in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax. PMID:22438738

  16. Gene expression profiling of breast cancer survivability by pooled cDNA microarray analysis using logistic regression, artificial neural networks and decision trees

    PubMed Central

    2013-01-01

    Background Microarray technology can acquire information about thousands of genes simultaneously. We analyzed published breast cancer microarray databases to predict five-year recurrence and compared the performance of three data mining algorithms of artificial neural networks (ANN), decision trees (DT) and logistic regression (LR) and two composite models of DT-ANN and DT-LR. The collection of microarray datasets from the Gene Expression Omnibus, four breast cancer datasets were pooled for predicting five-year breast cancer relapse. After data compilation, 757 subjects, 5 clinical variables and 13,452 genetic variables were aggregated. The bootstrap method, Mann–Whitney U test and 20-fold cross-validation were performed to investigate candidate genes with 100 most-significant p-values. The predictive powers of DT, LR and ANN models were assessed using accuracy and the area under ROC curve. The associated genes were evaluated using Cox regression. Results The DT models exhibited the lowest predictive power and the poorest extrapolation when applied to the test samples. The ANN models displayed the best predictive power and showed the best extrapolation. The 21 most-associated genes, as determined by integration of each model, were analyzed using Cox regression with a 3.53-fold (95% CI: 2.24-5.58) increased risk of breast cancer five-year recurrence… Conclusions The 21 selected genes can predict breast cancer recurrence. Among these genes, CCNB1, PLK1 and TOP2A are in the cell cycle G2/M DNA damage checkpoint pathway. Oncologists can offer the genetic information for patients when understanding the gene expression profiles on breast cancer recurrence. PMID:23506640

  17. Augmentation of French grunt diet description using combined visual and DNA-based analyses

    USGS Publications Warehouse

    Hargrove, John S.; Parkyn, Daryl C.; Murie, Debra J.; Demopoulos, Amanda W.J.; Austin, James D.

    2012-01-01

    Trophic linkages within a coral-reef ecosystem may be difficult to discern in fish species that reside on, but do not forage on, coral reefs. Furthermore, dietary analysis of fish can be difficult in situations where prey is thoroughly macerated, resulting in many visually unrecognisable food items. The present study examined whether the inclusion of a DNA-based method could improve the identification of prey consumed by French grunt, Haemulon flavolineatum, a reef fish that possesses pharyngeal teeth and forages on soft-bodied prey items. Visual analysis indicated that crustaceans were most abundant numerically (38.9%), followed by sipunculans (31.0%) and polychaete worms (5.2%), with a substantial number of unidentified prey (12.7%). For the subset of prey with both visual and molecular data, there was a marked reduction in the number of unidentified sipunculans (visual – 31.1%, combined &ndash 4.4%), unidentified crustaceans (visual &ndash 15.6%, combined &ndash 6.7%), and unidentified taxa (visual &ndash 11.1%, combined &ndash 0.0%). Utilising results from both methodologies resulted in an increased number of prey placed at the family level (visual &ndash 6, combined &ndash 33) and species level (visual &ndash 0, combined &ndash 4). Although more costly than visual analysis alone, our study demonstrated the feasibility of DNA-based identification of visually unidentifiable prey in the stomach contents of fish.

  18. Perspectives on the combination of radiotherapy and targeted therapy with DNA repair inhibitors in the treatment of pancreatic cancer

    PubMed Central

    Yang, Shih-Hung; Kuo, Ting-Chun; Wu, Hsu; Guo, Jhe-Cyuan; Hsu, Chiun; Hsu, Chih-Hung; Tien, Yu-Wen; Yeh, Kun-Huei; Cheng, Ann-Lii; Kuo, Sung-Hsin

    2016-01-01

    Pancreatic cancer is highly lethal. Current research that combines radiation with targeted therapy may dramatically improve prognosis. Cancerous cells are characterized by unstable genomes and activation of DNA repair pathways, which are indicated by increased phosphorylation of numerous factors, including H2AX, ATM, ATR, Chk1, Chk2, DNA-PKcs, Rad51, and Ku70/Ku80 heterodimers. Radiotherapy causes DNA damage. Cancer cells can be made more sensitive to the effects of radiation (radiosensitization) through inhibition of DNA repair pathways. The synergistic effects, of two or more combined non-lethal treatments, led to co-administration of chemotherapy and radiosensitization in BRCA-defective cells and patients, with promising results. ATM/Chk2 and ATR/Chk1 pathways are principal regulators of cell cycle arrest, following DNA double-strand or single-strand breaks. DNA double-stranded breaks activate DNA-dependent protein kinase, catalytic subunit (DNA-PKcs). It forms a holoenzyme with Ku70/Ku80 heterodimers, called DNA-PK, which catalyzes the joining of nonhomologous ends. This is the primary repair pathway utilized in human cells after exposure to ionizing radiation. Radiosensitization, induced by inhibitors of ATM, ATR, Chk1, Chk2, Wee1, PP2A, or DNA-PK, has been demonstrated in preclinical pancreatic cancer studies. Clinical trials are underway. Development of agents that inhibit DNA repair pathways to be clinically used in combination with radiotherapy is warranted for the treatment of pancreatic cancer. PMID:27621574

  19. Perspectives on the combination of radiotherapy and targeted therapy with DNA repair inhibitors in the treatment of pancreatic cancer.

    PubMed

    Yang, Shih-Hung; Kuo, Ting-Chun; Wu, Hsu; Guo, Jhe-Cyuan; Hsu, Chiun; Hsu, Chih-Hung; Tien, Yu-Wen; Yeh, Kun-Huei; Cheng, Ann-Lii; Kuo, Sung-Hsin

    2016-08-28

    Pancreatic cancer is highly lethal. Current research that combines radiation with targeted therapy may dramatically improve prognosis. Cancerous cells are characterized by unstable genomes and activation of DNA repair pathways, which are indicated by increased phosphorylation of numerous factors, including H2AX, ATM, ATR, Chk1, Chk2, DNA-PKcs, Rad51, and Ku70/Ku80 heterodimers. Radiotherapy causes DNA damage. Cancer cells can be made more sensitive to the effects of radiation (radiosensitization) through inhibition of DNA repair pathways. The synergistic effects, of two or more combined non-lethal treatments, led to co-administration of chemotherapy and radiosensitization in BRCA-defective cells and patients, with promising results. ATM/Chk2 and ATR/Chk1 pathways are principal regulators of cell cycle arrest, following DNA double-strand or single-strand breaks. DNA double-stranded breaks activate DNA-dependent protein kinase, catalytic subunit (DNA-PKcs). It forms a holoenzyme with Ku70/Ku80 heterodimers, called DNA-PK, which catalyzes the joining of nonhomologous ends. This is the primary repair pathway utilized in human cells after exposure to ionizing radiation. Radiosensitization, induced by inhibitors of ATM, ATR, Chk1, Chk2, Wee1, PP2A, or DNA-PK, has been demonstrated in preclinical pancreatic cancer studies. Clinical trials are underway. Development of agents that inhibit DNA repair pathways to be clinically used in combination with radiotherapy is warranted for the treatment of pancreatic cancer. PMID:27621574

  20. DNA Repair in Human Cells Exposed to Combinations of Carcinogenic Agents

    SciTech Connect

    Setlow, R. B.; Ahmed, F. E.

    1980-01-01

    Normal human and XP2 fibroblasts were treated with UV plus UV-mimetic chemicals. The UV dose used was sufficient to saturate the UV excision repair system. Excision repair after combined treatments was estimated by unscheduled DNA synthesis, BrdUrd photolysis, and the loss of sites sensitive to a UV specific endonuclease. Since the repair of damage from UV and its mimetics is coordinately controlled we expected that there would be similar rate-limiting steps in the repair of UV and chemical damage and that after a combined treatment the total amount of repair would be the same as from UV or the chemicals separately. The expectation was not fulfilled. In normal cells repair after a combined treatment was additive whereas in XP cells repair after a combined treatment was usually less than after either agent separately. The chemicals tested were AAAF, DMBA-epoxide, 4NQO, and ICR-170.

  1. Assessment of a magnet system combining the advantages of cable-in-conduit forced-flow and pool-boiling magnets

    SciTech Connect

    Slack, D.; Hassenzahl, W.; Felker, B.; Chaplin, M.

    1993-10-06

    This paper presents an idea for a magnet system that could be used to advantage in tokamaks and other fusion engineering devices. Higher performance designs, specifically newer tokamaks such as those for the international Tokamak Engineering Reactor (ITER) and Tokamak Physics Experiment (TPX) use Cable in Conduit Conductor (CICC) forced flow coils to advantage to meet field and current density requirements. Pool boiling magnets lack structural integrity to resist high magnetic forces since helium cooling areas must surround each conductor. A second problem is that any leak can threaten the voltage standoff integrity of the magnet system. This is because a leak can result in low-pressure helium gas becoming trapped by limited conductance in the magnet bundle and low-pressure helium has poor dielectric strength. The system proposed here is basically a CICC system, with it`s inherent advantages, but bathed in higher pressure supercritical helium to eliminate the leak and voltage break-down problems. Schemes to simplify helium coolant plumbing with the proposed system are discussed. A brief historical review of related magnet systems is included. The advantages and disadvantages of using higher pressure, supercritical helium in combination with solid electrical insulation in a CICC system are discussed. Related electrical data from some previous works are compiled and discussed.

  2. Combining allele frequency uncertainty and population substructure corrections in forensic DNA calculations.

    PubMed

    Cowell, Robert

    2016-07-01

    In forensic DNA calculations of relatedness of individuals and in DNA mixture analyses, at least two sources of uncertainty are present concerning the allele frequencies used for evaluating genotype probabilities when evaluating likelihoods. They are: (i) imprecision in the estimates of the allele frequencies in the population by using an inevitably finite database of DNA profiles to estimate them; and (ii) the existence of population substructure. Green and Mortera [6] showed that these effects may be taken into account individually using a common Dirichlet model within a Bayesian network formulation, but that when taken in combination this is not the case; however they suggested an approximation that could be used. Here we develop a slightly different approximation that is shown to be exact in the case of a single individual. We demonstrate the numerical closeness of the approximation using a published database of allele counts, and illustrate the effect of incorporating the approximation into calculations of a recently published statistical model of DNA mixtures. PMID:27231804

  3. Safety and Comparative Immunogenicity of an HIV-1 DNA Vaccine in Combination with Plasmid Interleukin 12 and Impact of Intramuscular Electroporation for Delivery

    PubMed Central

    Kalams, Spyros A.; Parker, Scott D.; Elizaga, Marnie; Metch, Barbara; Edupuganti, Srilatha; Hural, John; De Rosa, Stephen; Carter, Donald K.; Rybczyk, Kyle; Frank, Ian; Fuchs, Jonathan; Koblin, Beryl; Kim, Denny H.; Joseph, Patrice; Keefer, Michael C.; Baden, Lindsey R.; Eldridge, John; Boyer, Jean; Sherwat, Adam; Cardinali, Massimo; Allen, Mary; Pensiero, Michael; Butler, Chris; Khan, Amir S.; Yan, Jian; Sardesai, Niranjan Y.; Kublin, James G.; Weiner, David B.

    2013-01-01

    Background. DNA vaccines have been very poorly immunogenic in humans but have been an effective priming modality in prime-boost regimens. Methods to increase the immunogenicity of DNA vaccines are needed. Methods. HIV Vaccine Trials Network (HVTN) studies 070 and 080 were multicenter, randomized, clinical trials. The human immunodeficiency virus type 1 (HIV-1) PENNVAX®-B DNA vaccine (PV) is a mixture of 3 expression plasmids encoding HIV-1 Clade B Env, Gag, and Pol. The interleukin 12 (IL-12) DNA plasmid expresses human IL-12 proteins p35 and p40. Study subjects were healthy HIV-1–uninfected adults 18–50 years old. Four intramuscular vaccinations were given in HVTN 070, and 3 intramuscular vaccinations were followed by electroporation in HVTN 080. Cellular immune responses were measured by intracellular cytokine staining after stimulation with HIV-1 peptide pools. Results. Vaccination was safe and well tolerated. Administration of PV plus IL-12 with electroporation had a significant dose-sparing effect and provided immunogenicity superior to that observed in the trial without electroporation, despite fewer vaccinations. A total of 71.4% of individuals vaccinated with PV plus IL-12 plasmid with electroporation developed either a CD4+ or CD8+ T-cell response after the second vaccination, and 88.9% developed a CD4+ or CD8+ T-cell response after the third vaccination. Conclusions. Use of electroporation after PV administration provided superior immunogenicity than delivery without electroporation. This study illustrates the power of combined DNA approaches to generate impressive immune responses in humans. PMID:23840043

  4. DISSOLVED FREE AMINO ACIDS, COMBINED AMINO ACIDS, AND DNA AS SOURCES OF CARBON AND NITROGEN TO MARINE BACTERIA

    EPA Science Inventory

    Utilization of naturally-occurring dissolved free and combined mino cids (DFAA and DCAA) and dissolved DNA FD-DNA) was studied in batch cultures of bacteria from 2 shallow marine environments. anta Rosa Sound (SRS), Florida, USA, and Flax Pond (FP), Long Island, New York, USA. n ...

  5. 53BP1 deficiency combined with telomere dysfunction activates ATR-dependent DNA damage response.

    PubMed

    Martínez, Paula; Flores, Juana M; Blasco, Maria A

    2012-04-16

    TRF1 protects mammalian telomeres from fusion and fragility. Depletion of TRF1 leads to telomere fusions as well as accumulation of γ-H2AX foci and activation of both the ataxia telangiectasia mutated (ATM)- and the ataxia telangiectasia and Rad3 related (ATR)-mediated deoxyribonucleic acid (DNA) damage response (DDR) pathways. 53BP1, which is also present at dysfunctional telomeres, is a target of ATM that accumulates at DNA double-strand breaks and favors nonhomologous end-joining (NHEJ) repair over ATM-dependent resection and homology-directed repair (homologous recombination [HR]). To address the role of 53BP1 at dysfunctional telomeres, we generated mice lacking TRF1 and 53BP1. 53BP1 deficiency significantly rescued telomere fusions in mouse embryonic fibroblasts (MEFs) lacking TRF1, but they showed evidence of a switch from the NHEJ- to HR-mediated repair of uncapped telomeres. Concomitantly, double-mutant MEFs showed evidence of hyperactivation of the ATR-dependent DDR. In intact mice, combined 53BP1/TRF1 deficiency in stratified epithelia resulted in earlier onset of DNA damage and increased CHK1 phosphorylation during embryonic development, leading to aggravation of skin phenotypes. PMID:22508511

  6. Determination of DNA adducts by combining acid-catalyzed hydrolysis and chromatographic analysis of the carcinogen-modified nucleobases.

    PubMed

    Leung, Elvis M K; Deng, Kailin; Wong, Tin-Yan; Chan, Wan

    2016-01-01

    The commonly used method of analyzing carcinogen-induced DNA adducts involves the hydrolysis of carcinogen-modified DNA samples by using a mixture of enzymes, followed by (32)P-postlabeling or liquid chromatography (LC)-based analyses of carcinogen-modified mononucleotides/nucleosides. In the present study, we report the development and application of a new approach to DNA adduct analysis by combining the H(+)/heat-catalyzed release of carcinogen-modified nucleobases and the use of LC-based methods to analyze DNA adducts. Results showed that heating the carcinogen-modified DNA samples at 70 °C for an extended period of 4 to 6 h in the presence of 0.05% HCl can efficiently induce DNA depurination, releasing the intact carcinogen-modified nucleobases for LC analyses. After optimizing the hydrolysis conditions, DNA samples with C8- and N (2) -modified 2'-deoxyguanosine, as well as N (6) -modified 2'-deoxyadenosine, were synthesized by reacting DNA with 1-nitropyrene, acetaldehyde, and aristolochic acids, respectively. These samples were then hydrolyzed, and the released nucleobase adducts were analyzed using LC-based analytical methods. Analysis results demonstrated a dose-dependent release of target DNA adducts from carcinogen-modified DNA samples, indicating that the developed H(+)/heat-catalyzed hydrolysis method was quantitative. Comparative studies with enzymatic digestion method on carcinogen-modified DNA samples revealed that the two hydrolysis methods did not yield systematically different results. PMID:26581621

  7. Combined sequencing of mRNA and DNA from human embryonic stem cells.

    PubMed

    Mertes, Florian; Kuhl, Heiner; Wruck, Wasco; Lehrach, Hans; Adjaye, James

    2016-06-01

    Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells) derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A) mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO) database under accession number GSE69471. PMID:27275414

  8. Combining natural and man-made DNA tracers to advance understanding of hydrologic flow pathway evolution

    NASA Astrophysics Data System (ADS)

    Dahlke, H. E.; Walter, M. T.; Lyon, S. W.; Rosqvist, G. N.

    2014-12-01

    Identifying and characterizing the sources, pathways and residence times of water and associated constituents is critical to developing improved understanding of watershed-stream connections and hydrological/ecological/biogeochemical models. To date the most robust information is obtained from integrated studies that combine natural tracers (e.g. isotopes, geochemical tracers) with controlled chemical tracer (e.g., bromide, dyes) or colloidal tracer (e.g., carboxilated microspheres, tagged clay particles, microorganisms) applications. In the presented study we explore how understanding of sources and flow pathways of water derived from natural tracer studies can be improved and expanded in space and time by simultaneously introducing man-made, synthetic DNA-based microtracers. The microtracer used were composed of polylactic acid (PLA) microspheres into which short strands of synthetic DNA and paramagnetic iron oxide nanoparticles are incorporated. Tracer experiments using both natural tracers and the DNA-based microtracers were conducted in the sub-arctic, glacierized Tarfala (21.7 km2) catchment in northern Sweden. Isotopic hydrograph separations revealed that even though storm runoff was dominated by pre-event water the event water (i.e. rainfall) contributions to streamflow increased throughout the summer season as glacial snow cover decreased. This suggests that glaciers are a major source of the rainwater fraction in streamflow. Simultaneous injections of ten unique DNA-based microtracers confirmed this hypothesis and revealed that the transit time of water traveling from the glacier surface to the stream decreased fourfold over the summer season leading to instantaneous rainwater contributions during storm events. These results highlight that integrating simultaneous tracer injections (injecting tracers at multiple places at one time) with traditional tracer methods (sampling multiple times at one place) rather than using either approach in isolation can

  9. Distribution of a limited Sir2 protein pool regulates the strength of yeast rDNA silencing and is modulated by Sir4p.

    PubMed Central

    Smith, J S; Brachmann, C B; Pillus, L; Boeke, J D

    1998-01-01

    Transcriptional silencing in Saccharomyces cerevisiae occurs at the silent mating-type loci HML and HMR, at telomeres, and at the ribosomal DNA (rDNA) locus RDN1. Silencing in the rDNA occurs by a novel mechanism that depends on a single Silent Information Regulator (SIR) gene, SIR2. SIR4, essential for other silenced loci, paradoxically inhibits rDNA silencing. In this study, we elucidate a regulatory mechanism for rDNA silencing based on the finding that rDNA silencing strength directly correlates with cellular Sir2 protein levels. The endogenous level of Sir2p was shown to be limiting for rDNA silencing. Furthermore, small changes in Sir2p levels altered rDNA silencing strength. In rDNA silencing phenotypes, sir2 mutations were shown to be epistatic to sir4 mutations, indicating that SIR4 inhibition of rDNA silencing is mediated through SIR2. Furthermore, rDNA silencing is insensitive to SIR3 overexpression, but is severely reduced by overexpression of full-length Sir4p or a fragment of Sir4p that interacts with Sir2p. This negative effect of SIR4 overexpression was overridden by co-overexpression of SIR2, suggesting that SIR4 directly inhibits the rDNA silencing function of SIR2. Finally, genetic manipulations of SIR4 previously shown to promote extended life span also resulted in enhanced rDNA silencing. We propose a simple model in which telomeres act as regulators of rDNA silencing by competing for limiting amounts of Sir2 protein. PMID:9649515

  10. A Pooling Genome-Wide Association Study Combining a Pathway Analysis for Typical Sporadic Parkinson's Disease in the Han Population of Chinese Mainland.

    PubMed

    Hu, Yakun; Deng, Libing; Zhang, Jie; Fang, Xin; Mei, Puming; Cao, Xuebing; Lin, Jiari; Wei, Yi; Zhang, Xiong; Xu, Renshi

    2016-09-01

    Genome-wide association studies (GWAS) on sporadic Parkinson's disease (sPD) are mainly conducted in European and American populations at present, and the Han populations of Chinese mainland (HPCM) almost have not been studied yet. Here, we conducted a pooling GWAS combining a pathway analysis with 862,198 autosomal single nucleotide polymorphisms of IlluminaHumanOmniZhongHua-8 in 250 sPD and 250 controls from HPCM precluded toxicant exposure, age, and heavy coffee drinking habit interference. We revealed that among the 22 potential loci implicated, PRDM2/KIAA1026 (kgp8090149), TSG1/MANEA (kgp154172), PDE10A (kgp8130520), MDGA2 (rs9323124), ATPBD4/LOC100288892 (kgp11333367), ZFP64/TSHZ2 (kgp4156164), PAQR3/ARD1B (kgp9482779), FLJ23172/FNDC3B (kgp760898), C18orf1 (kgp348599), FLJ43860/NCRNA00051 (kgp4105983), CYP1B1/C2orf58 (kgp11353523), WNT9A/LOC728728 (rs849898), ANXA1/LOC100130911 (rs10746953), FLJ35379/LOC100132423 (kgp9550589), PLEKHN1 (kgp7172368), DMRT2/SMARCA2 (kgp10769919), ZNF396/INO80C (rs1362858), C3orf67/LOC339902 (rs6783485), LOC285194/IGSF11 (rs1879553), FGF10/MRPS30 (rs13153459), BARX1/PTPDC1 (kgp6542803), and COL5 A2 (rs11186), the peak significance was at the kgp4105983 of FLJ43860 gene in chromosome 8, the first top strongest associated locus with sPD was PRDM2 (kgp8090149) in chromosome 1, and the 24 pathways including 100 significantly associated genes were strongly associated with sPD from HPCM. The 40 genes were shared by at least two pathways. The most possible associated pathways with sPD were axon guidance, ECM-receptor interaction, neuroactive ligand-receptor interaction, tight junction, focal adhesion, gap junction, long-term depression, drug metabolism-cytochrome P450, adherens junction, endocytosis, and protein digestion and absorption. Our results indicated that these loci, pathways, and their related genes might be involved in the pathogenesis of sPD from HPCM and provided some novel evidences for further searching the genetic

  11. A combination treatment with DNA methyltransferase inhibitors and suramin decreases invasiveness of breast cancer cells

    PubMed Central

    Borges, Sahra; Döppler, Heike R.

    2014-01-01

    The treatment of patients with invasive breast cancer remains a major issue because of the acquisition of drug resistance to conventional chemotherapy. Here we propose a new therapeutic strategy by combining DNA methyltransferase inhibitors (DMTIs) with suramin. Cytotoxic effects of suramin or combination treatment with DMTIs were determined in highly invasive breast cancer cell lines MDA-MB-231, BT-20 and HCC1954, or control cells. In addition, effects on cell invasion were determined in 3-dimensional cell culture assays. DMTI-mediated upregulation of Protein Kinase D1 (PKD1) expression was shown by Western blotting. Effects of suramin on PKD1 activity was determined in vitro and in cells. The importance of PKD1 in mediating the effects of such combination treatment in cell invasion was demonstrated using 3D cell culture assays. A proof of principal animal experiment was performed showing that PKD1 is critical for breast cancer growth. We show that when used in combination, suramin and DMTIs impair the invasive phenotype of breast cancer cells. We show that PKD1, a kinase that previously has been described as a suppressor of tumor cell invasion, is an interface for both FDA-approved drugs, since the additive effects observed are due to DMTI-mediated re-expression and suramin-induced activation of PKD1. Our data reveal a mechanism of how a combination treatment with non-toxic doses of suramin and DMTIs may be of therapeutic benefit for patients with aggressive, multi-drug resistant breast cancer. PMID:24510012

  12. DNA bases assembled on the Au(110)/electrolyte interface: a combined experimental and theoretical study.

    PubMed

    Salvatore, Princia; Nazmutdinov, Renat R; Ulstrup, Jens; Zhang, Jingdong

    2015-02-19

    Among the low-index single-crystal gold surfaces, the Au(110) surface is the most active toward molecular adsorption and the one with fewest electrochemical adsorption data reported. Cyclic voltammetry (CV), electrochemically controlled scanning tunneling microscopy (EC-STM), and density functional theory (DFT) calculations have been employed in the present study to address the adsorption of the four nucleobases adenine (A), cytosine (C), guanine (G), and thymine (T), on the Au(110)-electrode surface. Au(110) undergoes reconstruction to the (1 × 3) surface in electrochemical environment, accompanied by a pair of strong voltammetry peaks in the double-layer region in acid solutions. Adsorption of the DNA bases gives featureless voltammograms with lower double-layer capacitance, suggesting that all the bases are chemisorbed on the Au(110) surface. Further investigation of the surface structures of the adlayers of the four DNA bases by EC-STM disclosed lifting of the Au(110) reconstruction, specific molecular packing in dense monolayers, and pH dependence of the A and G adsorption. DFT computations based on a cluster model for the Au(110) surface were performed to investigate the adsorption energy and geometry of the DNA bases in different adsorbate orientations. The optimized geometry is further used to compute models for STM images which are compared with the recorded STM images. This has provided insight into the physical nature of the adsorption. The specific orientations of A, C, G, and T on Au(110) and the nature of the physical adsorbate/surface interaction based on the combination of the experimental and theoretical studies are proposed, and differences from nucleobase adsorption on Au(111)- and Au(100)-electrode surfaces are discussed. PMID:25611676

  13. Radiation damage to a DNA-binding protein. Combined circular dichroism and molecular dynamics simulation analysis.

    PubMed

    Mazier, S; Villette, S; Goffinont, S; Renouard, S; Maurizot, J C; Genest, D; Spotheim-Maurizot, M

    2008-11-01

    The E. coli lactose operon, the paradigm of gene expression regulation systems, is the best model for studying the effect of radiation on such systems. The operon function requires the binding of a protein, the repressor, to a specific DNA sequence, the operator. We have previously shown that upon irradiation the repressor loses its operator binding ability. The main radiation-induced lesions of the headpiece have been identified by mass spectrometry. All tyrosine residues are oxidized into 3,4-dihydroxyphenylalanine (DOPA). In the present study we report a detailed characterization of the headpiece radiation-induced modification. An original approach combining circular dichroism measurements and the analysis of molecular dynamics simulation of headpieces bearing DOPA-s instead of tyrosines has been applied. The CD measurements reveal an irreversible modification of the headpiece structure and stability. The molecular dynamics simulation shows a loss of stability shown by an increase in internal dynamics and allows the estimation of the modifications due to tyrosine oxidation for each structural element of the protein. The changes in headpiece structure and stability can explain at least in part the radiation-induced loss of binding ability of the repressor to the operator. This conclusion should hold for all proteins containing radiosensitive amino acids in their DNA-binding site. PMID:18959464

  14. DNA damage and radiosensitizers: M. luteus sensitive sites for misonidazole-TAN combination

    SciTech Connect

    Skov, K.A.; Palcic, B.; Skarsgard, L.D.

    1982-10-01

    It has been shown previously that TAN does not enhance production of single-strand breaks (SSB) in DNA of Chinese hamster cells irradiated under hypoxia. In contrast, misonidazole does enhance the yield of SSB, but this SSB enhancement by misonidazole is reduced if TAN is present with misonidazole during irradiation. It was also shown that each of these sensitizers enhances base/sugar damage, measured with the aid of bacterial enzymes (MLS). The results presented here for MLS damage in mammalian cells irradiated in the presence of the misonidazole-TAN combination indicate that the two drugs act independently in enhancing MLS damage, and hence they interact with different types of lesions to produce base/sugar damage. It is proposed that the protection by TAN in mammalian cells observed at the survival level is due to repair of some of that type of damage which would otherwise become a misonidazole-enhanced SSB.

  15. Swimming Pool Safety

    MedlinePlus

    ... insist that the following rules are followed: Keep toys away from the pool when the pool is ... after each use. No tricycles or other riding toys at poolside. No electrical appliances near the pool. ...

  16. LinguisticBelief and PoolEvidence

    SciTech Connect

    DARBY, JOHN

    2008-03-11

    LinguisticBelief allows the creation and analysis of combinations of linguistic variables with epistemic uncertainty for decision making. The model is solved using approximate reasoning to implement the belief/plausibility measure of uncertainty for combinations of variables expressed as purely linguistic fuzzy sets. PoolEvidence pools evidence for linguistic variables from many experts for input into LinguisticBelief.

  17. Combined metformin and resveratrol confers protection against UVC-induced DNA damage in A549 lung cancer cells via modulation of cell cycle checkpoints and DNA repair.

    PubMed

    Lee, Yong-Syu; Doonan, Barbara B; Wu, Joseph M; Hsieh, Tze-Chen

    2016-06-01

    Aging in humans is a multi-factorial cellular process that is associated with an increase in the risk of numerous diseases including diabetes, coronary heart disease and cancer. Aging is linked to DNA damage, and a persistent source of DNA damage is exposure to ultraviolet (UV) radiation. As such, identifying agents that confer protection against DNA damage is an approach that could reduce the public health burden of age-related disorders. Metformin and resveratrol have both shown effectiveness in preventing several age-related diseases; using human A549 cells, we investigated whether metformin or resveratrol, alone or combined, prevent UVC-induced DNA damage. We found that metformin inhibited UVC-induced upregulation of p53, as well as downregulated the expression of two DNA damage markers: γH2AX and p-chk2. Metformin also upregulated DNA repair as evidenced by the increase in expression of p53R2. Treatment with metformin also induced cell cycle arrest in UVC-induced cells, in correlation with a reduction in the levels of cyclin E/cdk2/Rb and cyclin B1/cdk1. Compared to metformin, resveratrol as a single agent showed less effectiveness in counteracting UVC-elicited cellular responses. However, resveratrol displayed synergism when combined with metformin as shown by the downregulation of p53/γH2AX/p-chk2. In conclusion, the results of the present study validate the effectiveness of metformin, alone or with the addition of resveratrol, in reducing the risk of aging by conferring protection against UV-induced DNA damage. PMID:27109601

  18. Biological evaluation of new nickel(II) metallates: Synthesis, DNA/protein binding and mitochondrial mediated apoptosis in human lung cancer cells (A549) via ROS hypergeneration and depletion of cellular antioxidant pool.

    PubMed

    Kalaivani, P; Saranya, S; Poornima, P; Prabhakaran, R; Dallemer, F; Vijaya Padma, V; Natarajan, K

    2014-07-23

    A series of novel nickel(II) thiosemicarbazone complexes(1-4) have been prepared and characterized by various spectral, analytical techniques and X-ray crystallography. Further, their efficacy to interact with CT-DNA/BSA has been explored. From the binding studies, it is inferred that complex 4 found to be more active than other complexes. The complexes bound with CT-DNA by intercalation mode. Moreover, static quenching was observed for their interaction with BSA. The new complexes were tested for their in vitro cytotoxicity against human lung adenocarcinoma (A549) cell line. The results showed that the new complexes exhibited significant degree of cytotoxicity at given experimental condition. Further, the results of LDH and NO release supported the cytotoxic nature of the complexes. The observed cytotoxicity of the complexes may be routed through ROS-hypergeneration and lipid-peroxidation with subsequent depletion of cellular antioxidant pool (GSH, SOD, CAT, GPx and GST) resulted in the reduction of mitochondrial-membrane potential, caspase-3 activation and DNA fragmentation. Thus, the data from the present study disclose that the complexes could induce apoptosis in A549 cells through mitochondrial mediated fashion and inhibited the migration of lung cancer cells and by metastasis. PMID:24946146

  19. DNA Diagnostics of Hereditary Hearing Loss: A Targeted Resequencing Approach Combined with a Mutation Classification System.

    PubMed

    Sommen, Manou; Schrauwen, Isabelle; Vandeweyer, Geert; Boeckx, Nele; Corneveaux, Jason J; van den Ende, Jenneke; Boudewyns, An; De Leenheer, Els; Janssens, Sandra; Claes, Kathleen; Verstreken, Margriet; Strenzke, Nicola; Predöhl, Friederike; Wuyts, Wim; Mortier, Geert; Bitner-Glindzicz, Maria; Moser, Tobias; Coucke, Paul; Huentelman, Matthew J; Van Camp, Guy

    2016-08-01

    Although there are nearly 100 different causative genes identified for nonsyndromic hearing loss (NSHL), Sanger sequencing-based DNA diagnostics usually only analyses three, namely, GJB2, SLC26A4, and OTOF. As this is seen as inadequate, there is a need for high-throughput diagnostic methods to detect disease-causing variations, including single-nucleotide variations (SNVs), insertions/deletions (Indels), and copy-number variations (CNVs). In this study, a targeted resequencing panel for hearing loss was developed including 79 genes for NSHL and selected forms of syndromic hearing loss. One-hundred thirty one presumed autosomal-recessive NSHL (arNSHL) patients of Western-European ethnicity were analyzed for SNVs, Indels, and CNVs. In addition, we established a straightforward variant classification system to deal with the large number of variants encountered. We estimate that combining prescreening of GJB2 with our panel leads to a diagnosis in 25%-30% of patients. Our data show that after GJB2, the most commonly mutated genes in a Western-European population are TMC1, MYO15A, and MYO7A (3.1%). CNV analysis resulted in the identification of causative variants in two patients in OTOA and STRC. One of the major challenges for diagnostic gene panels is assigning pathogenicity for variants. A collaborative database collecting all identified variants from multiple centers could be a valuable resource for hearing loss diagnostics. PMID:27068579

  20. A Synthetic Lethal Screen Identifies DNA Repair Pathways that Sensitize Cancer Cells to Combined ATR Inhibition and Cisplatin Treatments

    PubMed Central

    Mohni, Kareem N.; Thompson, Petria S.; Luzwick, Jessica W.; Glick, Gloria G.; Pendleton, Christopher S.; Lehmann, Brian D.; Pietenpol, Jennifer A.; Cortez, David

    2015-01-01

    The DNA damage response kinase ATR may be a useful cancer therapeutic target. ATR inhibition synergizes with loss of ERCC1, ATM, XRCC1 and DNA damaging chemotherapy agents. Clinical trials have begun using ATR inhibitors in combination with cisplatin. Here we report the first synthetic lethality screen with a combination treatment of an ATR inhibitor (ATRi) and cisplatin. Combination treatment with ATRi/cisplatin is synthetically lethal with loss of the TLS polymerase ζ and 53BP1. Other DNA repair pathways including homologous recombination and mismatch repair do not exhibit synthetic lethal interactions with ATRi/cisplatin, even though loss of some of these repair pathways sensitizes cells to cisplatin as a single-agent. We also report that ATRi strongly synergizes with PARP inhibition, even in homologous recombination-proficient backgrounds. Lastly, ATR inhibitors were able to resensitize cisplatin-resistant cell lines to cisplatin. These data provide a comprehensive analysis of DNA repair pathways that exhibit synthetic lethality with ATR inhibitors when combined with cisplatin chemotherapy, and will help guide patient selection strategies as ATR inhibitors progress into the cancer clinic. PMID:25965342

  1. A Preclinical Study Combining the DNA Repair Inhibitor Dbait with Radiotherapy for the Treatment of Melanoma1

    PubMed Central

    Biau, Julian; Devun, Flavien; Jdey, Wael; Kotula, Ewa; Quanz, Maria; Chautard, Emmanuel; Sayarath, Mano; Sun, Jian-Sheng; Verrelle, Pierre; Dutreix, Marie

    2014-01-01

    Melanomas are highly radioresistant tumors, mainly due to efficient DNA double-strand break (DSB) repair. Dbait (which stands for DNA strand break bait) molecules mimic DSBs and trap DNA repair proteins, thereby inhibiting repair of DNA damage induced by radiation therapy (RT). First, the cytotoxic efficacy of Dbait in combination with RT was evaluated in vitro in SK28 and 501mel human melanoma cell lines. Though the extent of RT-induced damage was not increased by Dbait, it persisted for longer revealing a repair defect. Dbait enhanced RT efficacy independently of RT doses. We further assayed the capacity of DT01 (clinical form of Dbait) to enhance efficacy of “palliative” RT (10 × 3 Gy) or “radical” RT (20 × 3 Gy), in an SK28 xenografted model. Inhibition of repair of RT-induced DSB by DT01 was revealed by the significant increase of micronuclei in tumors treated with combined treatment. Mice treated with DT01 and RT combination had significantly better tumor growth control and longer survival compared to RT alone with the “palliative” protocol [tumor growth delay (TGD) by 5.7-fold; median survival: 119 vs 67 days] or the “radical” protocol (TGD by 3.2-fold; median survival: 221 vs 109 days). Only animals that received the combined treatment showed complete responses. No additional toxicity was observed in any DT01-treated groups. This preclinical study provides encouraging results for a combination of a new DNA repair inhibitor, DT01, with RT, in the absence of toxicity. A first-in-human phase I study is currently under way in the palliative management of melanoma in-transit metastases (DRIIM trial). PMID:25379020

  2. Combined shared and distributed memory ab-initio computations of molecular-hydrogen systems in the correlated state: Process pool solution and two-level parallelism

    NASA Astrophysics Data System (ADS)

    Biborski, Andrzej; Kądzielawa, Andrzej P.; Spałek, Józef

    2015-12-01

    An efficient computational scheme devised for investigations of ground state properties of the electronically correlated systems is presented. As an example, (H2)n chain is considered with the long-range electron-electron interactions taken into account. The implemented procedure covers: (i) single-particle Wannier wave-function basis construction in the correlated state, (ii) microscopic parameters calculation, and (iii) ground state energy optimization. The optimization loop is based on highly effective process-pool solution - specific root-workers approach. The hierarchical, two-level parallelism was applied: both shared (by use of Open Multi-Processing) and distributed (by use of Message Passing Interface) memory models were utilized. We discuss in detail the feature that such approach results in a substantial increase of the calculation speed reaching factor of 300 for the fully parallelized solution. The scheme elaborated in detail reflects the situation in which the most demanding task is the single-particle basis optimization.

  3. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  4. Combined DNA extraction and antibody elution from filter papers for the assessment of malaria transmission intensity in epidemiological studies

    PubMed Central

    2013-01-01

    Background Informing and evaluating malaria control efforts relies on knowledge of local transmission dynamics. Serological and molecular tools have demonstrated great sensitivity to quantify transmission intensity in low endemic settings where the sensitivity of traditional methods is limited. Filter paper blood spots are commonly used a source of both DNA and antibodies. To enhance the operational practicability of malaria surveys, a method is presented for combined DNA extraction and antibody elution. Methods Filter paper blood spots were collected as part of a large cross-sectional survey in the Kenyan highlands. DNA was extracted using a saponin/chelex method. The eluate of the first wash during the DNA extraction process was used for antibody detection and compared with previously validated antibody elution procedures. Antibody elution efficiency was assessed by total IgG ELISA for malaria antigens apical membrane antigen-1 (AMA-1) and merozoite-surface protein-1 (MSP-142). The sensitivity of nested 18S rRNA and cytochrome b PCR assays and the impact of doubling filter paper material for PCR sensitivity were determined. The distribution of cell material and antibodies throughout filter paper blood spots were examined using luminescent and fluorescent reporter assays. Results Antibody levels measured after the combined antibody/DNA extraction technique were strongly correlated to those measured after standard antibody elution (p < 0.0001). Antibody levels for both AMA-1 and MSP-142 were generally slightly lower (11.3-21.4%) but age-seroprevalence patterns were indistinguishable. The proportion of parasite positive samples ranged from 12.9% to 19.2% in the different PCR assays. Despite strong agreement between outcomes of different PCR assays, none of the assays detected all parasite-positive individuals. For all assays doubling filter paper material for DNA extraction increased sensitivity. The concentration of cell and antibody material was not

  5. Laser microbeam - kinetic studies combined with molecule - structures reveal mechanisms of DNA repair

    NASA Astrophysics Data System (ADS)

    Altenberg, B.; Greulich, K. O.

    2011-10-01

    Kinetic studies on double strand DNA damages induced by a laser microbeam have allowed a precise definition of the temporal order of recruitment of repair molecules. The order is KU70 / KU80 - XRCC4 --NBS1 -- RAD51. These kinetic studies are now complemented by studies on molecular structures of the repair proteins, using the program YASARA which does not only give molecular structures but also physicochemical details on forces involved in binding processes. It turns out that the earliest of these repair proteins, the KU70 / KU80 heterodimer, has a hole with high DNA affinity. The next molecule, XRCC4, has a body with two arms, the latter with extremely high DNA affinity at their ends and a binding site for Ligase 4. Together with the laser microbeam results this provides a detailed view on the early steps of DNA double strand break repair. The sequence of DNA repair events is presented as a movie.

  6. A Combinational Clustering Based Method for cDNA Microarray Image Segmentation

    PubMed Central

    Shao, Guifang; Li, Tiejun; Zuo, Wangda; Wu, Shunxiang; Liu, Tundong

    2015-01-01

    Microarray technology plays an important role in drawing useful biological conclusions by analyzing thousands of gene expressions simultaneously. Especially, image analysis is a key step in microarray analysis and its accuracy strongly depends on segmentation. The pioneering works of clustering based segmentation have shown that k-means clustering algorithm and moving k-means clustering algorithm are two commonly used methods in microarray image processing. However, they usually face unsatisfactory results because the real microarray image contains noise, artifacts and spots that vary in size, shape and contrast. To improve the segmentation accuracy, in this article we present a combination clustering based segmentation approach that may be more reliable and able to segment spots automatically. First, this new method starts with a very simple but effective contrast enhancement operation to improve the image quality. Then, an automatic gridding based on the maximum between-class variance is applied to separate the spots into independent areas. Next, among each spot region, the moving k-means clustering is first conducted to separate the spot from background and then the k-means clustering algorithms are combined for those spots failing to obtain the entire boundary. Finally, a refinement step is used to replace the false segmentation and the inseparable ones of missing spots. In addition, quantitative comparisons between the improved method and the other four segmentation algorithms--edge detection, thresholding, k-means clustering and moving k-means clustering--are carried out on cDNA microarray images from six different data sets. Experiments on six different data sets, 1) Stanford Microarray Database (SMD), 2) Gene Expression Omnibus (GEO), 3) Baylor College of Medicine (BCM), 4) Swiss Institute of Bioinformatics (SIB), 5) Joe DeRisi’s individual tiff files (DeRisi), and 6) University of California, San Francisco (UCSF), indicate that the improved approach is

  7. A Combinational Clustering Based Method for cDNA Microarray Image Segmentation.

    PubMed

    Shao, Guifang; Li, Tiejun; Zuo, Wangda; Wu, Shunxiang; Liu, Tundong

    2015-01-01

    Microarray technology plays an important role in drawing useful biological conclusions by analyzing thousands of gene expressions simultaneously. Especially, image analysis is a key step in microarray analysis and its accuracy strongly depends on segmentation. The pioneering works of clustering based segmentation have shown that k-means clustering algorithm and moving k-means clustering algorithm are two commonly used methods in microarray image processing. However, they usually face unsatisfactory results because the real microarray image contains noise, artifacts and spots that vary in size, shape and contrast. To improve the segmentation accuracy, in this article we present a combination clustering based segmentation approach that may be more reliable and able to segment spots automatically. First, this new method starts with a very simple but effective contrast enhancement operation to improve the image quality. Then, an automatic gridding based on the maximum between-class variance is applied to separate the spots into independent areas. Next, among each spot region, the moving k-means clustering is first conducted to separate the spot from background and then the k-means clustering algorithms are combined for those spots failing to obtain the entire boundary. Finally, a refinement step is used to replace the false segmentation and the inseparable ones of missing spots. In addition, quantitative comparisons between the improved method and the other four segmentation algorithms--edge detection, thresholding, k-means clustering and moving k-means clustering--are carried out on cDNA microarray images from six different data sets. Experiments on six different data sets, 1) Stanford Microarray Database (SMD), 2) Gene Expression Omnibus (GEO), 3) Baylor College of Medicine (BCM), 4) Swiss Institute of Bioinformatics (SIB), 5) Joe DeRisi's individual tiff files (DeRisi), and 6) University of California, San Francisco (UCSF), indicate that the improved approach is

  8. Swimming pool cleaner poisoning

    MedlinePlus

    Swimming pool cleaner poisoning occurs when someone swallows this type of cleaner, touches it, or breathes in ... The harmful substances in swimming pool cleaner are: Bromine ... copper Chlorine Soda ash Sodium bicarbonate Various mild acids

  9. Swimming pool granuloma

    MedlinePlus

    A swimming pool granuloma is a long-term (chronic) skin infection. It is caused by the bacteria Mycobacterium marinum . ... A swimming pool granuloma occurs when water containing Mycobacterium marinum bacteria enters a break in the skin. Signs of ...

  10. Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata

    PubMed Central

    Fracassetti, Marco; Griffin, Philippa C.; Willi, Yvonne

    2015-01-01

    Sequencing pooled DNA of multiple individuals from a population instead of sequencing individuals separately has become popular due to its cost-effectiveness and simple wet-lab protocol, although some criticism of this approach remains. Here we validated a protocol for pooled whole-genome re-sequencing (Pool-seq) of Arabidopsis lyrata libraries prepared with low amounts of DNA (1.6 ng per individual). The validation was based on comparing single nucleotide polymorphism (SNP) frequencies obtained by pooling with those obtained by individual-based Genotyping By Sequencing (GBS). Furthermore, we investigated the effect of sample number, sequencing depth per individual and variant caller on population SNP frequency estimates. For Pool-seq data, we compared frequency estimates from two SNP callers, VarScan and Snape; the former employs a frequentist SNP calling approach while the latter uses a Bayesian approach. Results revealed concordance correlation coefficients well above 0.8, confirming that Pool-seq is a valid method for acquiring population-level SNP frequency data. Higher accuracy was achieved by pooling more samples (25 compared to 14) and working with higher sequencing depth (4.1× per individual compared to 1.4× per individual), which increased the concordance correlation coefficient to 0.955. The Bayesian-based SNP caller produced somewhat higher concordance correlation coefficients, particularly at low sequencing depth. We recommend pooling at least 25 individuals combined with sequencing at a depth of 100× to produce satisfactory frequency estimates for common SNPs (minor allele frequency above 0.05). PMID:26461136

  11. The science of pooling

    SciTech Connect

    Gilbert, E.

    1995-10-01

    The pooling of data from radon studies is described. Pooling refers to the analysis of original data from several studies, not meta-analysis in which summary measures from published data are analyzed. A main objective for pooling is to reduce uncertainty and to obtain more precise estimates of risk than would be available from any single study.

  12. Enhancement of DNA cancer vaccine efficacy by combination with anti-angiogenesis in regression of established subcutaneous B16 melanoma.

    PubMed

    Chan, Ray Chun-Fai; Gutierrez, Benjamin; Ichim, Thomas E; Lin, Feng

    2009-11-01

    Immunotherapy of cancer offers great promise, however translation into human studies has yielded relatively poor results to date. The concept of combining cancer vaccination with angiogenesis inhibition is appealing, due to favorable safety profile of both approaches, as well as possible biological synergies. Here we studied the anti-tumor effects of combining plasmid DNA (pDNA) vaccination and anti-angiogenesis in B16F10 murine model. By using electroporation-mediated gene/pDNA delivery, the anti-tumor efficacy of vaccination with pDNAs encoding gp100, TRP2 and Ii-PADRE was facilitated by administration of soluble form of EphB4 fused with human serum albumin (sEphB4-HSA), or by co-delivery of pDNAs encoding Angiostatin and/or Endostatin. In an optimized administration protocol, melanoma vaccination together with intratumoral delivery of pDNAs encoding Angiostatin and Endostatin resulted in 57% tumor-free survival over 90 days after challenge. These data support the general concept that suppression of angiogenesis may allow for enhanced efficacy of anti-tumor immunity, suggesting the synergetic effects of therapeutic pDNA vaccination and angiogenesis inhibition in cancer therapy. PMID:19787240

  13. Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

    PubMed Central

    Chaumeil, Julie; Micsinai, Mariann; Skok, Jane A.

    2013-01-01

    Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei. PMID:23407477

  14. Simultaneous measurement of DNA motor protein conformation and activity with combined optical trap and single-molecule fluorescence

    NASA Astrophysics Data System (ADS)

    Chemla, Yann

    2013-03-01

    We present single-molecule measurements of Superfamily 1 UvrD helicase DNA unwinding that reveal directly how helicase stoichiometry and conformation regulate motor activity. Using a new instrument that combines high resolution optical tweezers with single-molecule fluorescence microscopy, we record DNA unwinding activity with base pair-scale resolution (via optical tweezers) simultaneously with helicase stoichiometry and conformation (via fluorescence). Quantifying the fluorescence signal from labeled UvrD, we observe that pairs of UvrD molecules are required for long distance unwinding but that individual molecules exhibit limited, non-processive unwinding activity. UvrD is also known to exhibit two different conformations, `closed' and `open', based on the orientation of its 2B regulatory domain. The function of these conformations has remained elusive. Measuring the fluorescence of FRET labeled proteins, we detect directly the conformation of the 2B domain of individual UvrD molecules during unwinding activity. We observe that UvrD is in the `closed' conformation during DNA unwinding but surprisingly switches to the `open' conformation upon reversal of helicase direction, i.e. when UvrD switches strands and translocates on the opposing strand with the DNA junction rezipping behind it. We hypothesize that the 2B domain acts as a conformational switch that controls DNA unwinding vs. re-annealing. Work supported by NSF (PHY-082261, Center for the Physics of Living Cells) and NIH (R21 RR025341A)

  15. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA

    NASA Astrophysics Data System (ADS)

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e.

    2016-03-01

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4- [4-(N-methyl)styrene] -benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2.

  16. Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ

    PubMed Central

    Barends, Thomas R. M.; Brosi, Richard W. W.; Steinmetz, Andrea; Scherer, Anna; Hartmann, Elisabeth; Eschenbach, Jessica; Lorenz, Thorsten; Seidel, Ralf; Shoeman, Robert L.; Zimmermann, Sabine; Bittl, Robert; Schlichting, Ilme; Reinstein, Jochen

    2013-01-01

    Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ. PMID:23897477

  17. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA.

    PubMed

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e

    2016-03-01

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4-[4-(N-methyl)styrene]-benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2. PMID:26629954

  18. Indolicidin targets duplex DNA: structural and mechanistic insight through a combination of spectroscopy and microscopy.

    PubMed

    Ghosh, Anirban; Kar, Rajiv Kumar; Jana, Jagannath; Saha, Abhijit; Jana, Batakrishna; Krishnamoorthy, Janarthanan; Kumar, Dinesh; Ghosh, Surajit; Chatterjee, Subhrangsu; Bhunia, Anirban

    2014-09-01

    Indolicidin (IR13), a 13-residue antimicrobial peptide from the cathelicidin family, is known to exhibit a broad spectrum of antimicrobial activity against various microorganisms. This peptide inhibits bacterial DNA synthesis resulting in cell filamentation. However, the precise mechanism remains unclear and requires further investigation. The central PWWP motif of IR13 provides a unique structural element that can wrap around, and thus stabilize, duplex B-type DNA structures. Replacements of the central Trp-Trp pair with Ala-Ala, His-His, or Phe-Phe residues in the PxxP motif significantly affects the ability of the peptide to stabilize duplex DNA. Results of microscopy studies in conjunction with spectroscopic data confirm that the DNA duplex is stabilized by IR13, thereby inhibiting DNA replication and transcription. In this study we provide high-resolution structural information on the interaction between indolicidin and DNA, which will be beneficial for the design of novel therapeutic antibiotics based on peptide scaffolds. PMID:25044630

  19. Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants

    PubMed Central

    McManus, Margaret; Mick, Eric; Hudson, Richard; Mofenson, Lynne M.; Sullivan, John L.; Somasundaran, Mohan; Luzuriaga, Katherine

    2016-01-01

    The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log10 copies per million PBMC, p<0.001; ET: -1.04±0.11 log10 DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log10 DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log10 DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible. PMID:27104621

  20. Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants.

    PubMed

    McManus, Margaret; Mick, Eric; Hudson, Richard; Mofenson, Lynne M; Sullivan, John L; Somasundaran, Mohan; Luzuriaga, Katherine

    2016-01-01

    The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log10 copies per million PBMC, p<0.001; ET: -1.04±0.11 log10 DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log10 DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log10 DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible. PMID:27104621

  1. Enhancement of reverse transfection efficiency by combining stimulated DNA surface desorption and electroporation

    NASA Astrophysics Data System (ADS)

    Creasey, Rhiannon; Hook, Andrew; Thissen, Helmut; Voelcker, Nicolas H.

    2007-12-01

    Transfection cell microarrays (TCMs) are a high-throughput, miniaturised cell-culture system utilising reverse transfection, in which cells are seeded onto a DNA array resulting in localised regions of transfected cells. TCMs are useful for the analysis of gene expression, and can be used to identify genes involved in many cellular processes. This is of significant interest in fields such as tissue engineering, diagnostic screening, and drug testing [1, 2]. Low transfection efficiency has so far limited the application and utility of this technique. Recently, the transfection efficiency of TCMs was improved by an application of a high voltage for a short period of time to the DNA array resulting in the electroporation of cells attached to the surface [3, 4]. Furthermore, application of a low voltage for a longer period of time to the DNA array was shown to improve the transfection efficiency by stimulating the desorption of attached DNA, increasing the concentration of DNA available for cellular uptake [5]. In the present study, the optimisation of the uptake of adsorbed DNA vectors by adherent cells, utilising a voltage bias without compromising cell viability was investigated. This was achieved by depositing negatively charged DNA plasmids onto a positively charged allylamine plasma polymer (ALAPP) layer deposited on highly doped p-type silicon wafers either using a pipettor or a microarray contact printer. Surface-dependant human embryonic kidney (HEK 293 line) cells were cultured onto the DNA vector loaded ALAPP spots and the plasmid transfection events were detected by fluorescence microscopy. Cell viability assays, including fluorescein diacetate (FDA) / Hoechst DNA labelling, were carried out to determine the number of live adherent cells before and after application of a voltage. A protocol was developed to screen for voltage biases and exposure times in order to optimise transfection efficiency and cell viability. Cross-contamination between the microarray

  2. The Molecular Dissection of mtDNA Haplogroup H Confirms That the Franco-Cantabrian Glacial Refuge Was a Major Source for the European Gene Pool

    PubMed Central

    Achilli, Alessandro; Rengo, Chiara; Magri, Chiara; Battaglia, Vincenza; Olivieri, Anna; Scozzari, Rosaria; Cruciani, Fulvio; Zeviani, Massimo; Briem, Egill; Carelli, Valerio; Moral, Pedro; Dugoujon, Jean-Michel; Roostalu, Urmas; Loogväli, Eva-Liis; Kivisild, Toomas; Bandelt, Hans-Jürgen; Richards, Martin; Villems, Richard; Santachiara-Benerecetti, A. Silvana; Semino, Ornella; Torroni, Antonio

    2004-01-01

    Complete sequencing of 62 mitochondrial DNAs (mtDNAs) belonging (or very closely related) to haplogroup H revealed that this mtDNA haplogroup—by far the most common in Europe—is subdivided into numerous subhaplogroups, with at least 15 of them (H1–H15) identifiable by characteristic mutations. All the haplogroup H mtDNAs found in 5,743 subjects from 43 populations were then screened for diagnostic markers of subhaplogroups H1 and H3. This survey showed that both subhaplogroups display frequency peaks, centered in Iberia and surrounding areas, with distributions declining toward the northeast and southeast—a pattern extremely similar to that previously reported for mtDNA haplogroup V. Furthermore, the coalescence ages of H1 and H3 (∼11,000 years) are close to that previously reported for V. These findings have major implications for the origin of Europeans, since they attest that the Franco-Cantabrian refuge area was indeed the source of late-glacial expansions of hunter-gatherers that repopulated much of Central and Northern Europe from ∼15,000 years ago. This has also some implications for disease studies. For instance, the high occurrence of H1 and H3 in Iberia led us to re-evaluate the haplogroup distribution in 50 Spanish families affected by nonsyndromic sensorineural deafness due to the A1555G mutation. The survey revealed that the previously reported excess of H among these families is caused entirely by H3 and is due to a major, probably nonrecent, founder event. PMID:15382008

  3. Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

    PubMed Central

    Barakat, Tahsin Stefan; Gribnau, Joost

    2014-01-01

    Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected. PMID:24961515

  4. Sensitive Zn(2+) sensor based on biofunctionalized nanopores via combination of DNAzyme and DNA supersandwich structures.

    PubMed

    Liu, Nannan; Hou, Ruizuo; Gao, Pengcheng; Lou, Xiaoding; Xia, Fan

    2016-06-21

    The sensitivity of detection based on biofunctionalized nanopores is limited since the target-to-signal ratio is 1 : 1. Isothermal amplification is a promising amplification strategy at constant temperature due to its easy operation, quick results, PCR-like sensitivity, low cost and energy efficiency. In the present work, the isothermally amplified detection of Zn(2+) is achieved by using a DNA supersandwich structure and Zn(2+)-requiring DNAzymes. The DNA supersandwich structures, due to the multiple amplification of nucleic acids, heavily plug the nanopore. Simultaneously, the DNA supersandwich structures bond with the sessile probe (SP) of the substrate in the nanopore which partially hybridizes with DNAzymes. In the presence of Zn(2+), the Zn(2+)-requiring DNAzyme cleaves the SP into two fragments, while the DNA supersandwich structures are peeled off and the ionic pathway is unimpeded. A steep drop and a sequential complete recovery of the current occur in the I-V plot when the DNA supersandwich structures are decorated and peeled off. In the present system, the reliable detection limit of Zn(2+) is as low as 1 nM. Discrimination between different types of ions (Cu(2+), Hg(2+), Pb(2+)) is achieved. PMID:26911926

  5. Combined hopping?superexchange model of a hole transfer in DNA

    NASA Astrophysics Data System (ADS)

    Lakhno, V. D.; Sultanov, V. B.; Pettitt, B. Montgomery

    2004-12-01

    Relative hole transfer rates in DNA have been investigated in a number of nucleotide sequences experimentally. Calculation of the transfer rates in DNA is performed relying on the assumption that the transfer is realized as hopping of a hole on guanine sites, each hop being calculated on the basis of superexchange theory. It is shown that the medium reorganization energy and free energy changes play an important role in determining the transfer rate and the type of a nucleotide sequence. The results of the calculations are compared with experimental data covering a range of available sequences.

  6. Fabrication of DNA/RNA Hybrids Through Sequence-Specific Scission of Both Strands by pcPNA-S1 Nuclease Combination.

    PubMed

    Futai, Kazuki; Sumaoka, Jun; Komiyama, Makoto

    2016-05-01

    By combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with S1 nuclease, a tool for site-selective and dual-strand scission of DNA/RNA hybrids has been developed. Both of the DNA and the RNA strands in the hybrids are hydrolyzed at desired sites to provide unique sticky ends. The scission fragments are directly ligated with other DNA/RNA hybrids by using T4 DNA ligase, resulting in the formation of desired recombinant DNA/RNA hybrids. PMID:27057646

  7. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  8. Combination of Gefitinib and DNA Methylation Inhibitor Decitabine Exerts Synergistic Anti-Cancer Activity in Colon Cancer Cells

    PubMed Central

    Chen, Pin-jia; Huang, Guo-bin; Li, Bin; Zheng, De-qing; Yu, Xiu-rong; Luo, Xiao-yong

    2014-01-01

    Despite recent advances in the treatment of human colon cancer, the chemotherapy efficacy against colon cancer is still unsatisfactory. In the present study, effects of concomitant inhibition of the epidermal growth factor receptor (EGFR) and DNA methyltransferase were examined in human colon cancer cells. We demonstrated that decitabine (a DNA methyltransferase inhibitor) synergized with gefitinib (an EGFR inhibitor) to reduce cell viability and colony formation in SW1116 and LOVO cells. However, the combination of the two compounds displayed minimal toxicity to NCM460 cells, a normal human colon mucosal epithelial cell line. The combination was also more effective at inhibiting the AKT/mTOR/S6 kinase pathway. In addition, the combination of decitabine with gefitinib markedly inhibited colon cancer cell migration. Furthermore, gefitinib synergistically enhanced decitabine-induced cytotoxicity was primarily due to apoptosis as shown by Annexin V labeling that was attenuated by z-VAD-fmk, a pan caspase inhibitor. Concomitantly, cell apoptosis resulting from the co-treatment of gefitinib and decitabine was accompanied by induction of BAX, cleaved caspase 3 and cleaved PARP, along with reduction of Bcl-2 compared to treatment with either drug alone. Interestingly, combined treatment with these two drugs increased the expression of XIAP-associated factor 1 (XAF1) which play an important role in cell apoptosis. Moreover, small interfering RNA (siRNA) depletion of XAF1 significantly attenuated colon cancer cells apoptosis induced by the combination of the two drugs. Our findings suggested that gefitinib in combination with decitabine exerted enhanced cell apoptosis in colon cancer cells were involved in mitochondrial-mediated pathway and induction of XAF1 expression. In conclusion, based on the observations from our study, we suggested that the combined administration of these two drugs might be considered as a novel therapeutic regimen for treating colon cancer. PMID

  9. Modified method for combined DNA and RNA isolation from peanut and other oil seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isolation of good quality RNA and DNA from seeds is difficult due to high levels of polysaccharides, polyphenols, and lipids that can degrade or co-precipitate with nucleic acids. Standard RNA extraction methods utilizing guanidinium-phenol-chloroform extraction has not shown to be successful. RNA...

  10. Proposed Strategy for Selection Against Recessive Genetic Defects Through a Combination of Inbreeding and DNA Markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recessive genetic defects are currently on the minds of many cattle breeders. The relatively rapid development of diagnostic DNA tests for recessive defects appears to be a major recent technological advancement. However, the attitude of breeders and breed associations toward recessive defects seems...

  11. Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA

    NASA Astrophysics Data System (ADS)

    Kurihara, Kensuke; Tamura, Mieko; Shohda, Koh-Ichiroh; Toyota, Taro; Suzuki, Kentaro; Sugawara, Tadashi

    2011-10-01

    The construction of a protocell from a materials point of view is important in understanding the origin of life. Both self-reproduction of a compartment and self-replication of an informational substance have been studied extensively, but these processes have typically been carried out independently, rather than linked to one another. Here, we demonstrate the amplification of DNA (encapsulated guest) within a self-reproducible cationic giant vesicle (host). With the addition of a vesicular membrane precursor, we observe the growth and spontaneous division of the giant vesicles, accompanied by distribution of the DNA to the daughter giant vesicles. In particular, amplification of the DNA accelerated the division of the giant vesicles. This means that self-replication of an informational substance has been linked to self-reproduction of a compartment through the interplay between polyanionic DNA and the cationic vesicular membrane. Our self-reproducing giant vesicle system therefore represents a step forward in the construction of an advanced model protocell.

  12. Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA.

    PubMed

    Kurihara, Kensuke; Tamura, Mieko; Shohda, Koh-Ichiroh; Toyota, Taro; Suzuki, Kentaro; Sugawara, Tadashi

    2011-10-01

    The construction of a protocell from a materials point of view is important in understanding the origin of life. Both self-reproduction of a compartment and self-replication of an informational substance have been studied extensively, but these processes have typically been carried out independently, rather than linked to one another. Here, we demonstrate the amplification of DNA (encapsulated guest) within a self-reproducible cationic giant vesicle (host). With the addition of a vesicular membrane precursor, we observe the growth and spontaneous division of the giant vesicles, accompanied by distribution of the DNA to the daughter giant vesicles. In particular, amplification of the DNA accelerated the division of the giant vesicles. This means that self-replication of an informational substance has been linked to self-reproduction of a compartment through the interplay between polyanionic DNA and the cationic vesicular membrane. Our self-reproducing giant vesicle system therefore represents a step forward in the construction of an advanced model protocell. PMID:21941249

  13. Immune responses induced by DNA vaccines bearing Spike gene of PEDV combined with porcine IL-18

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, a highly contagious enteric disease of swine. The Spike (S) protein is one of the main structural proteins of PEDV capable of inducing neutralizing antibodies in vivo. Herein, we generated three distinct DNA ...

  14. Quantitation of DNA methyltransferase activity via chronocoulometry in combination with rolling chain amplification.

    PubMed

    Ji, Jingjing; Liu, Yuanjian; Wei, Wei; Zhang, Yuanjian; Liu, Songqin

    2016-11-15

    In this paper, a rolling chain amplification (RCA) strategy was proposed for chronocoulometric detection of DNA methyltransferase (MTase) activity. Briefly, after the double DNA helix structure was assembled on the surface of gold electrode, it was first methylated by M. SssI MTase and then RCA was realized in the presence of E. coli and phi29 DNA polymerase. Successively, numerous hexaammineruthenium (III) chloride ([Ru(NH3)6)(3+), RuHex) were adsorbed on replicons by electrostatic interaction and generated a large electrochemical readout, the signal was "on". On the contrary, in the absence of M. SssI MTase, the methylated CpG site in the unmethylated double DNA helix structure could be specifically recognized and cleaved by HpaII, resulting in a disconnection of RCA from the electrode. This led seldom RuHex to be absorbed onto the surface of electrode, the signal was "off". Based on the proposed strategy, the activity of M. SssI MTase was assayed in the range of 0.5-60U/mL with a detection limit of 0.09U/mL (S/N=3). In addition, the inhibition of procaine and epicatechin on M. SssI MTase activity was evaluated. When the proposed method was applied in complex matrix such as human serum samples, acceptable accuracy, precision and high sensitivity were achieved. Therefore, the proposed method was a potential useful mean for clinical diagnosis and drug development. PMID:27155113

  15. Combined effect of Cd and Pb spiked field soils on bioaccumulation, DNA damage, and peroxidase activities in Trifolium repens.

    PubMed

    Lanier, C; Bernard, F; Dumez, S; Leclercq, J; Lemière, S; Vandenbulcke, F; Nesslany, F; Platel, A; Devred, I; Cuny, D; Deram, A

    2016-01-01

    The present study was designed to investigate the combined effects of Cd and Pb on accumulation and genotoxic potential in white clover (Trifolium repens). For this purpose, T. repens was exposed to contaminated soils (2.5-20 mg kg(-1) cadmium (Cd), 250-2000 mg kg(-1) lead (Pb) and a mixture of these two heavy metals) for 3, 10 and 56 days. The resulting bioaccumulation of Cd and Pb, DNA damage (comet assay) and peroxidase activities (APOX and GPOX) were determined. The exposure time is a determinant factor in experiments designed to measure the influence of heavy metal contamination. The accumulation of Cd or Pb resulting from exposure to the two-metal mixture does not appear to depend significantly on whether the white clover is exposed to soil containing one heavy metal or both. However, when T. repens is exposed to a Cd/Pb mixture, the percentage of DNA damage is lower than when the plant is exposed to monometallic Cd. DNA damage is close to that observed in the case of monometallic Pb exposure. Peroxidase activity cannot be associated with DNA damage under these experimental conditions. PMID:26396009

  16. Combined analysis of DNA methylome and transcriptome reveal novel candidate genes with susceptibility to bovine Staphylococcus aureus subclinical mastitis

    PubMed Central

    Song, Minyan; He, Yanghua; Zhou, Huangkai; Zhang, Yi; Li, Xizhi; Yu, Ying

    2016-01-01

    Subclinical mastitis is a widely spread disease of lactating cows. Its major pathogen is Staphylococcus aureus (S. aureus). In this study, we performed genome-wide integrative analysis of DNA methylation and transcriptional expression to identify candidate genes and pathways relevant to bovine S. aureus subclinical mastitis. The genome-scale DNA methylation profiles of peripheral blood lymphocytes in cows with S. aureus subclinical mastitis (SA group) and healthy controls (CK) were generated by methylated DNA immunoprecipitation combined with microarrays. We identified 1078 differentially methylated genes in SA cows compared with the controls. By integrating DNA methylation and transcriptome data, 58 differentially methylated genes were shared with differently expressed genes, in which 20.7% distinctly hypermethylated genes showed down-regulated expression in SA versus CK, whereas 14.3% dramatically hypomethylated genes showed up-regulated expression. Integrated pathway analysis suggested that these genes were related to inflammation, ErbB signalling pathway and mismatch repair. Further functional analysis revealed that three genes, NRG1, MST1 and NAT9, were strongly correlated with the progression of S. aureus subclinical mastitis and could be used as powerful biomarkers for the improvement of bovine mastitis resistance. Our studies lay the groundwork for epigenetic modification and mechanistic studies on susceptibility of bovine mastitis. PMID:27411928

  17. Molecular phylogeny of Diabrotica beetles (Coleoptera: Chrysomelidae) inferred from analysis of combined mitochondrial and nuclear DNA sequences.

    PubMed

    Clark, T L; Meinke, L J; Foster, J E

    2001-08-01

    The phylogenetic relationships of thirteen Diabrotica (representing virgifera and fucata species groups) and two outgroup Acalymma beetle species (Coleoptera: Chrysomelidae) were inferred from the phylogenetic analysis of a combined data set of 1323 bp of mitochondrial DNA (mtDNA) cytochrome oxidase subunit 1 (COI) and the entire second internal transcribed spacer region (ITS-2) of nuclear ribosomal DNA of 362 characters. Species investigated were D. adelpha, D. balteata, D. barberi, D. cristata, D. lemniscata, D. longicornis, D. porracea, D. speciosa, D. undecimpunctata howardi, D. u. undecimpunctata, D. virgifera virgifera, D. v. zeae, D. viridula, and outgroup A. blandulum and A. vittatum. Maximum parsimony (MP), minimum evolution (ME), and maximum likelihood (ML) analyses of combined COI and ITS-2 sequences clearly place species into their traditional morphological species groups with MP and ME analyses resulting in identical topologies. Results generally confer with a prior work based on allozyme data, but within the virgifera species group, D. barberi and D. longicornis strongly resolve as sister taxa as well as monophyletic with the neotropical species, D. viridula, D. cristata and D. lemniscata also resolve as sister taxa. Both relationships are not in congruence with the prior allozyme-based hypothesis. Within the fucata species group, D. speciosa and D. balteata resolve as sister taxa. Results also strongly supported the D. virgifera and D. undecimpunctata subspecies complexes. Our proposed phylogeny provides some insight into current hypotheses regarding distribution status and evolution of various life history traits for Diabrotica. PMID:11520353

  18. Aggravated DNA damage as a basis for enhanced glioma cell killing by MJ-66 in combination with minocycline

    PubMed Central

    Hour, Mann-Jen; Liu, Wei-Ting; Lu, I-Chen; Kuo, Sheng-Chu; Gean, Po-Wu

    2014-01-01

    Despite recent advances in the treatment of malignant glomas, the prognosis of patients remains very poor and more efficient therapeutic approaches are urgently needed. In the present study, we investigated whether 2-(naphthalene-1-yl)-6-pyrrolidinyl-4-quinazolinone (MJ-66), a synthetic quinazolinone analog, induces glioma cell death through DNA damage. Treatment of C6 glioma cells with MJ-66 resulted in a time-dependent increase in γ-H2AX and increased the appearance of nuclear γ-H2AX foci. MJ-66 interfered with G2/M DNA damage checkpoint through increasing phosphorylated levels of Chk1 and Cdc25C. UCN-01, a Chk1 inhibitor, reversed MJ-66-induced activation of Cdc25C and caspase 3. MJ-66 inhibited tumor growth and prolonged survival time in intracranial glioma xenograft model. The combination of MJ-66 and Mino enhanced DNA damage and synergistically inhibited tumor growth and prolonged survival time in intracranial glioma xenograft model. These results suggest that the combination of MJ-66 and Mino may be developed as a new therapeutic strategy against malignant gliomas. PMID:25232489

  19. Genetic variants in combination with early partial improvement as a clinical utility predictor of treatment outcome in major depressive disorder: the result of two pooled RCTs.

    PubMed

    Kato, M; Serretti, A; Nonen, S; Takekita, Y; Wakeno, M; Azuma, J; Kinoshita, T

    2015-01-01

    Pharmacogenetics may allow for a personalized treatment, but a combination with clinical variables may further enhance prediction. In particular, in the present paper, we investigated early partial improvement (EPI) defined as 20% or more improvement by rating scales 2  weeks after treatment, in combination with selected gene variants as a predictor of treatment outcome in patients with major depressive disorder. Two randomized controlled trials with 168 Japanese depressed patients were used. A stepwise multiple linear regression model with HAM-D score change at week 6 as the dependent variable and genotypes, EPI, baseline HAM-D score, age and sex as independent variables was performed in paroxetine, fluvoxamine and milnacipran, respectively, to estimate the prediction of HAM-D change at week 6. In the paroxetine sample, only EPI (P<0.001) was significantly associated with HAM-D change (n=81, R(2)=0.25, P<0.001). In the fluvoxamine sample, 5-HTTLPR La/Lg, S (P=0.029), FGF2 rs1449683C/T (P=0.013) and EPI (P=0.003) were associated with HAM-D change (n=42, R(2)=0.43, P<0.001). In the milnacipran sample, HTR-1A-1019C/G (P=0.001), ADRA2A-1297C/G (P=0.028) and EPI (P<0.001) were associated with outcome (n=45, R(2)=0.71, P<0.001). EPI in combination with genetic variants could be a useful predictor of treatment outcome and could strengthen the practical use of pharmacogenetic data in clinical practice. PMID:25710119

  20. Genetic variants in combination with early partial improvement as a clinical utility predictor of treatment outcome in major depressive disorder: the result of two pooled RCTs

    PubMed Central

    Kato, M; Serretti, A; Nonen, S; Takekita, Y; Wakeno, M; Azuma, J; Kinoshita, T

    2015-01-01

    Pharmacogenetics may allow for a personalized treatment, but a combination with clinical variables may further enhance prediction. In particular, in the present paper, we investigated early partial improvement (EPI) defined as 20% or more improvement by rating scales 2  weeks after treatment, in combination with selected gene variants as a predictor of treatment outcome in patients with major depressive disorder. Two randomized controlled trials with 168 Japanese depressed patients were used. A stepwise multiple linear regression model with HAM-D score change at week 6 as the dependent variable and genotypes, EPI, baseline HAM-D score, age and sex as independent variables was performed in paroxetine, fluvoxamine and milnacipran, respectively, to estimate the prediction of HAM-D change at week 6. In the paroxetine sample, only EPI (P<0.001) was significantly associated with HAM-D change (n=81, R2=0.25, P<0.001). In the fluvoxamine sample, 5-HTTLPR La/Lg, S (P=0.029), FGF2 rs1449683C/T (P=0.013) and EPI (P=0.003) were associated with HAM-D change (n=42, R2=0.43, P<0.001). In the milnacipran sample, HTR-1A-1019C/G (P=0.001), ADRA2A-1297C/G (P=0.028) and EPI (P<0.001) were associated with outcome (n=45, R2=0.71, P<0.001). EPI in combination with genetic variants could be a useful predictor of treatment outcome and could strengthen the practical use of pharmacogenetic data in clinical practice. PMID:25710119

  1. Cold pool dissipation

    NASA Astrophysics Data System (ADS)

    Grant, Leah D.; Heever, Susan C.

    2016-02-01

    The mechanisms by which sensible heat fluxes (SHFs) alter cold pool characteristics and dissipation rates are investigated in this study using idealized two-dimensional numerical simulations and an environment representative of daytime, dry, continental conditions. Simulations are performed with no SHFs, SHFs calculated using a bulk formula, and constant SHFs for model resolutions with horizontal (vertical) grid spacings ranging from 50 m (25 m) to 400 m (200 m). In the highest resolution simulations, turbulent entrainment of environmental air into the cold pool is an important mechanism for dissipation in the absence of SHFs. Including SHFs enhances cold pool dissipation rates, but the processes responsible for the enhanced dissipation differ depending on the SHF formulation. The bulk SHFs increase the near-surface cold pool temperatures, but their effects on the overall cold pool characteristics are small, while the constant SHFs influence the near-surface environmental stability and the turbulent entrainment rates into the cold pool. The changes to the entrainment rates are found to be the most significant of the SHF effects on cold pool dissipation. SHFs may also influence the timing of cold pool-induced convective initiation by altering the environmental stability and the cold pool intensity. As the model resolution is coarsened, cold pool dissipation is found to be less sensitive to SHFs. Furthermore, the coarser resolution simulations not only poorly but sometimes wrongly represent the SHF impacts on the cold pools. Recommendations are made regarding simulating the interaction of cold pools with convection and the land surface in cloud-resolving models.

  2. Combined first pass and gated blood pool radionuclide studies in the hemodynamic-cardiac evaluation of patients with low cardiac output

    SciTech Connect

    Abi-Mansour, P.; Fouad, F.M.; Sheeler, L.R.; Bravo, E.L.; MacIntyre, W.J.; Tarazi, R.C.

    1984-01-01

    Cardiac output (CO) is frequently used in the evaluation of cardiac function but low CO does not necessarily reflect heart failure. Similarly, low ejection fraction (EF) can be present in compensated heart diseases. In order to evaluate cardiac performance in relation to systematic hemodynamics, the authors used a multifactorial approach for the determination of CO, EF, pulmonary mean transit time (MTT), ratio of cardiopulmonary volume over total blood volume (CPV/TBV as an index of venous tone) all obtained from a single injection of 99m Tc-HSA. Four different conditions associated with low CO (less than or equal to 2.1 L/min/m/sup 2/) were evaluated. The combined use of CO, EF, MTT and CPV/TBV allowed a better understanding of the myocardial and peripheral circulatory factors associated with low CO states. This is helpful in the selection and follow-up of appropriate therapeutic intervention.

  3. Berberine in combination with cisplatin suppresses breast cancer cell growth through induction of DNA breaks and caspase-3-dependent apoptosis.

    PubMed

    Zhao, Yuwan; Jing, Zuolei; Li, Yan; Mao, Weifeng

    2016-07-01

    Berberine (BBR) is an isoquinoline alkaloid extracted from medicinal plants such as Hydrastis canadensis, Berberis aristata and Coptis chinensis. BBR displays a number of beneficial roles in the treatment of various types of cancers, yet the precise mechanisms of its action remain unclear. Cisplatin is an effective cancer chemotherapeutic agent and functions by generating DNA damage, promoting DNA damage-induced cell cycle arrest and apoptosis; however, its efficacy is challenged by the resistance of tumor cells in clinical application. The aim of the present study was to investigate the effects of BBR in combination with cisplatin on human breast cancer cells. MTT assay showed that BBR inhibited breast cancer MCF-7 cell growth with a 50% inhibitory concentration (IC50) value of 52.178±1.593 µM and the IC50 value of cisplatin was 49.541±1.618 µM, while in combination with 26 µM BBR, the IC50 value of cisplatin was 5.759±0.76 µM. BBR sensitized the MCF-7 cells to cisplatin in a time- and dose-dependent manner. After treatment of BBR and cisplatin, the cellular pro-apoptotic capase-3 and cleaved capspase-3 and caspase-9 were upregulated and the anti-apoptotic Bcl-2 was downregulated. Importantly, BBR restrained the expression of cellular PCNA, and immunofluoresence analysis of γH2AX showed that BBR increased the DNA damages induced by cisplatin. Taken together, the results demonstrated that BBR sensitized MCF-7 cells to cisplatin through induction of DNA breaks and caspase-3-dependent apoptosis. PMID:27177238

  4. Enhanced Performance of Plasmid DNA Polyplexes Stabilized by a Combination of Core Hydrophobicity and Surface PEGylation

    PubMed Central

    Adolph, Elizabeth J.; Nelson, Christopher E.; Werfel, Thomas A.; Guo, Ruijing; Davidson, Jeffrey M.; Duvall, Craig L.

    2014-01-01

    Nonviral gene therapy has high potential for safely promoting tissue restoration and for treating various genetic diseases. One current limitation is that conventional transfection reagents such as polyethylenimine (PEI) form electrostatically stabilized plasmid DNA (pDNA) polyplexes with poor colloidal stability. In this study, a library of poly(ethylene glycol-b-(dimethylaminoethyl methacrylate-co-butyl methacrylate)) [poly(EG-b-(DMAEMA-co-BMA))] polymers were synthesized and screened for improved colloidal stability and nucleic acid transfection following lyophilization. When added to pDNA in the appropriate pH buffer, the DMAEMA moieties initiate formation of electrostatic polyplexes that are internally stabilized by hydrophobic interactions of the core BMA blocks and sterically stabilized against aggregation by a PEG corona. The BMA content was varied from 0% to 60% in the second polymer block in order to optimally tune the balance of electrostatic and hydrophobic interactions in the polyplex core, and polymers with 40 and 50 mol% BMA achieved the highest transfection efficiency. Diblock copolymers were more stable than PEI in physiologic buffers. Consequently, diblock copolymer polyplexes aggregated more slowly and followed a reaction-limited colloidal aggregation model, while fast aggregation of PEI polyplexes was governed by a diffusion-limited model. Polymers with 40% BMA did not aggregate significantly after lyophilization and produced up to 20-fold higher transfection efficiency than PEI polyplexes both before and after lyophilization. Furthermore, poly(EG-b-(DMAEMA-co-BMA)) polyplexes exhibited pH-dependent membrane disruption in a red blood cell hemolysis assay and endosomal escape as observed by confocal microscopy.Lyophilized polyplexes made with the lead candidate diblock copolymer (40% BMA) also successfully transfected cells in vitro following incorporation into gas-foamed polymeric scaffolds. In summary, the enhanced colloidal stability

  5. Mitochondrial DNA deletion in a patient with combined features of Leigh and Pearson syndromes

    SciTech Connect

    Blok, R.B.; Thorburn, D.R.; Danks, D.M.

    1994-09-01

    We describe a heteroplasmic 4237 bp mitochondrial DNA (mtDNA) deletion in an 11 year old girl who has suffered from progressive illness since birth. She has some features of Leigh syndrome (global developmental delay with regression, brainstem dysfunction and lactic acidosis), together with other features suggestive of Pearson syndrome (history of pancytopenia and failure to thrive). The deletion was present at a level greater than 50% in skeletal muscle, but barely detectable in skin fibroblasts following Southern blot analysis, and only observed in blood following PCR analysis. The deletion spanned nt 9498 to nt 13734, and was flanked by a 12 bp direct repeat. Genes for cytochrome c oxidase subunit III, NADH dehydrogenase subunits 3, 4L, 4 and 5, and tRNAs for glycine, arginine, histidine, serine({sup AGY}) and leucine({sup CUN}) were deleted. Southern blotting also revealed an altered Apa I restriction site which was shown by sequence analysis to be caused by G{r_arrow}A nucleotide substitution at nt 1462 in the 12S rRNA gene. This was presumed to be a polymorphism. No abnormalities of mitochondrial ultrastructure, distribution or of respiratory chain enzyme complexes I-IV in skeletal muscle were observed. Mitochondrial disorders with clinical features overlapping more than one syndrome have been reported previously. This case further demonstrates the difficulty in correlating observed clinical features with a specific mitochondrial DNA mutation.

  6. A Fatal Combination: A Thymidylate Synthase Inhibitor with DNA Damaging Activity

    PubMed Central

    Ligasová, Anna; Strunin, Dmytro; Friedecký, David; Adam, Tomáš; Koberna, Karel

    2015-01-01

    2′-deoxy-5-ethynyluridine (EdU) has been previously shown to be a cell poison whose toxicity depends on the particular cell line. The reason is not known. Our data indicates that different efficiency of EdU incorporation plays an important role. The EdU-mediated toxicity was elevated by the inhibition of 2′-deoxythymidine 5′-monophosphate synthesis. EdU incorporation resulted in abnormalities of the cell cycle including the slowdown of the S phase and a decrease in DNA synthesis. The slowdown but not the cessation of the first cell division after EdU administration was observed in all of the tested cell lines. In HeLa cells, a 10 μM EdU concentration led to the cell death in the 100% of cells probably due to the activation of an intra S phase checkpoint in the subsequent S phase. Our data also indicates that this EdU concentration induces interstrand DNA crosslinks in HeLa cells. We suppose that these crosslinks are the primary DNA damage resulting in cell death. According to our results, the EdU-mediated toxicity is further increased by the inhibition of thymidylate synthase by EdU itself at its higher concentrations. PMID:25671308

  7. Combined inhibition of DNA methyltransferase and histone deacetylase restores caspase-8 expression and sensitizes SCLC cells to TRAIL.

    PubMed

    Kaminskyy, Vitaliy O; Surova, Olga V; Vaculova, Alena; Zhivotovsky, Boris

    2011-10-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising drug for the treatment of tumors; however, a number of cancer cells are resistant to this cytokine. Among the mechanisms of resistance of small cell lung carcinomas (SCLCs) to TRAIL is the lack of caspase-8 expression. Although methylation of the caspase-8 promoter has been suggested as the main mechanism of caspase-8 silencing, we showed that reduction of the enzymes involved in DNA methylation, DNA methyltransferases (DNMT) 1, 3a and 3b, was not sufficient to significantly restore caspase-8 expression in SCLC cells, signifying that other mechanisms are involved in caspase-8 silencing. We found that combination of the DNMT inhibitor decitabine with an inhibitor of histone deacetylase (HDAC) significantly increased caspase-8 expression in SCLC cells at the RNA and protein levels. Among all studied HDAC inhibitors, valproic acid (VPA) and CI-994 showed prolonged effects on histone acetylation, while combination with decitabine produced the most prominent effects on caspase-8 re-expression. Moreover, a significant reduction of survivin and cIAP-1 proteins level was observed after treatment with VPA. The combination of two drugs sensitized SCLC cells to TRAIL-induced apoptosis, involving mitochondrial apoptotic pathway and was accompanied by Bid cleavage, activation of Bax, and release of cytochrome c. Both initiator caspase-8 and -9 were required for the sensitization of SCLC cells to TRAIL. Thus, efficient restoration of caspase-8 expression in SCLC cells is achieved when a combination of DNMT and HDAC inhibitors is used, suggesting a combination of decitabine and VPA or CI-994 as a potential treatment for sensitization of SCLC cells lacking caspase-8 to TRAIL. PMID:21771726

  8. Effects of combinations of ROS scavengers on oxidative DNA damage caused by visible-light-activated camphorquinone/N,N-dimethyl-p-toluidine.

    PubMed

    Lee, Seungbum; Pagoria, Dustin; Raigrodski, Ariel; Geurtsen, Werner

    2007-11-01

    The objective of this investigation was to analyze whether various combinations of the ROS scavengers glutathione (GSH), N-acetyl-cysteine (NAC), and vitamins C and E decrease DNA damage due to visible-light-irradiated (VL-irradiated) camphorquinone/N,N-dimethyl-p-toluidine (CQ/DMT) compared with individual vitamin C or E. PhiX-174 RF plasmid DNA was used to determine single and double strand breaks as parameters of DNA damage. Individual ROS scavengers and combinations of the antioxidants were added to plasmid DNA treated with VL-irradiated CQ/DMT/Cu (II). After incubation, DNA was loaded into a 1% agarose gel. Following electrophoresis, gels stained with 0.5 microg/mL ethidium bromide were photographed under ultraviolet illumination and analyzed with NIH ImageJ software. Results were evaluated between groups for statistical significance using Student's paired t-test (p < 0.05). Glutathione significantly reduced oxidative DNA damage at all test concentrations when combined with vitamin C or vitamin E. The concentration of damaged DNA observed in the presence of combinations of GSH with vitamin C or vitamin E was significantly lower compared with all other combinations of antioxidants investigated in our study (p < 0.05). In contrast to GSH, NAC was not able to compensate the pro-oxidative effects of vitamin C and vitamin E. Only at a concentration of 2 mM, NAC combined with vitamin C efficiently prevented CQ/DMT/Cu (II)-associated DNA damage. Our data indicate that solely the combinations of GSH with vitamin C or vitamin E significantly reduce the severity of oxidative DNA damage caused by CQ/DMT, whereas NAC may even increase the pro-oxidant activity of vitamin C and vitamin E. PMID:17443666

  9. Electrochemiluminescence signal amplification combined with a conformation-switched hairpin DNA probe for determining the methylation level and position in the Hsp53 tumor suppressor gene.

    PubMed

    Zhang, Hui; Li, Meixing; Fan, Mengxing; Gu, Jinxing; Wu, Ping; Cai, Chenxin

    2014-03-18

    We report a new strategy for detection of the methylation level and position in the Hsp53 tumor suppressor gene based on the electrochemiluminescence signal amplification combined with a conformation-switched hairpin DNA probe for improving selectivity. PMID:24501739

  10. 13 CFR 120.611 - Pools backing Pool Certificates.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Pools backing Pool Certificates. 120.611 Section 120.611 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION BUSINESS LOANS Secondary Market Certificates § 120.611 Pools backing Pool Certificates. (a) Pool characteristics. As...

  11. Combination of cascade chemical reactions with graphene-DNA interaction to develop new strategy for biosensor fabrication.

    PubMed

    Zhu, Xiaoli; Sun, Liya; Chen, Yangyang; Ye, Zonghuang; Shen, Zhongming; Li, Genxi

    2013-09-15

    Graphene, a single atom thick and two dimensional carbon nano-material, has been proven to possess many unique properties, one of which is the recent discovery that it can interact with single-stranded DNA through noncovalent π-π stacking. In this work, we demonstrate that a new strategy to fabricate many kinds of biosensors can be developed by combining this property with cascade chemical reactions. Taking the fabrication of glucose sensor as an example, while the detection target, glucose, may regulate the graphene-DNA interaction through three cascade chemical reactions, electrochemical techniques are employed to detect the target-regulated graphene-DNA interaction. Experimental results show that in a range from 5μM to 20mM, the glucose concentration is in a natural logarithm with the logarithm of the amperometric response, suggesting a best detection limit and detection range. The proposed biosensor also shows favorable selectivity, and it has the advantage of no need for labeling. What is more, by controlling the cascade chemical reactions, detection of a variety of other targets may be achieved, thus the strategy proposed in this work may have a wide application potential in the future. PMID:23542067

  12. A computer programme for estimation of genetic risk in X linked disorders, combining pedigree and DNA probe data with other conditional information.

    PubMed Central

    Sarfarazi, M; Williams, H

    1986-01-01

    A computer programme is presented for calculating the recurrence risk in X linked disorders, combining pedigree and DNA probe data with other conditional information such as carrier detection tests. The methods of computation are shown in the given examples. The programme can be used with either a single DNA probe or two 'flanking' DNA probes for both familial and isolated case pedigrees. For isolated case families the mutation rate at the disease locus can be taken into account in conjunction with the DNA probe data. PMID:3754009

  13. Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM)

    PubMed Central

    Coghlan, Megan L.; Maker, Garth; Crighton, Elly; Haile, James; Murray, Dáithí C.; White, Nicole E.; Byard, Roger W.; Bellgard, Matthew I.; Mullaney, Ian; Trengove, Robert; Allcock, Richard J. N.; Nash, Christine; Hoban, Claire; Jarrett, Kevin; Edwards, Ross; Musgrave, Ian F.; Bunce, Michael

    2015-01-01

    Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines. PMID:26658160

  14. Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM)

    NASA Astrophysics Data System (ADS)

    Coghlan, Megan L.; Maker, Garth; Crighton, Elly; Haile, James; Murray, Dáithí C.; White, Nicole E.; Byard, Roger W.; Bellgard, Matthew I.; Mullaney, Ian; Trengove, Robert; Allcock, Richard J. N.; Nash, Christine; Hoban, Claire; Jarrett, Kevin; Edwards, Ross; Musgrave, Ian F.; Bunce, Michael

    2015-12-01

    Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.

  15. Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM).

    PubMed

    Coghlan, Megan L; Maker, Garth; Crighton, Elly; Haile, James; Murray, Dáithí C; White, Nicole E; Byard, Roger W; Bellgard, Matthew I; Mullaney, Ian; Trengove, Robert; Allcock, Richard J N; Nash, Christine; Hoban, Claire; Jarrett, Kevin; Edwards, Ross; Musgrave, Ian F; Bunce, Michael

    2015-01-01

    Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines. PMID:26658160

  16. Swimming pool. View of aisle between swimming pool and seating ...

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

    Swimming pool. View of aisle between swimming pool and seating area. Non-original spa pool is partially visible on right. - Jewish Community Center of San Francisco, 3200 California Street, San Francisco, San Francisco County, CA

  17. The combination of FLT3 and DNA methyltransferase inhibition is synergistically cytotoxic to FLT3/ITD acute myeloid leukemia cells.

    PubMed

    Chang, E; Ganguly, S; Rajkhowa, T; Gocke, C D; Levis, M; Konig, H

    2016-05-01

    Effective treatment regimens for elderly acute myeloid leukemia (AML) patients harboring internal tandem duplication mutations in the FMS-like tyrosine kinase-3 (FLT3) gene (FLT3/ITD) are lacking and represent a significant unmet need. Recent data on the effects of FLT3 tyrosine kinase inhibitors on FLT3/ITD(+) AML showed promising clinical activity, including in elderly patients. DNA methyltransferase (DNMT) inhibitors such as decitabine (5-aza-2-deoxycytidine, DEC) and 5-azacitidine (AZA) demonstrated clinical benefit in AML, are well tolerated and are associated with minimal increases in FLT3 ligand, which can represent a potential resistance mechanism to FLT3 inhibitors. In addition, both FLT3 and DNMT inhibition are associated with the induction of terminal differentiation of myeloid blasts. Consequently, there is a strong theoretical rationale for combining FLT3 and DNMT inhibition for FLT3/ITD(+) AML. We therefore sought to study the anti-leukemic effects of DEC, AZA and FLT3 inhibitors, either as single agents or in combination, on AML cell lines and primary cells derived from newly diagnosed and relapsed AML patients. Our studies indicate that combined treatment using FLT3 inhibition and hypomethylation confers synergistic anti-leukemic effects, including apoptosis, growth inhibition and differentiation. The simultaneous administration of AZA and FLT3 inhibition appears to be the most efficacious combination in this regard. These drugs may provide a novel therapeutic approach for FLT3/ITD(+) AML, in particular for older patients. PMID:26686245

  18. Synthesis of DNA containing the simian virus 40 origin of replication by the combined action of DNA polymerases alpha and delta.

    PubMed Central

    Lee, S H; Eki, T; Hurwitz, J

    1989-01-01

    Proliferating-cell nuclear antigen (PCNA) mediates the replication of simian virus 40 (SV40) DNA by reversing the effects of a protein that inhibits the elongation reaction. Two other protein fractions, activator I and activator II, were also shown to play important roles in this process. We report that activator II isolated from HeLa cell extracts is a PCNA-dependent DNA polymerase delta that is required for efficient replication of DNA containing the SV40 origin of replication. PCNA-dependent DNA polymerase delta on a DNA singly primed phi X174 single-stranded circular DNA template required PCNA, a complex of the elongation inhibitor and activator I, and the single-stranded DNA-binding protein essential for SV40 DNA replication. DNA polymerase delta, in contrast to DNA polymerase alpha, hardly used RNA-primed DNA templates. These results indicate that both DNA polymerase alpha and delta are involved in SV40 DNA replication in vitro and their activity depends on PCNA, the elongation inhibitor, and activator I. Images PMID:2571990

  19. Weld pool phenomena

    SciTech Connect

    David, S.A.; Vitek, J.M.; Zacharia, T.; DebRoy, T.

    1994-09-01

    During welding, the composition, structure and properties of the welded structure are affected by the interaction of the heat source with the metal. The interaction affects the fluid flow, heat transfer and mass transfer in the weld pool, and the solidification behavior of the weld metal. In recent years, there has been a growing recognition of the importance of the weld pool transport processes and the solid state transformation reactions in determining the composition, structure and properties of the welded structure. The relation between the weld pool transport processes and the composition and structure is reviewed. Recent applications of various solidification theories to welding are examined to understand the special problems of weld metal solidification. The discussion is focussed on the important problems and issues related to weld pool transport phenomena and solidification. Resolution of these problems would be an important step towards a science based control of composition, structure and properties of the weld metal.

  20. Vitamin D Pooling Project

    Cancer.gov

    The Vitamin D Pooling Project of Rarer Cancers brought together investigators from 10 cohorts to conduct a large prospective epidemiologic study of the association between vitamin D status and seven rarer cancers.

  1. Pools for the Handicapped.

    ERIC Educational Resources Information Center

    American School and University, 1979

    1979-01-01

    Three institutions in Ohio now stress hydrotherapy and water recreation as important parts of individual educational programs for the handicapped. Specially designed and adapted pools provide freedom of movement and ego building as well as physical education and recreation. (Author)

  2. Swimming pool granuloma

    MedlinePlus

    Aquarium granuloma; Fish tank granuloma ... Risks include exposure to swimming pools, salt water aquariums, or ocean fish. ... Wash hands and arms thoroughly after cleaning aquariums. Or, wear rubber gloves when cleaning.

  3. The energetic basis of the DNA double helix: a combined microcalorimetric approach

    PubMed Central

    Vaitiekunas, Paulius; Crane-Robinson, Colyn; Privalov, Peter L.

    2015-01-01

    Microcalorimetric studies of DNA duplexes and their component single strands showed that association enthalpies of unfolded complementary strands into completely folded duplexes increase linearly with temperature and do not depend on salt concentration, i.e. duplex formation results in a constant heat capacity decrement, identical for CG and AT pairs. Although duplex thermostability increases with CG content, the enthalpic and entropic contributions of an AT pair to duplex formation exceed that of a CG pair when compared at the same temperature. The reduced contribution of AT pairs to duplex stabilization comes not from their lower enthalpy, as previously supposed, but from their larger entropy contribution. This larger enthalpy and particularly the greater entropy results from water fixed by the AT pair in the minor groove. As the increased entropy of an AT pair exceeds that of melting ice, the water molecule fixed by this pair must affect those of its neighbors. Water in the minor groove is, thus, orchestrated by the arrangement of AT groups, i.e. is context dependent. In contrast, water hydrating exposed nonpolar surfaces of bases is responsible for the heat capacity increment on dissociation and, therefore, for the temperature dependence of all thermodynamic characteristics of the double helix. PMID:26304541

  4. Circular Dichroism of DNA G-Quadruplexes: Combining Modeling and Spectroscopy To Unravel Complex Structures.

    PubMed

    Gattuso, Hugo; Spinello, Angelo; Terenzi, Alessio; Assfeld, Xavier; Barone, Giampaolo; Monari, Antonio

    2016-03-31

    We report on the comparison between the computational and experimental determination of electronic circular dichroism spectra of different guanine quadruplexes obtained from human telomeric sequences. In particular the difference between parallel, antiparallel, and hybrid structures is evidenced, as well as the induction of transitions between the polymorphs depending on the solution environment. Extensive molecular dynamics simulations (MD) are used to probe the conformational space of the different quadruplexes, and subsequently state-of-the-art hybrid quantum mechanics/molecular mechanics (QM/MM) techniques coupled with excitonic semiempirical Hamiltonian are used to simulate the macromolecular induced circular dichroism. The coupling of spectroscopy and molecular simulation allows an efficient one-to-one mapping between structures and optical properties, offering a way to disentangle the rich, yet complicated, quantity of information embedded in circular dichroism spectra. We show that our methodology is robust and efficient and allows us to take into account subtle conformational changes. As such, it could be used as an efficient tool to investigate structural modification upon DNA/drug interactions. PMID:26943487

  5. Pool-riffle Maintenance in Mountain Streams

    NASA Astrophysics Data System (ADS)

    Chartrand, S. M.

    2015-12-01

    Pool-riffles are maintained through a combination of at least several mechanisms that operate and interact over a range of temporal and spatial scales. Velocity or shear reversal is subsumed within several of these mechanisms, however a growing body of work suggests that (1) flow convergence into pools, (2) structuring of riffle crest sediments, and (3) local feedbacks between flood stage bedform evolution and hydrodynamics may be disproportionately important. We additionally propose that temporal and spatial patterns of sediment sorting across pool-riffles may also provide some level of bedform maintenance. A comprehensive understanding of these maintenance mechanisms is needed. We will report results of several flume experiments for autogenic pool-riffles. The experiments examined pool-riffle maintenance processes under variable flood and sediment supply conditions. A focus of our work is to characterize spatial and temporal patterns of pool-riffle sediment sorting, and to examine this in relation to temporal patterns of bedform evolution. The experiments represent a 5:1 scale-model of a prototype reach of a pool-riffle stream located within the University of British Columbia Malcolm Knapp Research Forest, Maple Ridge, BC.

  6. Synergistic effects of combined DNA methyltransferase inhibition and MBD2 depletion on breast cancer cells; MBD2 depletion blocks 5-aza-2ʹ-deoxycytidine-triggered invasiveness

    PubMed Central

    Cheishvili, David; Chik, Flora; Li, Chen Chen; Bhattacharya, Bishnu; Suderman, Matthew; Arakelian, Ani; Hallett, Michael; Rabbani, Shafaat A.; Szyf, Moshe

    2014-01-01

    5-Aza-2ʹ-deoxycytidine (5-azaCdR) not only inhibits growth of non-invasive breast cancer cells but also increases their invasiveness through induction of pro-metastatic genes. Methylated DNA binding protein 2 (MBD2) is involved in silencing methylated tumor suppressor genes as well as activation of pro-metastatic genes. In this study, we show that a combination of MBD2 depletion and DNA methyltransferases (DNMT) inhibition in breast cancer cells results in a combined effect in vitro and in vivo, enhancing tumor growth arrest on one hand, while inhibiting invasiveness triggered by 5-azaCdR on the other hand. The combined treatment of MBD2 depletion and 5-azaCdR suppresses and augments distinct gene networks that are induced by DNMT inhibition alone. These data point to a potential new approach in targeting the DNA methylation machinery by combination of MBD2 and DNMT inhibitors. PMID:25178277

  7. Combinations of various CpG motifs cloned into plasmid backbone modulate and enhance protective immunity of viral replicon DNA anthrax vaccines.

    PubMed

    Yu, Yun-Zhou; Ma, Yao; Xu, Wen-Hui; Wang, Shuang; Sun, Zhi-Wei

    2015-08-01

    DNA vaccines are generally weak stimulators of the immune system. Fortunately, their efficacy can be improved using a viral replicon vector or by the addition of immunostimulatory CpG motifs, although the design of these engineered DNA vectors requires optimization. Our results clearly suggest that multiple copies of three types of CpG motifs or combinations of various types of CpG motifs cloned into a viral replicon vector backbone with strong immunostimulatory activities on human PBMC are efficient adjuvants for these DNA vaccines to modulate and enhance protective immunity against anthrax, although modifications with these different CpG forms in vivo elicited inconsistent immune response profiles. Modification with more copies of CpG motifs elicited more potent adjuvant effects leading to the generation of enhanced immunity, which indicated a CpG motif dose-dependent enhancement of antigen-specific immune responses. Notably, the enhanced and/or synchronous adjuvant effects were observed in modification with combinations of two different types of CpG motifs, which provides not only a contribution to the knowledge base on the adjuvant activities of CpG motifs combinations but also implications for the rational design of optimal DNA vaccines with combinations of CpG motifs as "built-in" adjuvants. We describe an efficient strategy to design and optimize DNA vaccines by the addition of combined immunostimulatory CpG motifs in a viral replicon DNA plasmid to produce strong immune responses, which indicates that the CpG-modified viral replicon DNA plasmid may be desirable for use as vector of DNA vaccines. PMID:25265876

  8. Epigenetic age predictions based on buccal swabs are more precise in combination with cell type-specific DNA methylation signatures.

    PubMed

    Eipel, Monika; Mayer, Felix; Arent, Tanja; Ferreira, Marcelo R P; Birkhofer, Carina; Gerstenmaier, Uwe; Costa, Ivan G; Ritz-Timme, Stefanie; Wagner, Wolfgang

    2016-05-01

    Aging is reflected by highly reproducible DNA methylation (DNAm) changes that open new perspectives for estimation of chronological age in legal medicine. DNA can be harvested non-invasively from cells at the inside of a person's cheek using buccal swabs - but these specimens resemble heterogeneous mixtures of buccal epithelial cells and leukocytes with different epigenetic makeup. In this study, we have trained an age predictor based on three age-associated CpG sites (associated with the genesPDE4C, ASPA, and ITGA2B) for swab samples to reach a mean absolute deviation (MAD) between predicted and chronological age of 4.3 years in a training set and of 7.03 years in a validation set. Subsequently, the composition of buccal epithelial cells versus leukocytes was estimated by two additional CpGs (associated with the genes CD6 and SERPINB5). Results of this "Buccal-Cell-Signature" correlated with cell counts in cytological stains (R2 = 0.94). Combination of cell type-specific and age-associated CpGs into one multivariate model enabled age predictions with MADs of 5.09 years and 5.12 years in two independent validation sets. Our results demonstrate that the cellular composition in buccal swab samples can be determined by DNAm at two cell type-specific CpGs to improve epigenetic age predictions. PMID:27249102

  9. Epigenetic age predictions based on buccal swabs are more precise in combination with cell type-specific DNA methylation signatures

    PubMed Central

    Eipel, Monika; Mayer, Felix; Arent, Tanja; Ferreira, Marcelo R. P.; Birkhofer, Carina; Gerstenmaier, Uwe; Costa, Ivan G.; Ritz-Timme, Stefanie; Wagner, Wolfgang

    2016-01-01

    Aging is reflected by highly reproducible DNA methylation (DNAm) changes that open new perspectives for estimation of chronological age in legal medicine. DNA can be harvested non-invasively from cells at the inside of a person's cheek using buccal swabs – but these specimens resemble heterogeneous mixtures of buccal epithelial cells and leukocytes with different epigenetic makeup. In this study, we have trained an age predictor based on three age-associated CpG sites (associated with the genes PDE4C, ASPA, and ITGA2B) for swab samples to reach a mean absolute deviation (MAD) between predicted and chronological age of 4.3 years in a training set and of 7.03 years in a validation set. Subsequently, the composition of buccal epithelial cells versus leukocytes was estimated by two additional CpGs (associated with the genes CD6 and SERPINB5). Results of this “Buccal-Cell-Signature” correlated with cell counts in cytological stains (R2 = 0.94). Combination of cell type-specific and age-associated CpGs into one multivariate model enabled age predictions with MADs of 5.09 years and 5.12 years in two independent validation sets. Our results demonstrate that the cellular composition in buccal swab samples can be determined by DNAm at two cell type-specific CpGs to improve epigenetic age predictions. PMID:27249102

  10. SHAMS: Combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids

    PubMed Central

    Turner, Kevin B.; Yi-Brunozzi, Hye Young; Brinson, Robert G.; Marino, John P.; Fabris, Daniele; Le Grice, Stuart F.J.

    2009-01-01

    Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) has gained popularity as a facile method of examining RNA structure both in vitro and in vivo, exploiting accessibility of the ribose 2′-OH to acylation by N-methylisatoic anhydride (NMIA) in unpaired or flexible configurations. Subsequent primer extension terminates at the site of chemical modification, and these products are fractionated by high-resolution gel electrophoresis. When applying SHAPE to investigate structural features associated with the wild-type and analog-substituted polypurine tract (PPT)–containing RNA/DNA hybrids, their size (20–25 base pairs) rendered primer extension impractical. As an alternative method of detection, we reasoned that chemical modification could be combined with tandem mass spectrometry, relying on the mass increment of RNA fragments containing the NMIA adduct (Mr = 133 Da). Using this approach, we demonstrate both specific modification of the HIV-1 PPT RNA primer and variations in its acylation pattern induced by replacing template nucleotides with a non-hydrogen-bonding thymine isostere. Our selective 2′-hydroxyl acylation analyzed by mass spectrometry strategy (SHAMS) should find utility when examining the structure of small RNA fragments or RNA/DNA hybrids where primer extension cannot be performed. PMID:19535461

  11. Nonsense mutation at Tyr-4046 in the DNA-dependent protein kinase catalytic subunit of severe combined immune deficiency mice

    PubMed Central

    Araki, Ryoko; Fujimori, Akira; Hamatani, Kiyohiro; Mita, Kazuei; Saito, Toshiyuki; Mori, Masahiko; Fukumura, Ryutaro; Morimyo, Mitsuoki; Muto, Masahiro; Itoh, Masahiro; Tatsumi, Kouichi; Abe, Masumi

    1997-01-01

    The severe combined immune deficiency (SCID) mouse was reported as an animal model for human immune deficiency. Through the course of several studies, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) gene came to be considered a candidate for the SCID-responsible gene. We isolated an ORF of the murine DNA-PKcs gene from SCID mice and their parent strain C.B-17 mice and determined the DNA sequences. The ORF of the murine DNA-PKcs gene contained 4128-aa residues and had 78.9% homology with the human DNA-PKcs gene. A particularly important finding is that a T to A transversion results in the substitution of termination codon in SCID mice for the Tyr-4046 in C.B-17 mice. No other mutation was detected in the ORF of the gene. The generality of this transversion was confirmed using four individual SCID and wild-type mice. The substitution took place in the phosphatidylinositol 3-kinase domain, and the mutated gene encodes the truncated products missing 83 residues of wild-type DNA-PKcs products. Furthermore, the quantity of DNA-PKcs transcript in wild-type and SCID cells was almost equal. These observations indicate that the DNA-PKcs gene is the SCID-responsible gene itself and that the detected mutation leads to the SCID aberration. PMID:9122213

  12. Combination of PDT and a DNA demethylating agent produces anti-tumor immune response in a mouse tumor model

    NASA Astrophysics Data System (ADS)

    Mroz, Pawel; Hamblin, Michael R.

    2009-06-01

    Epigenetic mechanisms, which involve DNA methylation and histone modifications, result in the heritable silencing of genes without a change in their coding sequence. However, these changes must be actively maintained after each cell division rendering them a promising target for pharmacologic inhibition. DNA methyltransferase inhibitors like 5-aza-deoxycytidine (5-aza-dC) induce and/or up-regulate the expression of MAGE-type antigens in human and mice cancer cells. Photodynamic therapy (PDT) has been shown to be an effective locally ablative anti-cancer treatment that has the additional advantage of stimulating tumor-directed immune response. We studied the effects of a new therapy that combined the demethylating agent 5-aza-dC with PDT in the breast cancer model 4T1 syngenic to immunocompetent BALB/c mice. PDT was used as a locally ablating tumor treatment that is capable of eliciting strong and tumor directed immune response while 5-aza-dC pretreatment was used promote de novo induction of the expression of P1A.protein. This is the mouse homolog of human MAGE family antigens and is reported to function as a tumor rejection antigen in certain mouse tumors. This strategy led to an increase in PDT-mediated immune response and better treatment outcome. These results strongly suggest that the MAGE family antigens are important target for PDT mediated immune response but that their expression can be silenced by epigenetic mechanisms. Therefore the possibility that PDT can be combined with epigenetic strategies to elicit anti-tumor immunity in MAGE-positive tumor models is highly clinically significant and should be studied in detail.

  13. Targeting DNA repair with combination veliparib (ABT-888) and temozolomide in patients with metastatic castration-resistant prostate cancer

    PubMed Central

    Carducci, Michael A.; Slovin, Susan; Cetnar, Jeremy; Qian, Jiang; McKeegan, Evelyn M.; Refici-Buhr, Marion; Chyla, Brenda; Shepherd, Stacie P.; Giranda, Vincent L.; Alumkal, Joshi J.

    2015-01-01

    Summary Androgen receptor-mediated transcription is directly coupled with the induction of DNA damage, and castration-resistant tumor cells exhibit increased activity of poly (ADP-ribose) polymerase (PARP)-1, a DNA repair enzyme. This study assessed the efficacy and safety of low dose oral PARP inhibitor veliparib (ABT-888) and temozolomide (TMZ) in docetaxel-pretreated patients with metastatic castration-resistant prostate cancer (mCRPC) in a single-arm, open-label, pilot study. Patients with mCRPC progressing on at least one docetaxel-based therapy and prostate specific antigen (PSA) ≥2 ng/mL were treated with veliparib 40 mg twice daily on days 1–7 and TMZ once daily (150 mg/m2/day cycle 1; if well tolerated then 200 mg/m2/day cycle 2 onwards) on days 1–5 q28 days. Patients received 2 (median) treatment cycles (range, 1–9). The primary end-point was confirmed PSA response rate (decline≥30 %). Twenty-six eligible patients were enrolled, 25 evaluable for PSA response. Median baseline PSA was 170 ng/mL. Two patients had a confirmed PSA response (8.0 %; 95 % CI: 1.0– 26.0), 13 stable PSA, and 10 PSA progression. The median progression-free survival was 9 weeks (95 % CI: 7.9–17) and median overall survival 39.6 weeks (95 % CI: 26.6–not estimable). The most frequent treatment-emergent adverse events (AEs) were thrombocytopenia (77 %), anemia (69 %), fatigue (50 %), neutropenia (42 %), nausea (38 %), and constipation (23 %). Grade 3/4 AEs occurring in >10 % of patients were thrombocytopenia (23 %) and anemia (15 %). Veliparib and TMZ combination was well tolerated but with modest activity. Biomarker analysis supported the proof of concept that this combination has some antitumor activity in mCRPC. PMID:24764124

  14. Acoustic detection of DNA conformation in genetic assays combined with PCR.

    PubMed

    Papadakis, G; Tsortos, A; Kordas, A; Tiniakou, I; Morou, E; Vontas, J; Kardassis, D; Gizeli, E

    2013-01-01

    Application of PCR to multiplexing assays is not trivial; it requires multiple fluorescent labels for amplicon detection and sophisticated software for data interpretation. Alternative PCR-free methods exploiting new concepts in nanotechnology exhibit high sensitivities but require multiple labeling and/or amplification steps. Here, we propose to simplify the problem of simultaneous analysis of multiple targets in genetic assays by detecting directly the conformation, rather than mass, of target amplicons produced in the same PCR reaction. The new methodology exploits acoustic wave devices which are shown to be able to characterize in a fully quantitative manner multiple double stranded DNAs of various lengths. The generic nature of the combined acoustic/PCR platform is shown using real samples and, specifically, during the detection of SNP genotyping in Anopheles gambiae and gene expression quantification in treated mice. The method possesses significant advantages to TaqMan assay and real-time PCR regarding multiplexing capability, speed, simplicity and cost. PMID:23778520

  15. Acoustic detection of DNA conformation in genetic assays combined with PCR

    PubMed Central

    Papadakis, G.; Tsortos, A.; Kordas, A.; Tiniakou, I.; Morou, E.; Vontas, J.; Kardassis, D.; Gizeli, E.

    2013-01-01

    Application of PCR to multiplexing assays is not trivial; it requires multiple fluorescent labels for amplicon detection and sophisticated software for data interpretation. Alternative PCR-free methods exploiting new concepts in nanotechnology exhibit high sensitivities but require multiple labeling and/or amplification steps. Here, we propose to simplify the problem of simultaneous analysis of multiple targets in genetic assays by detecting directly the conformation, rather than mass, of target amplicons produced in the same PCR reaction. The new methodology exploits acoustic wave devices which are shown to be able to characterize in a fully quantitative manner multiple double stranded DNAs of various lengths. The generic nature of the combined acoustic/PCR platform is shown using real samples and, specifically, during the detection of SNP genotyping in Anopheles gambiae and gene expression quantification in treated mice. The method possesses significant advantages to TaqMan assay and real-time PCR regarding multiplexing capability, speed, simplicity and cost. PMID:23778520

  16. Genetic variants in DNA repair pathways and risk of upper aerodigestive tract cancers: combined analysis of data from two genome-wide association studies in European populations.

    PubMed

    Babron, Marie-Claude; Kazma, Rémi; Gaborieau, Valérie; McKay, James; Brennan, Paul; Sarasin, Alain; Benhamou, Simone

    2014-07-01

    DNA repair pathways are good candidates for upper aerodigestive tract cancer susceptibility because of their critical role in maintaining genome integrity. We have selected 13 pathways involved in DNA repair representing 212 autosomal genes. To assess the role of these pathways and their associated genes, two European data sets from the International Head and Neck Cancer Epidemiology consortium were pooled, totaling 1954 cases and 3121 controls, with documented demographic, lifetime alcohol and tobacco consumption information. We applied an innovative approach that tests single nucleotide polymorphism (SNP)-sets within DNA repair pathways and then within genes belonging to the significant pathways. We showed an association between the polymerase pathway and oral cavity/pharynx cancers (P-corrected = 4.45 × 10(-) (2)), explained entirely by the association with one SNP, rs1494961 (P = 2.65 × 10(-) (4)), a missense mutation V306I in the second exon of HELQ gene. We also found an association between the cell cycle regulation pathway and esophagus cancer (P-corrected = 1.48 × 10(-) (2)), explained by three SNPs located within or near CSNK1E gene: rs1534891 (P = 1.27 × 10(-) (4)), rs7289981 (P = 3.37 × 10(-) (3)) and rs13054361 (P = 4.09 × 10(-) (3)). As a first attempt to investigate pathway-level associations, our results suggest a role of specific DNA repair genes/pathways in specific upper aerodigestive tract cancer sites. PMID:24658182

  17. Vernal Pool Lessons and Activities.

    ERIC Educational Resources Information Center

    Childs, Nancy; Colburn, Betsy

    This curriculum guide accompanies Certified: A Citizen's Step-by-Step Guide to Protecting Vernal Pools which is designed to train volunteers in the process of identifying vernal pool habitat so that as many of these pools as possible can be certified by the Massachusetts Natural Heritage and Endangered Species Program. Vernal pools are a kind of…

  18. Treatment of metastatic breast cancer by combination of chemotherapy and photothermal ablation using doxorubicin-loaded DNA wrapped gold nanorods.

    PubMed

    Wang, Dangge; Xu, Zhiai; Yu, Haijun; Chen, Xianzhi; Feng, Bing; Cui, Zhirui; Lin, Bin; Yin, Qi; Zhang, Zhiwen; Chen, Chunying; Wang, Jun; Zhang, Wen; Li, Yaping

    2014-09-01

    Despite the exciting advances in cancer therapy over past decades, tumor metastasis remains the dominate reason for cancer-related mortality. In present work, DNA-wrapped gold nanorods with doxorubicin (DOX)-loading (GNR@DOX) were developed for treatment of metastatic breast cancer via a combination of chemotherapy and photothermal ablation. The GNR@DOX nanoparticles induced significant temperature elevation and DOX release upon irradiation with near infrared (NIR) light as shown in the test tube studies. It was found that GNR@DOX nanoparticles in combination with laser irradiation caused higher cytotoxicity than free DOX in 4T1 breast cancer cells. Animal experiment with an orthotropic 4T1 mammary tumor model demonstrated that GNR@DOX nanoplatform significantly reduced the growth of primary tumors and suppressed their lung metastasis. The Hematoxylin and Eosin (H&E) and immunohistochemistry (IHC) staining assays confirmed that the tumor growth inhibition and metastasis prevention of GNR@DOX nanoparticles were attributed to their abilities to induce cellular apoptosis/necrosis and ablate intratumoral blood vessels. All these results suggested a considerable potential of GNR@DOX nanoplatform for treatment of metastatic breast cancer. PMID:24996756

  19. Determining the Architecture of a Protein-DNA Complex by Combining FeBABE Cleavage Analyses, 3-D Printed Structures, and the ICM Molsoft Program.

    PubMed

    James, Tamara; Hsieh, Meng-Lun; Knipling, Leslie; Hinton, Deborah

    2015-01-01

    Determining the structure of a protein-DNA complex can be difficult, particularly if the protein does not bind tightly to the DNA, if there are no homologous proteins from which the DNA binding can be inferred, and/or if only portions of the protein can be crystallized. If the protein comprises just a part of a large multi-subunit complex, other complications can arise such as the complex being too large for NMR studies, or it is not possible to obtain the amounts of protein and nucleic acids needed for crystallographic analyses. Here, we describe a technique we used to map the position of an activator protein relative to the DNA within a large transcription complex. We determined the position of the activator on the DNA from data generated using activator proteins that had been conjugated at specific residues with the chemical cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). These analyses were combined with 3-D models of the available structures of portions of the activator protein and B-form DNA to obtain a 3-D picture of the protein relative to the DNA. Finally, the Molsoft program was used to refine the position, revealing the architecture of the protein-DNA within the transcription complex. PMID:26404142

  20. Ultra-low Doping on Two-Dimensional Transition Metal Dichalcogenides using DNA Nanostructure Doped by a Combination of Lanthanide and Metal Ions

    PubMed Central

    Kang, Dong-Ho; Dugasani, Sreekantha Reddy; Park, Hyung-Youl; Shim, Jaewoo; Gnapareddy, Bramaramba; Jeon, Jaeho; Lee, Sungjoo; Roh, Yonghan; Park, Sung Ha; Park, Jin-Hong

    2016-01-01

    Here, we propose a novel DNA-based doping method on MoS2 and WSe2 films, which enables ultra-low n- and p-doping control and allows for proper adjustments in device performance. This is achieved by selecting and/or combining different types of divalent metal and trivalent lanthanide (Ln) ions on DNA nanostructures, using the newly proposed concept of Co-DNA (DNA functionalized by both divalent metal and trivalent Ln ions). The available n-doping range on the MoS2 by Ln-DNA is between 6 × 109 and 2.6 × 1010 cm−2. The p-doping change on WSe2 by Ln-DNA is adjusted between −1.0 × 1010 and −2.4 × 1010 cm−2. In Eu3+ or Gd3+-Co-DNA doping, a light p-doping is observed on MoS2 and WSe2 (~1010 cm−2). However, in the devices doped by Tb3+ or Er3+-Co-DNA, a light n-doping (~1010 cm−2) occurs. A significant increase in on-current is also observed on the MoS2 and WSe2 devices, which are, respectively, doped by Tb3+- and Gd3+-Co-DNA, due to the reduction of effective barrier heights by the doping. In terms of optoelectronic device performance, the Tb3+ or Er3+-Co-DNA (n-doping) and the Eu3+ or Gd3+-Co-DNA (p-doping) improve the MoS2 and WSe2 photodetectors, respectively. We also show an excellent absorbing property by Tb3+ ions on the TMD photodetectors. PMID:26838524

  1. Ultra-low Doping on Two-Dimensional Transition Metal Dichalcogenides using DNA Nanostructure Doped by a Combination of Lanthanide and Metal Ions.

    PubMed

    Kang, Dong-Ho; Dugasani, Sreekantha Reddy; Park, Hyung-Youl; Shim, Jaewoo; Gnapareddy, Bramaramba; Jeon, Jaeho; Lee, Sungjoo; Roh, Yonghan; Park, Sung Ha; Park, Jin-Hong

    2016-01-01

    Here, we propose a novel DNA-based doping method on MoS2 and WSe2 films, which enables ultra-low n- and p-doping control and allows for proper adjustments in device performance. This is achieved by selecting and/or combining different types of divalent metal and trivalent lanthanide (Ln) ions on DNA nanostructures, using the newly proposed concept of Co-DNA (DNA functionalized by both divalent metal and trivalent Ln ions). The available n-doping range on the MoS2 by Ln-DNA is between 6 × 10(9) and 2.6 × 10(10 ) cm(-2). The p-doping change on WSe2 by Ln-DNA is adjusted between -1.0 × 10(10) and -2.4 × 10(10 ) cm(-2). In Eu(3+) or Gd(3+)-Co-DNA doping, a light p-doping is observed on MoS2 and WSe2 (~10(10 ) cm(-2)). However, in the devices doped by Tb(3+) or Er(3+)-Co-DNA, a light n-doping (~10(10 ) cm(-2)) occurs. A significant increase in on-current is also observed on the MoS2 and WSe2 devices, which are, respectively, doped by Tb(3+)- and Gd(3+)-Co-DNA, due to the reduction of effective barrier heights by the doping. In terms of optoelectronic device performance, the Tb(3+) or Er(3+)-Co-DNA (n-doping) and the Eu(3+) or Gd(3+)-Co-DNA (p-doping) improve the MoS2 and WSe2 photodetectors, respectively. We also show an excellent absorbing property by Tb(3+) ions on the TMD photodetectors. PMID:26838524

  2. Ultra-low Doping on Two-Dimensional Transition Metal Dichalcogenides using DNA Nanostructure Doped by a Combination of Lanthanide and Metal Ions

    NASA Astrophysics Data System (ADS)

    Kang, Dong-Ho; Dugasani, Sreekantha Reddy; Park, Hyung-Youl; Shim, Jaewoo; Gnapareddy, Bramaramba; Jeon, Jaeho; Lee, Sungjoo; Roh, Yonghan; Park, Sung Ha; Park, Jin-Hong

    2016-02-01

    Here, we propose a novel DNA-based doping method on MoS2 and WSe2 films, which enables ultra-low n- and p-doping control and allows for proper adjustments in device performance. This is achieved by selecting and/or combining different types of divalent metal and trivalent lanthanide (Ln) ions on DNA nanostructures, using the newly proposed concept of Co-DNA (DNA functionalized by both divalent metal and trivalent Ln ions). The available n-doping range on the MoS2 by Ln-DNA is between 6 × 109 and 2.6 × 1010 cm-2. The p-doping change on WSe2 by Ln-DNA is adjusted between -1.0 × 1010 and -2.4 × 1010 cm-2. In Eu3+ or Gd3+-Co-DNA doping, a light p-doping is observed on MoS2 and WSe2 (~1010 cm-2). However, in the devices doped by Tb3+ or Er3+-Co-DNA, a light n-doping (~1010 cm-2) occurs. A significant increase in on-current is also observed on the MoS2 and WSe2 devices, which are, respectively, doped by Tb3+- and Gd3+-Co-DNA, due to the reduction of effective barrier heights by the doping. In terms of optoelectronic device performance, the Tb3+ or Er3+-Co-DNA (n-doping) and the Eu3+ or Gd3+-Co-DNA (p-doping) improve the MoS2 and WSe2 photodetectors, respectively. We also show an excellent absorbing property by Tb3+ ions on the TMD photodetectors.

  3. Evidence of Subclinical mtDNA Alterations in HIV-Infected Pregnant Women Receiving Combination Antiretroviral Therapy Compared to HIV-Negative Pregnant Women

    PubMed Central

    Money, Deborah M.; Wagner, Emily C.; Maan, Evelyn J.; Chaworth-Musters, Tessa; Gadawski, Izabelle; van Schalkwyk, Julie E.; Forbes, John C.; Burdge, David R.; Albert, Arianne Y. K.; Lohn, Zoe; Côté, Hélène C. F.

    2015-01-01

    Introduction Combination antiretroviral therapy (cART) can effectively prevent vertical transmission of HIV but there is potential risk of adverse maternal, foetal or infant effects. Specifically, the effect of cART use during pregnancy on mitochondrial DNA (mtDNA) content in HIV-positive (HIV+) women is unclear. We sought to characterize subclinical alterations in peripheral blood mtDNA levels in cART-treated HIV+ women during pregnancy and the postpartum period. Methods This prospective longitudinal observational cohort study enrolled both HIV+ and HIV-negative (HIV-) pregnant women. Clinical data and blood samples were collected at three time points in pregnancy (13-<23 weeks, 23-<30 weeks, 30–40 weeks), and at delivery and six weeks post-partum in HIV+ women. Peripheral blood mtDNA to nuclear DNA (nDNA) ratio was measured by qPCR. Results Over a four year period, 63 HIV+ and 42 HIV- women were enrolled. HIV+ women showed significantly lower mtDNA/nDNA ratios compared to HIV- women during pregnancy (p = 0.003), after controlling for platelet count and repeated measurements using a multivariable mixed-effects model. Ethnicity, gestational age (GA) and substance use were also significantly associated with mtDNA/nDNA ratio (p≤0.02). Among HIV+ women, higher CD4 nadir was associated with higher mtDNA/nDNA ratios (p<0.0001), and these ratio were significantly lower during pregnancy compared to the postpartum period (p<0.0001). Conclusions In the context of this study, it was not possible to distinguish between mtDNA effects related to HIV infection versus cART therapy. Nevertheless, while mtDNA levels were relatively stable over time in both groups during pregnancy, they were significantly lower in HIV+ women compared to HIV- women. Although no immediate clinical impact was observed on maternal or infant health, lower maternal mtDNA levels may exert long-term effects on women and children and remain a concern. Improved knowledge of such subclinical alterations is

  4. Thread Pool Interface (TPI)

    Energy Science and Technology Software Center (ESTSC)

    2008-04-01

    Thread Pool Interface (TpI) provides a simple interface for running functions written in C or C++ in a thread-parallel mode. Application or library codes may need to perform operations thread-parallel on machines with multicore processors. the TPI library provides a simple mechanism for managing thread activation, deactivation, and thread-parallel execution of application-provided subprograms.

  5. The Future of Pooling.

    ERIC Educational Resources Information Center

    Young, Peter C.; Fone, Martin

    1997-01-01

    Discusses seven propositions underlying the strategies that insurance pools can, will, and must pursue: (1) risk management versus risk financing; (2) elimination of windfall advantages; (3) the maintenance of market-dominant status; (4) cost leadership; (5) client focus; (6) innovation and diversification; and (7) leadership challenges. A sidebar…

  6. Data Pool Description

    Atmospheric Science Data Center

    2016-04-29

    ... "Internet Options", "Advanced" tab, "Enable FTP folder view (outside of Internet Explorer)" under "Browsing"  • Use IE7 for FTP ... FTP mode is required to access the Data Pool from the command line as "ftp -p l5eil01.larc.nasa.gov" , where the system translates ...

  7. The use of biodegradable polymeric nanoparticles in combination with a low-pressure gene gun for transdermal DNA delivery.

    PubMed

    Lee, Po-Wei; Peng, Shu-Fen; Su, Chun-Jen; Mi, Fwu-Long; Chen, Hsin-Lung; Wei, Ming-Cheng; Lin, Hao-Jan; Sung, Hsing-Wen

    2008-02-01

    Gold particles have been used as a carrier for transdermal gene delivery, which may cause adverse side effects when accumulated. In this study, biodegradable nanoparticles, composed of chitosan (CS) and poly-gamma-glutamic acid (gamma-PGA), were prepared by an ionic-gelation method for transdermal DNA delivery (CS/gamma-PGA/DNA) using a low-pressure gene gun. The conventional CS/DNA without the incorporation of gamma-PGA was used as a control. Small-angle X-ray scattering (SAXS) was used to examine the internal structures of test nanoparticles, while identification of their constituents was conducted by Fourier transformed infrared (FT-IR) spectroscopy. The CS/gamma-PGA/DNA were spherical in shape with a relatively homogeneous size distribution. In contrast, CS/DNA had a heterogeneous size distribution with a donut, rod or pretzel shape. Both test nanoparticles were able to effectively retain the encapsulated DNA and protect it from nuclease degradation. As compared with CS/DNA, CS/gamma-PGA/DNA improved their penetration depth into the mouse skin and enhanced gene expression. These observations may be attributed to the fact that CS/gamma-PGA/DNA were more compact in their internal structures and had a greater density than their CS/DNA counterparts, thus having a larger momentum to penetrate into the skin barrier. The results revealed that CS/gamma-PGA/DNA may substitute gold particles as a DNA carrier for transdermal gene delivery. PMID:18001831

  8. Combined immunization using DNA-Sm14 and DNA-Hsp65 increases CD8+ memory T cells, reduces chronic pathology and decreases egg viability during Schistosoma mansoni infection

    PubMed Central

    2014-01-01

    Background Schistosomiasis is one of the most important neglected diseases found in developing countries and affects 249 million people worldwide. The development of an efficient vaccination strategy is essential for the control of this disease. Previous work showed partial protection induced by DNA-Sm14 against Schistosoma mansoni infection, whereas DNA-Hsp65 showed immunostimulatory properties against infectious diseases, autoimmune diseases, cancer and antifibrotic properties in an egg-induced granuloma model. Methods C57BL/6 mice received 4 doses of DNA-Sm14 (100 μg/dose) and DNA-Hsp65 (100 μg/dose), simultaneously administrated, or DNA-Sm14 alone, once a week, during four weeks. Three groups were included: 1- Control (no immunization); 2- DNA-Sm14; 3- DNA-Sm14/DNA-Hsp65. Two weeks following last immunization, animals were challenged subcutaneously with 30 cercariae. Fifteen, 48 and 69 days after infection splenocytes were collected to evaluate the number of CD8+ memory T cells (CD44highCD62low) using flow cytometry. Forty-eight days after challenge adult worms were collected by portal veins perfusion and intestines were collected to analyze the intestinal egg viability. Histological, immunohistochemical and soluble quantification of collagen and α-SMA accumulation were performed on the liver. Results In the current work, we tested a new vaccination strategy using DNA-Sm14 with DNA-Hsp65 to potentiate the protection against schistosomiasis. Combined vaccination increased the number of CD8+ memory T cells and decreased egg viability on the intestinal wall of infected mice. In addition, simultaneous vaccination with DNA-Sm14/DNA-Hsp65 reduced collagen and α-SMA accumulation during the chronic phase of granuloma formation. Conclusion Simultaneous vaccination with DNA-Sm14/DNA-Hsp65 showed an immunostimulatory potential and antifibrotic property that is associated with the reduction of tissue damage on Schistosoma mansoni experimental infection. PMID

  9. Site-specific T-DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and ϕC31 integrase.

    PubMed

    De Paepe, Annelies; De Buck, Sylvie; Nolf, Jonah; Van Lerberge, Els; Depicker, Ann

    2013-07-01

    Random T-DNA integration into the plant host genome can be problematic for a variety of reasons, including potentially variable transgene expression as a result of different integration positions and multiple T-DNA copies, the risk of mutating the host genome and the difficulty of stacking well-defined traits. Therefore, recombination systems have been proposed to integrate the T-DNA at a pre-selected site in the host genome. Here, we demonstrate the capacity of the ϕC31 integrase (INT) for efficient targeted T-DNA integration. Moreover, we show that the iterative site-specific integration system (ISSI), which combines the activities of the CRE recombinase and INT, enables the targeting of genes to a pre-selected site with the concomitant removal of the resident selectable marker. To begin, plants expressing both the CRE and INT recombinase and containing the target attP site were constructed. These plants were supertransformed with a T-DNA vector harboring the loxP site, the attB sites, a selectable marker and an expression cassette encoding a reporter protein. Three out of the 35 transformants obtained (9%) showed transgenerational site-specific integration (SSI) of this T-DNA and removal of the resident selectable marker, as demonstrated by PCR, Southern blot and segregation analysis. In conclusion, our results show the applicability of the ISSI system for precise and targeted Agrobacterium-mediated integration, allowing the serial integration of transgenic DNA sequences in plants. PMID:23574114

  10. Combined chemoassay and mass spectrometric approach to study the reactive potential of electrophiles towards deoxynucleosides as model for DNA.

    PubMed

    Schmied-Tobies, Maria I H; Paschke, Heidrun; Reemtsma, Thorsten

    2016-05-01

    The modification of DNA by adduct formation is a potential molecular initiating event of genotoxicity. A chemoassay was established to study adduct formation of electrophiles with deoxynucleosides. Liquid chromatography-mass spectrometry was used to determine the reactivity of the model electrophiles para-benzoquinone, hydroquinone, and 1,4-naphthoquinone with deoxynucleoside (deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC) and thymidine (dT)) to detect formation of adducts via constant neutral loss scan of deoxyribose (116 Da), and to elucidate adduct structures using high resolution mass spectrometry. Of the four deoxynucleosides dG was most susceptible, followed by dC and para-benzoquinone was the most reactive electrophile. With this approach five dG and four dC adducts were detected, formed by Michael addition and subsequent condensation. Also oxidation occurred with reactive oxygen species (ROS). Three of the adducts formed by benzoquinone have not been reported before. This chemoassay combined with mass spectrometry offers a way (a) to screen a large number of chemicals for their genotoxic potential, (b) to determine novel adducts that may be searched for in in vitro and in vivo studies and thus (c) to better understand the reaction of electrophiles with nucleobases. PMID:26945242

  11. Preclinical evaluation of antineoplastic activity of inhibitors of DNA methylation (5-aza-2'-deoxycytidine) and histone deacetylation (trichostatin A, depsipeptide) in combination against myeloid leukemic cells.

    PubMed

    Shaker, Sepideh; Bernstein, Mark; Momparler, Louise F; Momparler, Richard L

    2003-05-01

    During the development of leukemia, genes that suppress growth and induce differentiation can be silenced by aberrant DNA methylation and by changes in chromatin structure that involve histone deacetylation. It has been reported that a positive interaction between DNA methylation and histone deacetylation takes place to inhibit transcription. Based on this observation, our working hypothesis was that a combination of inhibitors of these processes should produce an enhancement of their antineoplastic activity on leukemic cells. The cytosine nucleoside analog, 5-aza-2'-deoxycytidine (5AZA), is a potent inhibitor of DNA methylation, which can activate tumor suppressor genes in leukemic cells that have been silenced by aberrant methylation. In clinical trials, 5AZA was demonstrated to be an active antileukemic agent. Histone deacetylase inhibitors (HDI) can also activate gene expression in leukemic cell lines by producing changes in chromatin configuration, and show antineoplastic activity in preclinical studies. In this report, we investigated the in vitro antineoplastic activity of 5AZA, alone and in combination with the HDI, trichostatin A (TSA) and depsipeptide (FR901228, depsi), on the human myeloid leukemic cell lines, HL-60 and KG1a. The results showed that the combination of 5AZA with TSA or depsi produced a greater inhibition of growth and DNA synthesis and a greater loss of clonogenicity than either agent alone. These results suggest that 5AZA used in combination with HDI may be an interesting chemotherapeutic regimen to investigate in patients with acute myeloid leukemia that is resistant to conventional chemotherapy. PMID:12620295

  12. A combined DNA vaccine encoding BCSP31, SOD, and L7/L12 confers high protection against Brucella abortus 2308 by inducing specific CTL responses.

    PubMed

    Yu, Da-Hai; Hu, Xi-Dan; Cai, Hong

    2007-06-01

    We constructed a combined DNA vaccine comprising genes encoding the antigens BCSP31, superoxide dismutase (SOD), and L7/L12 and evaluated its immunogenicity and protective efficacy. Immunization of mice with the combined DNA vaccine offered high protection against Brucella abortus (B. abortus) infection. The vaccine induced a vigorous specific immunoglobulin G (IgG) response, with higher IgG2a than IgG1 titers. Cytokine profiling performed at the same time showed a biased Th1-type immune response with significantly increased interferon-gamma and tumor necrosis factor-alpha stimulation. CD8(+), but not CD4(+), T cells accumulated at significantly higher levels after administration of the vaccine. Granzyme B-producing CD8(+) T cells were significantly higher in number in samples prepared from combined DNA-vaccinated mice compared with S19-vaccinated mice, demonstrating that the cytotoxicity lysis pathway is involved in the response to Brucella infection. The success of our combined DNA vaccine in a mouse model suggests its potential efficacy against brucellosis infection in large animals. PMID:17570767

  13. A strategy of tumor treatment in mice with doxorubicin-cyclophosphamide combination based on dendritic cell activation by human double-stranded DNA preparation

    PubMed Central

    2010-01-01

    Background Immunization of mice with tumor homogenate after combined treatment with cyclophosphamide (CP) and double-stranded DNA (dsDNA) preparation is effective at inhibition of growth of tumor challenged after the treatment. It was assumed that this inhibition might be due to activation of the antigen-presenting cells. The purpose was to develop improved antitumor strategy using mice. We studied the combined action of cytostatics doxorubicin (Dox) plus CP with subsequent dsDNA preparation on tumor growth. Methods Three-month old CBA/Lac mice were used in the experiments. Mice were injected with CP and human dsDNA preparation. The percentage of mature dendritic cells (DCs) was estimated by staining of mononuclear cells isolated from spleen and bone marrow 3, 6, and 9 days later with monoclonal antibodies CD34, CD80, and CD86. In the next set of experiments, mice were given intramuscularly injections of 1-3 × 105 tumor cells. Four days later, they were injected intravenously with 6-6.7 mg/kg Dox and intraperitoneally with 100-200 mg/kg CP; 200 mkg human DNA was injected intraperitoneally after CP administration. Differences in tumor size between groups were analyzed for statistical significance by Student's t-test. The MTT-test was done to determine the cytotoxic index of mouse leucocytes from treated groups. Results The conducted experiments showed that combined treatment with CP and dsDNA preparation produce an increase in the total amount of mature DCs in vivo. Treatment of tumor bearers with preparation of fragmented dsDNA on the background of pretreatment with Dox plus CP demonstrated a strong suppression of tumor growth in two models. RLS, a weakly immunogenic, resistant to alkalyting cytostatics tumor, grew 3.4-fold slower when compared with the control (p < 0.001). In experiment with Krebs-2 tumor, only 2 of the 10 mice in the Dox+CP+DNA group had a palpable tumor on day 16. The cytotoxic index of leucocytes was 86.5% in the Dox+CP+DNA group, but it was 0

  14. Allergic to Pool Water

    PubMed Central

    2012-01-01

    To identify the allergy problem of a 36-year old swimming instructor, who experiences heavy itching and rashes whenever she comes in contact with pool water. Patch tests were performed with European standard series and materials from the work floor. A positive patch test to aluminum chloride and flocculant was observed. Occupational dermatitis is, based on a contact allergy to aluminum chloride in the flocculant. PMID:22993713

  15. 17 CFR 4.22 - Reporting to pool participants.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... strategy. Unrealized gains or losses on open regulated commodity positions presented in the Statement of...-commodity positions, provided that the gains or losses to be combined are part of a related trading strategy... particular pool, it shall be presumed that the particular pool continues to invest in another...

  16. 17 CFR 4.22 - Reporting to pool participants.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... are part of a related trading strategy. Unrealized gains or losses on open regulated commodity... combined are part of a related trading strategy. (f)(1)(i) In the event the commodity pool operator finds... continues to invest in another collective investment vehicle and the commodity pool operator may claim...

  17. Swimming Pools and Molluscum Contagiosum

    MedlinePlus

    ... Travelers' Health: Smallpox & Other Orthopoxvirus-Associated Infections Poxvirus Swimming Pools Recommend on Facebook Tweet Share Compartir The ... often ask if molluscum virus can spread in swimming pools. There is also concern that it can ...

  18. Novel strategy combining SYBR Green I with carbon nanotubes for highly sensitive detection of Salmonella typhimurium DNA.

    PubMed

    Mao, Pingdao; Ning, Yi; Li, Wenkai; Peng, Zhihui; Chen, Yongzhe; Deng, Le

    2014-01-10

    A simple, selective, sensitive and label-free fluorescent method for detecting trpS-harboring Salmonella typhimurium was developed in this study. This assay used the non-covalent interaction of single-stranded DNA (ssDNA) probes with SWNTs, since SWNTs can quench fluorescence. Fluorescence recovery (78% with 1.8 nM target DNA) was detected in the presence of target DNA as ssDNA probes detached from SWNTs hybridized with target DNA, and the resulting double-stranded DNA (dsDNA) intercalated with SYBR Green I (SG) dyes. The increasing fluorescence intensity reached 4.54-fold. In contrast, mismatched oligonucleotides (1- or 3-nt difference to the target DNA) did not contribute to significant fluorescent recovery, which demonstrated the specificity of the assay. The increasing fluorescence intensity increased 3.15-fold when purified PCR products containing complementary sequences of trpS gene were detected. These results confirmed the ability to use this assay for detecting real samples. PMID:24267562

  19. Using minimum DNA marker loci for accurate population classification in rice (Oryza sativa L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using few DNA markers to classify genetic background of a germplasm pool will help breeders make a quick decision while saving time and resources. WHICHLOCI is a computer program that selects the best combination of loci for population assignment through empiric analysis of molecular marker data. Th...

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

    PubMed

    Schmidt, Gunilla Veslemøy; Mellerup, Anders; Christiansen, Lasse Engbo; Ståhl, Marie; Olsen, John Elmerdahl; Angen, Øystein

    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 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 samples, and therefore differences in antibiotic resistance levels between samples were more readily detected. To our knowledge, this is the first study to describe sampling and pooling methods for qPCR quantification of antibiotic resistance genes in total DNA extracted from swine feces. PMID:26114765

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

    PubMed Central

    Mellerup, Anders; Ståhl, Marie

    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 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 samples, and therefore differences in antibiotic resistance levels between samples were more readily detected. To our knowledge, this is the first study to describe sampling and pooling methods for qPCR quantification of antibiotic resistance genes in total DNA extracted from swine feces. PMID:26114765

  2. Energy pooling upconversion in organic molecular systems.

    PubMed

    LaCount, Michael D; Weingarten, Daniel; Hu, Nan; Shaheen, Sean E; van de Lagemaat, Jao; Rumbles, Garry; Walba, David M; Lusk, Mark T

    2015-04-30

    A combination of molecular quantum electrodynamics, perturbation theory, and ab initio calculations was used to create a computational methodology capable of estimating the rate of three-body singlet upconversion in organic molecular assemblies. The approach was applied to quantify the conditions under which such relaxation rates, known as energy pooling, become meaningful for two test systems, stilbene-fluorescein and hexabenzocoronene-oligothiophene. Both exhibit low intramolecular conversion, but intermolecular configurations exist in which pooling efficiency is at least 90% when placed in competition with more conventional relaxation pathways. For stilbene-fluorescein, the results are consistent with data generated in an earlier experimental investigation. Exercising these model systems facilitated the development of a set of design rules for the optimization of energy pooling. PMID:25793313

  3. Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

    PubMed Central

    Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R.; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J.

    2013-01-01

    For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding. PMID:23592960

  4. Combined Stimulation of IL-2 and 4-1BB Receptors Augments the Antitumor Activity of E7 DNA Vaccines by Increasing Ag-Specific CTL Responses

    PubMed Central

    Kim, Ha; Kwon, Byungsuk; Sin, Jeong-Im

    2013-01-01

    Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer. PMID:24391824

  5. Combined stimulation of IL-2 and 4-1BB receptors augments the antitumor activity of E7 DNA vaccines by increasing Ag-specific CTL responses.

    PubMed

    Kim, Ha; Kwon, Byungsuk; Sin, Jeong-Im

    2013-01-01

    Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer. PMID:24391824

  6. Targeting BRCA1-BER deficient breast cancer by ATM or DNA-PKcs blockade either alone or in combination with cisplatin for personalized therapy.

    PubMed

    Albarakati, Nada; Abdel-Fatah, Tarek M A; Doherty, Rachel; Russell, Roslin; Agarwal, Devika; Moseley, Paul; Perry, Christina; Arora, Arvind; Alsubhi, Nouf; Seedhouse, Claire; Rakha, Emad A; Green, Andrew; Ball, Graham; Chan, Stephen; Caldas, Carlos; Ellis, Ian O; Madhusudan, Srinivasan

    2015-01-01

    BRCA1, a key factor in homologous recombination (HR) repair may also regulate base excision repair (BER). Targeting BRCA1-BER deficient cells by blockade of ATM and DNA-PKcs could be a promising strategy in breast cancer. We investigated BRCA1, XRCC1 and pol β protein expression in two cohorts (n = 1602 sporadic and n = 50 germ-line BRCA1 mutated) and mRNA expression in two cohorts (n = 1952 and n = 249). Artificial neural network analysis for BRCA1-DNA repair interacting genes was conducted in 249 tumours. Pre-clinically, BRCA1 proficient and deficient cells were DNA repair expression profiled and evaluated for synthetic lethality using ATM and DNA-PKcs inhibitors either alone or in combination with cisplatin. In human tumours, BRCA1 negativity was strongly associated with low XRCC1, and low pol β at mRNA and protein levels (p < 0.0001). In patients with BRCA1 negative tumours, low XRCC1 or low pol β expression was significantly associated with poor survival in univariate and multivariate analysis compared to high XRCC1 or high pol β expressing BRCA1 negative tumours (ps < 0.05). Pre-clinically, BRCA1 negative cancer cells exhibit low mRNA and low protein expression of XRCC1 and pol β. BRCA1-BER deficient cells were sensitive to ATM and DNA-PKcs inhibitor treatment either alone or in combination with cisplatin and synthetic lethality was evidenced by DNA double strand breaks accumulation, cell cycle arrest and apoptosis. We conclude that XRCC1 and pol β expression status in BRCA1 negative tumours may have prognostic significance. BRCA1-BER deficient cells could be targeted by ATM or DNA-PKcs inhibitors for personalized therapy. PMID:25205036

  7. DNA Damage and Inhibition of Akt Pathway in MCF-7 Cells and Ehrlich Tumor in Mice Treated with 1,4-Naphthoquinones in Combination with Ascorbate

    PubMed Central

    Ourique, Fabiana; Kviecinski, Maicon R.; Felipe, Karina B.; Correia, João Francisco Gomes; Farias, Mirelle S.; Castro, Luiza S. E. P. W.; Grinevicius, Valdelúcia M. A. S.; Valderrama, Jaime; Rios, David; Benites, Julio; Buc Calderon, Pedro; Pedrosa, Rozangela Curi

    2015-01-01

    The aim of this study was to enhance the understanding of the antitumor mechanism of 1,4-naphthoquinones and ascorbate. Juglone, phenylaminonaphthoquinone-7, and 9 (Q7/Q9) were evaluated for effects on CT-DNA and DNA of cancer cells. Evaluations in MCF-7 cells are DNA damage, ROS levels, viability, and proliferation. Proteins from MCF-7 lysates were immunoblotted for verifying PARP integrity, γH2AX, and pAkt. Antitumor activity was measured in Ehrlich ascites carcinoma-bearing mice. The same markers of molecular toxicity were assessed in vivo. The naphthoquinones intercalate into CT-DNA and caused oxidative cleavage, which is increased in the presence of ascorbate. Treatments caused DNA damage and reduced viability and proliferation of MCF-7 cells. Effects were potentiated by ascorbate. No PARP cleavage was observed. Naphthoquinones, combined with ascorbate, caused phosphorylation of H2AX and inhibited pAkt. ROS were enhanced in MCF-7 cells, particularly by the juglone and Q7 plus ascorbate. Ehrlich carcinoma was inhibited by juglone, Q7, or Q9, but the potentiating effect of ascorbate was reproduced in vivo only in the cases of juglone and Q7, which caused up to 60% inhibition of tumor and the largest extension of survival. Juglone and Q7 plus ascorbate caused enhanced ROS and DNA damage and inhibited pAkt also in Ehrlich carcinoma cells. PMID:25793019

  8. Combined exposure to nano-silica and lead induced potentiation of oxidative stress and DNA damage in human lung epithelial cells.

    PubMed

    Lu, Chun-Feng; Yuan, Xiao-Yan; Li, Li-Zhong; Zhou, Wei; Zhao, Jun; Wang, Yi-Mei; Peng, Shuang-Qing

    2015-12-01

    Growing evidence has confirmed that exposure to ambient particulate matters (PM) is associated with increased morbidity and mortality of cardiovascular and pulmonary diseases. Ambient PM is a complex mixture of particles and air pollutants. Harmful effects of PM are specifically associated with ultrafine particles (UFPs) that can adsorb high concentrations of toxic air pollutants and are easily inhaled into the lungs. However, combined effects of UFPs and air pollutants on human health remain unclear. In the present study, we elucidated the combined toxicity of silica nanoparticles (nano-SiO2), a typical UFP, and lead acetate (Pb), a typical air pollutant. Lung adenocarcinoma A549 cells were exposed to nano-SiO2 and Pb alone or their combination, and their combined toxicity was investigated by focusing on cellular oxidative stress and DNA damage. Factorial analyses were performed to determine the potential interactions between nano-SiO2 and Pb. Our results showed that exposure of A549 cells to a modest cytotoxic concentration of Pb alone induced oxidative stress, as evidenced by elevated reactive oxygen species generation and lipid peroxidation, and reduced glutathione content and superoxide dismutase and glutathione peroxidase activities. In addition, exposure of A549 cells to Pb alone induced DNA damage, as evaluated by alkaline comet assay. Exposure of A549 cells to non-cytotoxic concentration of nano-SiO2 did not induce cellular oxidative stress and DNA damage. However, exposure to the combination of nano-SiO2 and Pb potentiated oxidative stress and DNA damage in A549 cells. Factorial analyses indicated that the potentiation of combined toxicity of nano-SiO2 and Pb was induced by additive or synergistic interactions. PMID:26432026

  9. Combining combing and secondary ion mass spectrometry to study DNA on chips using (13)C and (15)N labeling.

    PubMed

    Cabin-Flaman, Armelle; Monnier, Anne-Francoise; Coffinier, Yannick; Audinot, Jean-Nicolas; Gibouin, David; Wirtz, Tom; Boukherroub, Rabah; Migeon, Henri-Noël; Bensimon, Aaron; Jannière, Laurent; Ripoll, Camille; Norris, Victor

    2016-01-01

    Dynamic secondary ion mass spectrometry ( D-SIMS) imaging of combed DNA - the combing, imaging by SIMS or CIS method - has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to (13)C-labeling via the detection and quantification of the (13)C (14)N (-) recombinant ion and the use of the (13)C: (12)C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS. PMID:27429742

  10. A dual amplification strategy for DNA detection combining bio-barcode assay and metal-enhanced fluorescence modality.

    PubMed

    Zhou, Zhenpeng; Li, Tian; Huang, Hongduan; Chen, Yang; Liu, Feng; Huang, Chengzhi; Li, Na

    2014-11-11

    Silver-enhanced fluorescence was coupled with a bio-barcode assay to facilitate a dual amplification assay to demonstrate a non-enzymatic approach for simple and sensitive detection of DNA. In the assay design, magnetic nanoparticles seeded with silver nanoparticles were modified with the capture DNA, and silver nanoparticles were modified with the binding of ssDNA and the fluorescently labeled barcode dsDNA. Upon introduction of the target DNA, a sandwich structure was formed because of the hybridization reaction. By simple magnetic separation, silver-enhanced fluorescence of barcode DNAs could be readily measured without the need of a further step to liberate barcode DNAs from silver nanoparticles, endowing the method with simplicity and high sensitivity with a detection limit of 1 pM. PMID:25233044

  11. Combining combing and secondary ion mass spectrometry to study DNA on chips using 13C and 15N labeling

    PubMed Central

    Cabin-Flaman, Armelle; Monnier, Anne-Francoise; Coffinier, Yannick; Audinot, Jean-Nicolas; Gibouin, David; Wirtz, Tom; Boukherroub, Rabah; Migeon, Henri-Noël; Bensimon, Aaron; Jannière, Laurent; Ripoll, Camille; Norris, Victor

    2016-01-01

    Dynamic secondary ion mass spectrometry ( D-SIMS) imaging of combed DNA – the combing, imaging by SIMS or CIS method – has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to 13C-labeling via the detection and quantification of the 13C 14N - recombinant ion and the use of the 13C: 12C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS. PMID:27429742

  12. Quantifying the effect of surface ligands on dendron-DNA interactions: insights into multivalency through a combined experimental and theoretical approach.

    PubMed

    Jones, Simon P; Pavan, Giovanni M; Danani, Andrea; Pricl, Sabrina; Smith, David K

    2010-04-19

    We report the synthesis, DNA binding ability and preliminary gene delivery profiles of dendrons with different amine surface groups, 1,3-diaminopropane (DAP), N,N-di-(3-aminopropyl)-N-(methyl)amine (DAPMA) and spermine (SPM). By using a combination of ethidium bromide displacement, gel electrophoresis and transfection assays, it is shown that the dendrons with SPM groups are the most effective DNA binders, while the DAPMA-functionalised dendrons were the most effective systems for gene delivery (although the gene delivery profiles were still modest). In order to provide deeper insight into the experimental data, we performed a molecular dynamics simulation of the interactions between the dendrons and DNA. The results of these simulations demonstrated that, in general terms, the enthalpic contribution to binding was roughly proportional to the dendron surface charge, but that dendrons with DAP (and DAPMA) surface amines had significant entropic costs of binding to DNA. In the case of DAP, this is a consequence of the fact that the entire dendron structure has to be organised in order for each individual monoamine charge to make effective contact with DNA. For SPM, however, each surface ligand is already a multivalent triamine, therefore, each individual charge has a much lower entropic cost of binding. For DAPMA, we observed that strong binding of the hindered tertiary amine to the DNA double helix led to ligand back-folding and significant geometric distortion of DNA. Although this weakens the overall binding, we suggest that this distortion might be an explanation for the experimentally observed enhanced gene delivery, in which DNA compaction is an important step. Overall, this paper demonstrates how structure-activity relationships can be developed for multivalent dendritic ligands and provides insights into the thermodynamics of multivalent interactions. PMID:20235240

  13. Combining denaturing gradient gel electrophoresis of 16S rDNA V3 region and 16S-23S rDNA spacer region polymorphism analyses for the identification of staphylococci from Italian fermented sausages.

    PubMed

    Blaiotta, Giuseppe; Pennacchia, Carmelina; Ercolini, Danilo; Moschetti, Giancarlo; Villani, Francesco

    2003-09-01

    Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (PCR-DGGE) and 16S-23S rDNA intergenic spacer region polymorphism (ISR-PCR) analyses were tested as tool for differentiation of staphylococcal strains commonly isolated from fermented sausages. Variable V3 regions of 25 staphylococcal reference strains and 96 wild strains of species belonging to the genera Staphylococcus, Micrococcus and Kocuria were analyzed. PCR-DGGE profiles obtained were species-specific for S. sciuri, S. haemolyticus, S. hominis, S. auricularis, S. condimenti, S. kloosi, S. vitulus, S. succinus, S. pasteuri, S. capitis and S. (Macrococcus) caseolyticus. Moreover, 7 groups could be distinguished gathering the remaining species as result of the separation of the V3 rDNA amplicons in DGGE. Furthermore, the combination of the results obtained by PCR-DGGE and ISR-PCR analyses allowed a clear differentiation of all the staphylococcal species analysed, with exception of the pairs S. equorum-S. cohnii and S. carnosus-S. schleiferi. The suitability of both molecular techniques and of the combination their results for the identification of staphylococci was validated analysing partial nucleotide sequence of the 16S rDNA of a representative number of wild strains. PMID:14529185

  14. Secondary pool boiling effects

    NASA Astrophysics Data System (ADS)

    Kruse, C.; Tsubaki, A.; Zuhlke, C.; Anderson, T.; Alexander, D.; Gogos, G.; Ndao, S.

    2016-02-01

    A pool boiling phenomenon referred to as secondary boiling effects is discussed. Based on the experimental trends, a mechanism is proposed that identifies the parameters that lead to this phenomenon. Secondary boiling effects refer to a distinct decrease in the wall superheat temperature near the critical heat flux due to a significant increase in the heat transfer coefficient. Recent pool boiling heat transfer experiments using femtosecond laser processed Inconel, stainless steel, and copper multiscale surfaces consistently displayed secondary boiling effects, which were found to be a result of both temperature drop along the microstructures and nucleation characteristic length scales. The temperature drop is a function of microstructure height and thermal conductivity. An increased microstructure height and a decreased thermal conductivity result in a significant temperature drop along the microstructures. This temperature drop becomes more pronounced at higher heat fluxes and along with the right nucleation characteristic length scales results in a change of the boiling dynamics. Nucleation spreads from the bottom of the microstructure valleys to the top of the microstructures, resulting in a decreased surface superheat with an increasing heat flux. This decrease in the wall superheat at higher heat fluxes is reflected by a "hook back" of the traditional boiling curve and is thus referred to as secondary boiling effects. In addition, a boiling hysteresis during increasing and decreasing heat flux develops due to the secondary boiling effects. This hysteresis further validates the existence of secondary boiling effects.

  15. Combination of MIDGE-Th1 DNA vaccines with the cationic lipid SAINT-18: studies on formulation, biodistribution and vector clearance.

    PubMed

    Endmann, Anne; Oswald, Detlef; Riede, Oliver; Talman, Eduard G; Vos, Roelien E; Schroff, Matthias; Kleuss, Christiane; Ruiters, Marcel H J; Juhls, Christiane

    2014-06-01

    We have previously shown that the combination of MIDGE-Th1 DNA vectors with the cationic lipid SAINT-18 increases the immune response to the encoded antigen in mice. Here, we report on experiments to further optimize and characterize this approach. We evaluated different formulations of MIDGE-Th1 vectors with SAINT-18 by assessing their influence on the transfection efficiency in cell culture and on the immune response in mice. We found that high amounts of SAINT-18 in formulations with a w/w ratio MIDGE Th1/SAINT-18 of 1:4.8 are beneficial for cell transfection in vitro. In contrast, the formulation of HBsAg-encoding MIDGE-Th1 DNA vectors with the lowest amount of SAINT-18 (w/w ratio MIDGE Th1/SAINT-18 of 1:0.5) resulted in the highest serum IgG1 and IgG2a levels after intradermal immunization of mice. Consequently, latter formulation was selected for a comparative biodistribution study in rats. Following intradermal administration of both naked and formulated MIDGE-Th1 DNA, the vectors localized primarily at the site of injection. Vector DNA levels decreased substantially over the two months duration of the study. When administered in combination with SAINT-18, the vectors were found in significantly higher amounts in draining lymph nodes in comparison to administration of naked MIDGE-Th1 DNA. We propose that the high immune responses induced by MIDGE-Th1/SAINT-18 lipoplexes are mediated by enhanced transfection of cells in vivo, resulting in stronger antigen expression and presentation. Importantly, the combination of MIDGE-Th1 vectors with SAINT-18 was well tolerated in mice and rats and is expected to be safe in human clinical applications. PMID:24681271

  16. Identification of invasive fungal diseases in immunocompromised patients by combining an Aspergillus specific PCR with a multifungal DNA-microarray from primary clinical samples.

    PubMed

    Boch, T; Reinwald, M; Postina, P; Cornely, O A; Vehreschild, J J; Heußel, C P; Heinz, W J; Hoenigl, M; Eigl, S; Lehrnbecher, T; Hahn, J; Claus, B; Lauten, M; Egerer, G; Müller, M C; Will, S; Merker, N; Hofmann, W-K; Buchheidt, D; Spiess, B

    2015-12-01

    The increasing incidence of invasive fungal diseases (IFD), most of all invasive aspergillosis (IA) in immunocompromised patients emphasises the need to improve the diagnostic tools for detection of fungal pathogens. We investigated the diagnostic performance of a multifungal DNA-microarray detecting 15 different fungi [Aspergillus, Candida, Fusarium, Mucor, Rhizopus, Scedosporium and Trichosporon species (spp.)] in addition to an Aspergillus specific polymerase chain reaction (PCR) assay. Biopsies, bronchoalveolar lavage and peripheral blood samples of 133 immunocompromised patients (pts) were investigated by a multifungal DNA-microarray as well as a nested Aspergillus specific PCR assay. Patients had proven (n = 18), probable (n = 29), possible (n = 48) and no IFD (n = 38) and were mostly under antifungal therapy at the time of sampling. The results were compared to culture, histopathology, imaging and serology, respectively. For the non-Aspergillus IFD the microarray analysis yielded in all samples a sensitivity of 64% and a specificity of 80%. Best results for the detection of all IFD were achieved by combining DNA-microarray and Aspergillus specific PCR in biopsy samples (sensitivity 79%; specificity 71%). The molecular assays in combination identify genomic DNA of fungal pathogens and may improve identification of causative pathogens of IFD and help overcoming the diagnostic uncertainty of culture and/or histopathology findings, even during antifungal therapy. PMID:26497302

  17. One simple DNA extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid on-field detection of genetically modified organisms.

    PubMed

    Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

    2013-01-01

    Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs. PMID:23181490

  18. The effect of two pre-cryopreservation single layer colloidal centrifugation protocols in combination with different freezing extenders on the fragmentation dynamics of thawed equine sperm DNA

    PubMed Central

    2012-01-01

    Background Variability among stallions in terms of semen cryopreservation quality renders it difficult to arrive at a standardized cryopreservation method. Different extenders and processing techniques (such us colloidal centrifugation) are used in order to optimize post-thaw sperm quality. Sperm chromatin integrity analysis is an effective tool for assessing such quality. The aim of the present study was to compare the effect of two single layer colloidal centrifugation protocols (prior to cryopreservation) in combination with three commercial freezing extenders on the post-thaw chromatin integrity of equine sperm samples at different post-thaw incubation (37°C) times (i.e., their DNA fragmentation dynamics). Results Post-thaw DNA fragmentation levels in semen samples subjected to either of the colloidal centrifugation protocols were significantly lower (p<0.05) immediately after thawing and after 4 h of incubation at 37°C compared to samples that underwent standard (control) centrifugation. The use of InraFreeze® extender was associated with significantly less DNA fragmentation than the use of Botu-Crio® extender at 6 h of incubation, and than the use of either Botu-Crio® or Gent® extender at 24 h of incubation (p<0.05). Conclusions These results suggest that single layer colloidal centrifugation performed with extended or raw semen prior to cryopreservation reduces DNA fragmentation during the first four hours after thawing. Further studies are needed to determine the influence of freezing extenders on equine sperm DNA fragmentation dynamics. PMID:23217215

  19. Vernal pool hydroperiod: simulations of long-term dynamics

    NASA Astrophysics Data System (ADS)

    Pyke, C. R.; Chadwick, O.

    2001-12-01

    Vernal pools are shallow, seasonal wetlands formed by the accumulation of seasonal rains on impermeable substrates. Vernal pools are part of a large set of ephemeral wetlands including prairie potholes, karst sinks, playa lakes, and savanna rain-pools. Research has shown the ecological importance of hydroperiod in the species richness of crustacean communities, the persistence and spatial distribution of amphibians, and plant community composition. Despite the well-known ecological importance of vernal pool hydroperiod, existing observational records are very short (typically < 5 years), unreplicated, and frequently lost in the informal gray literature. These issues suggest the need for a process-based model of vernal pool hydroperiod that can be applied to hind-cast historical patterns of hydroperiod variability based on meteorological records. This research developed a water balanced-based model for the simulation of vernal pool hydrology based on historical meteorological records for coastal vernal pools in Santa Barbara, California. Stochastic weather generation was also applied to evaluate the potential statistical distribution of vernal pool hydroperiod over long periods of time. The model suggests that hydroperiod is particularly sensitive to maximum pool depth, soil thickness, and soil water holding capacity. Results suggest that combinations of these factors can change flooding duration and return interval by more than 50% under identical weather conditions. These findings have significant implications for wetland restoration and management.

  20. Pathways controlling dNTP pools to maintain genome stability.

    PubMed

    Rudd, Sean G; Valerie, Nicholas C K; Helleday, Thomas

    2016-08-01

    Artificially modified nucleotides, in the form of nucleoside analogues, are widely used in the treatment of cancers and various other diseases, and have become important tools in the laboratory to characterise DNA repair pathways. In contrast, the role of endogenously occurring nucleotide modifications in genome stability is little understood. This is despite the demonstration over three decades ago that the cellular DNA precursor pool is orders of magnitude more susceptible to modification than the DNA molecule itself. More recently, underscoring the importance of this topic, oxidation of the cellular nucleotide pool achieved through targeting the sanitation enzyme MTH1, appears to be a promising anti-cancer strategy. This article reviews our current understanding of modified DNA precursors in genome stability, with a particular focus upon oxidised nucleotides, and outlines some important outstanding questions. PMID:27311542

  1. DNA: Polymer and molecular code

    NASA Astrophysics Data System (ADS)

    Shivashankar, G. V.

    1999-10-01

    The thesis work focusses upon two aspects of DNA, the polymer and the molecular code. Our approach was to bring single molecule micromanipulation methods to the study of DNA. It included a home built optical microscope combined with an atomic force microscope and an optical tweezer. This combined approach led to a novel method to graft a single DNA molecule onto a force cantilever using the optical tweezer and local heating. With this method, a force versus extension assay of double stranded DNA was realized. The resolution was about 10 picoN. To improve on this force measurement resolution, a simple light backscattering technique was developed and used to probe the DNA polymer flexibility and its fluctuations. It combined the optical tweezer to trap a DNA tethered bead and the laser backscattering to detect the beads Brownian fluctuations. With this technique the resolution was about 0.1 picoN with a millisecond access time, and the whole entropic part of the DNA force-extension was measured. With this experimental strategy, we measured the polymerization of the protein RecA on an isolated double stranded DNA. We observed the progressive decoration of RecA on the l DNA molecule, which results in the extension of l , due to unwinding of the double helix. The dynamics of polymerization, the resulting change in the DNA entropic elasticity and the role of ATP hydrolysis were the main parts of the study. A simple model for RecA assembly on DNA was proposed. This work presents a first step in the study of genetic recombination. Recently we have started a study of equilibrium binding which utilizes fluorescence polarization methods to probe the polymerization of RecA on single stranded DNA. In addition to the study of material properties of DNA and DNA-RecA, we have developed experiments for which the code of the DNA is central. We studied one aspect of DNA as a molecular code, using different techniques. In particular the programmatic use of template specificity makes

  2. Solar heater for swimming pools

    SciTech Connect

    Babcock, H.W.

    1984-12-04

    A solar heater for swimming pools is provided having one or more heating panels installable on a roof or the like and arranged to discharge into a pool equipped with an apron without need for disturbing or obstructing the apron. This is accomplished by the provision of an elevated bistable dumper adjacent the perimeter of the apron having a dispensing spout normally inclined upwardly but pivoting at intervals to discharge into the pool across the apron without obstructing it. Water to be heated is diverted from the pool filtering system to the solar heater via a pressure regulator and a solar responsive flow control.

  3. Synaptic vesicle pools: an update.

    PubMed

    Denker, Annette; Rizzoli, Silvio O

    2010-01-01

    During the last few decades synaptic vesicles have been assigned to a variety of functional and morphological classes or "pools". We have argued in the past (Rizzoli and Betz, 2005) that synaptic activity in several preparations is accounted for by the function of three vesicle pools: the readily releasable pool (docked at active zones and ready to go upon stimulation), the recycling pool (scattered throughout the nerve terminals and recycling upon moderate stimulation), and finally the reserve pool (occupying most of the vesicle clusters and only recycling upon strong stimulation). We discuss here the advancements in the vesicle pool field which took place in the ensuing years, focusing on the behavior of different pools under both strong stimulation and physiological activity. Several new findings have enhanced the three-pool model, with, for example, the disparity between recycling and reserve vesicles being underlined by the observation that the former are mobile, while the latter are "fixed". Finally, a number of altogether new concepts have also evolved such as the current controversy on the identity of the spontaneously recycling vesicle pool. PMID:21423521

  4. Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum.

    PubMed

    Cheng, Po-Ching; Lin, Ching-Nan; Peng, Shih-Yi; Kang, Tsung-Fu; Lee, Kin-Mu

    2016-02-01

    Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42-44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (pcDNA/SjGST), IL-12 expressing-plasmid (pIL-12), and rSjGST. Co-vaccination with pcDNA/SjGST, pIL-12, and rSjGST led to a reduction in worm burden, hepatic egg burden, and the size of liver tissue granulomas than that in the untreated infection controls. In addition, we detected high levels of specific IgG, IgG1, and IgG2a against the rSjGST antigen in infected mice vaccinated with this combination of pcDNA/SjGST, pIL-12, and rSjGST. Moreover, high expression levels of Th2 cytokines, including IL-4 and IL-10, were also detected in this group, without diminished levels of IL-12, INF-γ, and TNF-α cytokines that are related to parasite killing. In conclusion, we have developed a new vaccination regimen against S. japonicum infection and shown that co-immunization with pcDNA/SjGST vaccine, pIL-12, and rSjGST has significant anti-parasite, anti-hepatic egg and anti-pathology effects in mice. The efficacy of this vaccination method should be further validated in large animals such as water buffalo. This method may help to reduce the transmission of zoonotic schistosomiasis japonica. PMID:26891172

  5. Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum

    PubMed Central

    Cheng, Po-Ching; Lin, Ching-Nan; Peng, Shih-Yi; Kang, Tsung-Fu; Lee, Kin-Mu

    2016-01-01

    Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42–44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (pcDNA/SjGST), IL-12 expressing-plasmid (pIL-12), and rSjGST. Co-vaccination with pcDNA/SjGST, pIL-12, and rSjGST led to a reduction in worm burden, hepatic egg burden, and the size of liver tissue granulomas than that in the untreated infection controls. In addition, we detected high levels of specific IgG, IgG1, and IgG2a against the rSjGST antigen in infected mice vaccinated with this combination of pcDNA/SjGST, pIL-12, and rSjGST. Moreover, high expression levels of Th2 cytokines, including IL-4 and IL-10, were also detected in this group, without diminished levels of IL-12, INF-γ, and TNF-α cytokines that are related to parasite killing. In conclusion, we have developed a new vaccination regimen against S. japonicum infection and shown that co-immunization with pcDNA/SjGST vaccine, pIL-12, and rSjGST has significant anti-parasite, anti-hepatic egg and anti-pathology effects in mice. The efficacy of this vaccination method should be further validated in large animals such as water buffalo. This method may help to reduce the transmission of zoonotic schistosomiasis japonica. PMID:26891172

  6. Efficacy and safety of combined prolonged-release oxycodone and naloxone in the management of moderate/severe chronic non-malignant pain: results of a prospectively designed pooled analysis of two randomised, double-blind clinical trials

    PubMed Central

    2010-01-01

    Background Two randomised 12-week, double-blind, parallel-group, multicenter studies comparing oxycodone PR/naloxone PR and oxycodone PR alone on symptoms of opioid-induced bowel dysfunction in patients with moderate/severe non-malignant pain have been conducted. Methods These studies were prospectively designed to be pooled and the primary outcome measure of the pooled data analysis was to demonstrate non-inferiority in 12-week analgesic efficacy of oxycodone PR/naloxone PR versus oxycodone PR alone. Patients with opioid-induced constipation were switched to oxycodone PR and then randomised to fixed doses of oxycodone PR/naloxone PR (n = 292) or oxycodone PR (n = 295) for 12 weeks (20-80 mg/day). Results No statistically significant differences in analgesic efficacy were observed for the two treatments (p = 0.3197; non-inferiority p < 0.0001; 95% CI -0.07, 0.23) and there was no statistically significant difference in frequency of analgesic rescue medication use. Improvements in Bowel Function Index score were observed for oxycodone PR/naloxone PR by Week 1 and at every subsequent time point (-15.1; p < 0.0001; 95% CI -17.3, -13.0). AE incidence was similar for both groups (61.0% and 57.3% of patients with oxycodone PR/naloxone PR and oxycodone PR alone, respectively). Conclusions Results of this pooled analysis confirm that oxycodone PR/naloxone PR provides effective analgesia and suggest that oxycodone PR/naloxone PR improves bowel function without compromising analgesic efficacy. Trial registration numbers ClinicalTrials.gov identifier: NCT00412100 and NCT00412152 PMID:20920236

  7. Comet assay combined with p53 detection as a sensitive approach for DNA photoprotection assessment in vitro.

    PubMed

    Marrot, Laurent; Belaïdi, Jean-Philippe; Meunier, Jean-Roch

    2002-01-01

    A simple in vitro approach where sun formulations are spread on a quartz slide and placed over human skin cells in culture is proposed as a convenient test for photoprotection assessment at the DNA level. Using the comet assay, DNA strand breaks and oxidative DNA damage were detected. Then, accumulation of p53 protein was studied as a marker for UV-induced genotoxic stress. Such a method was used to compare formulations with different photostability. Spectroradiometry showed that a photo-unstable formulation lost its effectiveness in UVA screening when pre-irradiated by simulated sunlight. As a consequence, such a formulation was not as protective as a photostable one at the genomic level. PMID:12444957

  8. Integrated process for purification of plasmid DNA using aqueous two-phase systems combined with membrane filtration and lid bead chromatography.

    PubMed

    Kepka, Cecilia; Lemmens, Raf; Vasi, Jozsef; Nyhammar, Tomas; Gustavsson, Per-Erik

    2004-11-19

    An integrated process for purifying a 6.1 kilo base pair (kbp) plasmid from a clarified Escherichia coli cell lysate based on an ultra/diafiltration step combined with polymer/polymer aqueous two-phase system and a new type of chromatography is described. The process starts with a volume reduction (ultrafiltration) and buffer exchange (diafiltration) of the clarified lysate using a hollow fibre membrane system. The concentrated and desalted plasmid solution is then extracted in a thermoseparating aqueous two-phase system, where the contaminants (RNA and proteins) to a large extent are removed. While the buffer exchange (diafiltration) is necessary in order to extract the plasmid DNA exclusively to the top phase, experiments showed that the ultrafiltration step increased the productivity of the aqueous two-phase system by a factor of more than 10. The thermoseparated water phase was then subjected to a polishing step using lid bead chromatography. Lid beads are a new type of restricted access chromatography beads, here with a positively charged inner core that adsorbed the remaining RNA while its inert surface layer prevented adsorption of the plasmid DNA thus passing in the flow-through of the column. Differently-sized plasmid DNA in the range of 2.7-20.5 kbp were also partitioned in the aqueous two-phase system. Within this size range, all plasmid DNA was exclusively extracted to the top phase. The complete process is free of additives and easy scalable for use in large scale production of plasmid DNA. The overall process yield for plasmid DNA was 69%. PMID:15584230

  9. Erbb2 DNA vaccine combined with regulatory T cell deletion enhances antibody response and reveals latent low-avidity T cells: potential and limits of its therapeutic efficacy.

    PubMed

    Rolla, Simona; Ria, Francesco; Occhipinti, Sergio; Di Sante, Gabriele; Iezzi, Manuela; Spadaro, Michela; Nicolò, Chiara; Ambrosino, Elena; Merighi, Irene Fiore; Musiani, Piero; Forni, Guido; Cavallo, Federica

    2010-06-01

    Rat (r)Erbb2 transgenic BALB-neuT mice genetically predestined to develop multiple invasive carcinomas allow an assessment of the potential of a vaccine against the stages of cancer progression. Because of rErbb2 expression in the thymus and its overexpression in the mammary gland, CD8(+) T cell clones reacting at high avidity with dominant rErbb2 epitopes are deleted in these mice. In BALB-neuT mice with diffuse and invasive in situ lesions and almost palpable carcinomas, a temporary regulatory T cells depletion combined with anti-rErbb2 vaccine markedly enhanced the anti-rErbb2 Ab response and allowed the expansion of latent pools of low-avidity CD8(+) T cells bearing TCRs repertoire reacting with the rErbb2 dominant peptide. This combination of a higher Ab response and activation of a low-avidity cytotoxic response persistently blocked tumor progression at stages in which the vaccine alone was ineffective. However, when diffuse and invasive microscopic cancers become almost palpable, this combination was no longer able to secure a significant extension of mice survival. PMID:20435927

  10. Fetal Gene Therapy for Ornithine Transcarbamylase Deficiency by Intrahepatic Plasmid DNA-Micro-Bubble Injection Combined with Hepatic Ultrasound Insonation.

    PubMed

    Oishi, Yoshie; Kakimoto, Takashi; Yuan, Wenji; Kuno, Shuichi; Yamashita, Hiromasa; Chiba, Toshio

    2016-06-01

    We evaluated the therapeutic efficacy of hepatic transfection of plasmid DNA using micro-bubbles and ultrasound insonation for fetal correction of ornithine transcarbamylase (OTC) deficiency in mice. Twenty-three sparse-fur heterozygous pregnant mice (day 16 of gestation) were divided into three groups: injection of plasmid-DNA micro-bubble mixture into fetal liver with ultrasound insonation (Tr, n = 11); control group 1 (C1), injection of plasmid-DNA micro-bubble mixture into fetal liver with no insonation (n = 5); and control group 2 (C2), injection of saline-micro-bubble mixture into fetal liver with ultrasound insonation (n = 7). Levels of blood ammonia and urinary orotic acid were significantly lower in the Tr group than in the C1 and C2 groups (p < 0.05, p < 0.01, respectively), whereas OTC activity was not different between groups. Therefore, ultrasound insonation with micro-bubbles enhanced plasmid DNA transfection into fetal mouse liver, leading to one of the therapeutic methods in ammonia metabolism. This might provide more time for OTC-deficient infants until liver transplantation. PMID:26995155

  11. Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

    PubMed Central

    Catez, Frédéric; Rousseau, Antoine; Labetoulle, Marc; Lomonte, Patrick

    2014-01-01

    Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 µm tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue. PMID:24514006

  12. Membrane Destruction and DNA Binding of Staphylococcus aureus Cells Induced by Carvacrol and Its Combined Effect with a Pulsed Electric Field.

    PubMed

    Wang, Lang-Hong; Wang, Man-Sheng; Zeng, Xin-An; Zhang, Zhi-Hong; Gong, De-Ming; Huang, Yan-Bo

    2016-08-17

    Carvacrol (5-isopropyl-2-methylphenol, CAR) is an antibacterial ingredient that occurs naturally in the leaves of the plant Origanum vulgare. The antimicrobial mechanism of CAR against Staphylococcus aureus ATCC 43300 was investigated in the study. Analysis of the membrane fatty acids by gas chromatography-mass spectrometry (GC-MS) showed that exposure to CAR at low concentrations induced a marked increase in the level of unbranched fatty acids (from 34.90 ± 1.77% to 62.37 ± 4.26%). Moreover, CAR at higher levels severely damaged the integrity and morphologies of the S. aureus cell membrane. The DNA-binding properties of CAR were also investigated using fluorescence, circular dichroism, molecular modeling, and atomic-force microscopy. The results showed that CAR bound to DNA via the minor-groove mode, mildly perturbed the DNA secondary structure, and induced DNA molecules to be aggregated. Furthermore, a combination of CAR with a pulsed-electric field was found to exhibit strong synergistic effects on S. aureus. PMID:27420472

  13. Multifunctional pDNA-Conjugated Polycationic Au Nanorod-Coated Fe3 O4 Hierarchical Nanocomposites for Trimodal Imaging and Combined Photothermal/Gene Therapy.

    PubMed

    Hu, Yang; Zhou, Yiqiang; Zhao, Nana; Liu, Fusheng; Xu, Fu-Jian

    2016-05-01

    It is very desirable to design multifunctional nanocomposites for theranostic applications via flexible strategies. The synthesis of one new multifunctional polycationic Au nanorod (NR)-coated Fe3 O4 nanosphere (NS) hierarchical nanocomposite (Au@pDM/Fe3 O4 ) based on the ternary assemblies of negatively charged Fe3 O4 cores (Fe3 O4 -PDA), polycation-modified Au nanorods (Au NR-pDM), and polycations is proposed. For such nanocomposites, the combined near-infrared absorbance properties of Fe3 O4 -PDA and Au NR-pDM are applied to photoacoustic imaging and photothermal therapy. Besides, Fe3 O4 and Au NR components allow the nanocomposites to serve as MRI and CT contrast agents. The prepared positively charged Au@pDM/Fe3 O4 also can complex plasmid DNA into pDNA/Au@pDM/Fe3 O4 and efficiently mediated gene therapy. The multifunctional applications of pDNA/Au@pDM/Fe3 O4 nanocomposites in trimodal imaging and combined photothermal/gene therapy are demonstrated using a xenografted rat glioma nude mouse model. The present study demonstrates that the proper assembly of different inorganic nanoparticles and polycations is an effective strategy to construct new multifunctional theranostic systems. PMID:26996155

  14. Tidal Pools--Miniature Oceans

    ERIC Educational Resources Information Center

    Plake, Linda Perry

    1977-01-01

    A comprehensive discussion of the biological activity in tidal pools is provided. The importance of environmental factors such as oxygen supply, temperature, salinity, and light is detailed. Plants and animals that might be found in a tidal pool are identified and described. (BT)

  15. Synaptic Vesicle Pools: An Update

    PubMed Central

    Denker, Annette; Rizzoli, Silvio O.

    2010-01-01

    During the last few decades synaptic vesicles have been assigned to a variety of functional and morphological classes or “pools”. We have argued in the past (Rizzoli and Betz, 2005) that synaptic activity in several preparations is accounted for by the function of three vesicle pools: the readily releasable pool (docked at active zones and ready to go upon stimulation), the recycling pool (scattered throughout the nerve terminals and recycling upon moderate stimulation), and finally the reserve pool (occupying most of the vesicle clusters and only recycling upon strong stimulation). We discuss here the advancements in the vesicle pool field which took place in the ensuing years, focusing on the behavior of different pools under both strong stimulation and physiological activity. Several new findings have enhanced the three-pool model, with, for example, the disparity between recycling and reserve vesicles being underlined by the observation that the former are mobile, while the latter are “fixed”. Finally, a number of altogether new concepts have also evolved such as the current controversy on the identity of the spontaneously recycling vesicle pool. PMID:21423521

  16. Combination of DNA prime--adenovirus boost immunization with entecavir elicits sustained control of chronic hepatitis B in the woodchuck model.

    PubMed

    Kosinska, Anna D; Zhang, Ejuan; Johrden, Lena; Liu, Jia; Seiz, Pia L; Zhang, Xiaoyong; Ma, Zhiyong; Kemper, Thekla; Fiedler, Melanie; Glebe, Dieter; Wildner, Oliver; Dittmer, Ulf; Lu, Mengji; Roggendorf, Michael

    2013-01-01

    A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8+ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8+ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4+ and CD8+ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients. PMID:23785279

  17. Detection of Leishmania-specific DNA and surface antigens using a combination of functionalized magnetic beads and cadmium selenite quantum dots.

    PubMed

    Andreadou, Margarita; Liandris, Emmanouil; Gazouli, Maria; Mataragka, Antonia; Tachtsidis, Ilias; Goutas, Nikolaοs; Vlachodimitropoulos, Dimitrios; Ikonomopoulos, John

    2016-04-01

    Leishmaniosis is a zoonotic disease that affects millions of people especially in resource-poor settings. The development of reliable diagnostic assays that do not require dedicated equipment or highly trained personnel would improve early diagnosis and effective control. For this purpose, a combination of magnetic bead and cadmium selenite quantum dot probes was applied for the detection of Leishmania-specific surface antigens (proteins) and DNA. Both analytes are isolated from the solution using magnetic bead capture probes whereas the presence of the targeted molecules is demonstrated by quantum dot detection probes. The sensitivity and specificity of this method reached 100% based on an assessment performed on 55 cultured isolates of various microbial pathogens. The low limit of detection was 3125 ng/μl and 10(3)cells/ml for Leishmania DNA and protein, respectively. The method shows considerable potential for clinical application in human and veterinary medicine, especially in resource-poor settings. PMID:26658854

  18. Taxonomic Confusion Permits the Unchecked Invasion of Vernal Pools in California by Glyceria declinata

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chloroplast DNA molecular markers and recently established morphological characters were used to confirm the widespread invasion of California's vernal pools by European low Glyceria (Glyceria declinata). Morphological similarities between low Glyceria and western mannagrass (Glyceria occidentalis)...

  19. Phylogenetic analysis of the genus Sorghum based on combined sequence data from cpDNA regions and ITS generate well-supported trees with two major lineages

    PubMed Central

    Ng'uni, Dickson; Geleta, Mulatu; Fatih, Moneim; Bryngelsson, Tomas

    2010-01-01

    Background and Aims Wild Sorghum species provide novel traits for both biotic and abiotic stress resistance and yield for the improvement of cultivated sorghum. A better understanding of the phylogeny in the genus Sorghum will enhance use of the valuable agronomic traits found in wild sorghum. Methods Four regions of chloroplast DNA (cpDNA; psbZ-trnG, trnY-trnD, trnY-psbM and trnT-trnL) and the internal transcribed spacer (ITS) of nuclear ribosomal DNA were used to analyse the phylogeny of sorghum based on maximum-parsimony analyses. Key Results Parsimony analyses of the ITS and cpDNA regions as separate or combined sequence datasets formed trees with strong bootstrap support with two lineages: the Eu-sorghum species S. laxiflorum and S. macrospermum in one and Stiposorghum and Para-sorghum in the other. Within Eu-sorghum, S. bicolor-3, -11 and -14 originating from southern Africa form a distinct clade. S. bicolor-2, originally from Yemen, is distantly related to other S. bicolor accessions. Conclusions Eu-sorghum species are more closely related to S. macrospermum and S. laxiflorum than to any other Australian wild Sorghum species. S. macrospermum and S. laxiflorum are so closely related that it is inappropriate to classify them in separate sections. S. almum is closely associated with S. bicolor, suggesting that the latter is the maternal parent of the former given that cpDNA is maternally inherited in angiosperms. S. bicolor-3, -11 and -14, from southern Africa, are closely related to each other but distantly related to S. bicolor-2. PMID:20061309

  20. An epigenetic biomarker combination of PCDH17 and POU4F2 detects bladder cancer accurately by methylation analyses of urine sediment DNA in Han Chinese

    PubMed Central

    Li, Qiaoling; An, Dan; Fang, Lu; Lin, Youcheng; Hou, Yong; Xu, Abai; Fu, Yu; Lu, Wei; Chen, Xin; Chen, Mingwei; Zhang, Meng; Jiang, Huiling; Zhang, Chuanxia; Dong, Pei; Li, Chong; Chen, Jun; Yang, Guosheng; Liu, Chunxiao; Cai, Zhiming; Zhou, Fangjian; Wu, Song

    2016-01-01

    To develop a routine and effectual procedure of detecting bladder cancer (BlCa), an optimized combination of epigenetic biomarkers that work synergistically with high sensitivity and specificity is necessary. In this study, methylation levels of seven biomarkers (EOMES, GDF15, NID2, PCDH17, POU4F2, TCF21, and ZNF154) in 148 individuals—which including 58 urothelial cell carcinoma (UCC) patients, 20 infected urinary calculi (IUC) patients, 20 kidney cancer (KC) patients,20 prostate cancer (PC) patients, and 30 healthy volunteers (HV)—were quantified by qMSP using the urine sediment DNA. Receiver operating characteristic (ROC) curves were generated for each biomarker. The combining predictors of possible combinations were calculated through logistic regression model. Subsequently, ROC curves of the three best performing combinations were constructed. Then, we validated the three best performing combinations and POU4F2 in another 72 UCC, 21 IUC, 26 KC and 22 PC, and 23 HV urine samples. The combination of POU4F2/PCDH17 has yielded the highest sensitivity and specificity of 90.00% and 93.96% in all the 312 individuals, showing the capability of detecting BlCa effectively among pathologically varied sample groups. PMID:26700620

  1. NSCLC cells demonstrate differential mode of cell death in response to the combined treatment of radiation and a DNA-PKcs inhibitor

    PubMed Central

    Hsu, Feng-Ming; Zhang, Zhang; Tumati, Vasu; Lin, Yu-Fen; Chen, Benjamin P.C.; Saha, Debabrata

    2015-01-01

    The current standard of care for lung cancer consists of concurrent chemotherapy and radiation. Several studies have shown that the DNA-PKcs inhibitor NU7441 is a highly potent radiosensitizer, however, the mechanism of NU7441's anti-proliferation effect has not been fully elucidated. In this study, the combined effect of NU7441 and ionizing radiation (IR) in a panel of non-small cell lung cancer cell lines (A549, H460 and H1299) has been investigated. We found that NU7441 significantly enhances the effect of IR in all cell lines. The notable findings in response to this combined treatment are (i) prolonged delay in IR-induced DNA DSB repair, (ii) induced robust G2/M checkpoint, (iii) increased aberrant mitosis followed by mitotic catastrophe specifically in H1299, (iv) dramatically induced autophagy in A549 and (v) IR-induced senescence specifically in H460. H1299 cells show greater G2 checkpoint adaptation after combined treatment, which can be attributed to higher expression level of Plk1 compared to A549 and H460. The enhanced autophagy after NU7441 treatment in A549 is possibly due to the higher endogenous expression of pS6K compared to H1299 and H460 cells. In conclusion, choice of cell death pathway is dependent on the mutation status and other genetic factors of the cells treated. PMID:25714019

  2. Controls on Filling and Evacuation of Sediment in Waterfall Plunge Pools

    NASA Astrophysics Data System (ADS)

    Scheingross, J. S.; Lamb, M. P.

    2014-12-01

    Many waterfalls are characterized by the presence of deep plunge pools that experience periods of sediment fill and evacuation. These cycles of sediment fill are a first order control on the relative magnitude of lateral versus vertical erosion at the base of waterfalls, as vertical incision requires cover-free plunge pools to expose the bedrock floor, while lateral erosion can occur when pools are partially filled and plunge-pool walls are exposed. Currently, there exists no mechanistic model describing sediment transport through waterfall plunge pools, limiting our ability to predict waterfall retreat. To address this knowledge gap, we performed detailed laboratory experiments measuring plunge-pool sediment transport capacity (Qsc_pool) under varying waterfall and plunge-pool geometries, flow hydraulics, and sediment size. Our experimental plunge-pool sediment transport capacity measurements match well with a mechanistic model we developed which combines existing waterfall jet theory with a modified Rouse profile to predict sediment transport capacity as a function of water discharge and suspended sediment concentration at the plunge-pool lip. Comparing the transport capacity of plunge pools to lower gradient portions of rivers (Qsc_river) shows that, for transport limited conditions, plunge pools fill with sediment under modest water discharges when Qsc_river > Qsc_pool, and empty to bedrock under high discharges when Qsc_pool > Qsc_river. These results are consistent with field observations of sand-filled plunge pools with downstream boulder rims, implying filling and excavation of plunge pools over single-storm timescales. Thus, partial filling of waterfall plunge pools may provide a mechanism to promote lateral undercutting and retreat of waterfalls in homogeneous rock in which plunge-pool vertical incision occurs during brief large floods that expose bedrock, whereas lateral erosion may prevail during smaller events.

  3. Evaluation of human gene variant detection in amplicon pools by the GS-FLX parallel Pyrosequencer

    PubMed Central

    Bordoni, Roberta; Bonnal, Raoul; Rizzi, Ermanno; Carrera, Paola; Benedetti, Sara; Cremonesi, Laura; Stenirri, Stefania; Colombo, Alessio; Montrasio, Cristina; Bonalumi, Sara; Albertini, Alberto; Bernardi, Luigi Rossi; Ferrari, Maurizio; De Bellis, Gianluca

    2008-01-01

    Background A new priority in genome research is large-scale resequencing of genes to understand the molecular basis of hereditary disease and cancer. We assessed the ability of massively parallel pyrosequencing to identify sequence variants in pools. From a large collection of human PCR samples we selected 343 PCR products belonging to 16 disease genes and including a large spectrum of sequence variations previously identified by Sanger sequencing. The sequence variants included SNPs and small deletions and insertions (up to 44 bp), in homozygous or heterozygous state. Results The DNA was combined in 4 pools containing from 27 to 164 amplicons and from 8,9 to 50,8 Kb to sequence for a total of 110 Kb. Pyrosequencing generated over 80 million base pairs of data. Blind searching for sequence variations with a specifically designed bioinformatics procedure identified 465 putative sequence variants, including 412 true variants, 53 false positives (in or adjacent to homopolymeric tracts), no false negatives. All known variants in positions covered with at least 30× depth were correctly recognized. Conclusion Massively parallel pyrosequencing may be used to simplify and speed the search for DNA variations in PCR products. Our results encourage further studies to evaluate molecular diagnostics applications. PMID:18842124

  4. 13 CFR 120.1704 - Pool Loans eligible for Pooling.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ..., construction or renovation of an aquarium, zoo, golf course, or swimming pool; or (iv) To a business covered by... (“Other Gambling Industries”); golf courses—713910 (“Golf Courses and Country Clubs”); or aquariums...

  5. 13 CFR 120.1704 - Pool Loans eligible for Pooling.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ..., construction or renovation of an aquarium, zoo, golf course, or swimming pool; or (iv) To a business covered by... (“Other Gambling Industries”); golf courses—713910 (“Golf Courses and Country Clubs”); or aquariums...

  6. 13 CFR 120.1704 - Pool Loans eligible for Pooling.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., construction or renovation of an aquarium, zoo, golf course, or swimming pool; or (iv) To a business covered by... (“Other Gambling Industries”); golf courses—713910 (“Golf Courses and Country Clubs”); or aquariums...

  7. 13 CFR 120.1704 - Pool Loans eligible for Pooling.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., construction or renovation of an aquarium, zoo, golf course, or swimming pool; or (iv) To a business covered by... (“Other Gambling Industries”); golf courses—713910 (“Golf Courses and Country Clubs”); or aquariums...

  8. 13 CFR 120.1704 - Pool Loans eligible for Pooling.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ..., construction or renovation of an aquarium, zoo, golf course, or swimming pool; or (iv) To a business covered by... (“Other Gambling Industries”); golf courses—713910 (“Golf Courses and Country Clubs”); or aquariums...

  9. Pooling techniques for bioassay screening

    SciTech Connect

    Sun, L.C.; Baum, J.W.; Kaplan, E; Moorthy, A.R.

    1996-03-01

    Pooling techniques commonly are used to increase the throughput of samples used for screening purposes. While the advantages of such techniques are increased analytical efficiency and cost savings, the sensitivity of measurements decreases because it is inversely proportional to the number of samples in the pools. Consequently, uncertainties in estimates of dose and risk which are based on the results of pooled samples increase as the number of samples in the pools increases in all applications. However, sensitivities may not be seriously degraded, for example, in urinalysis, if the samples in the pools are of known time duration, or if the fraction of some attribute of the grab urine samples to that in a 24-hour composite is known (e.g., mass, specific gravity, creatinine, or volume, per 24-h interval). This paper presents square and cube pooling schemes that greatly increase throughput and can considerably reduce analytical costs (on a sample basis). The benefit-cost ratios for 5{times}5 square and 5{times}5{times}5 cube pooling schemes are 2.5 and 8.3, respectively. Three-dimensional and higher arrayed pooling schemes would result in even greater economies; however, significant improvements in analytical sensitivity are required to achieve these advantages. These are various other considerations for designing a pooling scheme, where the number of dimensions and of samples in the optimum array are influenced by: (1) the minimal detectable amount (MDA) of the analytical processes, (2) the screening dose-rate requirements, (3) the maximum masses or volumes of the composite samples that can be analyzed, (4) the information already available from results of composite analysis, and (5) the ability of an analytical system to guard against both false negative and false positive results. Many of these are beyond the scope of this paper but are being evaluated.

  10. Structure of Low-Lying Excited States of Guanine in DNA and Solution: Combined Molecular Mechanics and High-Level Coupled Cluster Studies

    DOE PAGESBeta

    Kowalski, Karol; Valiev, Marat

    2007-01-01

    High-level ab-initio equation-of-motion coupled-cluster methods with singles, doubles, and noniterative triples are used, in conjunction with the combined quantum mechanical molecular mechanics approach, to investigate the structure of low-lying excited states of the guanine base in DNA and solvated environments. Our results indicate that while the excitation energy of the first excited state is barely changed compared to its gas-phase counterpart, the excitation energy of the second excited state is blue-shifted by 0.24 eV.

  11. Inhibition of DNA Topoisomerase Type IIα (TOP2A) by Mitoxantrone and Its Halogenated Derivatives: A Combined Density Functional and Molecular Docking Study

    PubMed Central

    Abu Saleh, Md.; Solayman, Md.; Hoque, Mohammad Mazharol; Khan, Mohammad A. K.; Sarwar, Mohammed G.; Halim, Mohammad A.

    2016-01-01

    In this study, mitoxantrone and its halogenated derivatives have been designed by density functional theory (DFT) to explore their structural and thermodynamical properties. The performance of these drugs was also evaluated to inhibit DNA topoisomerase type IIα (TOP2A) by molecular docking calculation. Noncovalent interactions play significant role in improving the performance of halogenated drugs. The combined quantum and molecular mechanics calculations revealed that CF3 containing drug shows better preference in inhibiting the TOP2A compared to other modified drugs. PMID:27088089

  12. Multiplex pairwise assembly of array-derived DNA oligonucleotides

    PubMed Central

    Klein, Jason C.; Lajoie, Marc J.; Schwartz, Jerrod J.; Strauch, Eva-Maria; Nelson, Jorgen; Baker, David; Shendure, Jay

    2016-01-01

    While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192–252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131–250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput ‘Dial-Out PCR’ protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens. PMID:26553805

  13. Multiplex pairwise assembly of array-derived DNA oligonucleotides.

    PubMed

    Klein, Jason C; Lajoie, Marc J; Schwartz, Jerrod J; Strauch, Eva-Maria; Nelson, Jorgen; Baker, David; Shendure, Jay

    2016-03-18

    While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192-252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131-250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput 'Dial-Out PCR' protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens. PMID:26553805

  14. A model of binding on DNA microarrays: understanding the combined effect of probe synthesis failure, cross-hybridization, DNA fragmentation and other experimental details of affymetrix arrays

    PubMed Central

    2012-01-01

    Background DNA microarrays are used both for research and for diagnostics. In research, Affymetrix arrays are commonly used for genome wide association studies, resequencing, and for gene expression analysis. These arrays provide large amounts of data. This data is analyzed using statistical methods that quite often discard a large portion of the information. Most of the information that is lost comes from probes that systematically fail across chips and from batch effects. The aim of this study was to develop a comprehensive model for hybridization that predicts probe intensities for Affymetrix arrays and that could provide a basis for improved microarray analysis and probe development. The first part of the model calculates probe binding affinities to all the possible targets in the hybridization solution using the Langmuir isotherm. In the second part of the model we integrate details that are specific to each experiment and contribute to the differences between hybridization in solution and on the microarray. These details include fragmentation, wash stringency, temperature, salt concentration, and scanner settings. Furthermore, the model fits probe synthesis efficiency and target concentration parameters directly to the data. All the parameters used in the model have a well-established physical origin. Results For the 302 chips that were analyzed the mean correlation between expected and observed probe intensities was 0.701 with a range of 0.88 to 0.55. All available chips were included in the analysis regardless of the data quality. Our results show that batch effects arise from differences in probe synthesis, scanner settings, wash strength, and target fragmentation. We also show that probe synthesis efficiencies for different nucleotides are not uniform. Conclusions To date this is the most complete model for binding on microarrays. This is the first model that includes both probe synthesis efficiency and hybridization kinetics/cross-hybridization. These

  15. Formation of the southern Bay of Bengal cold pool

    NASA Astrophysics Data System (ADS)

    Das, Umasankar; Vinayachandran, P. N.; Behara, Ambica

    2015-12-01

    A pool of relatively cooler water, called here as the southern Bay of Bengal cold pool, exists around Sri Lanka and southern tip of India during the summer monsoon. This cold pool is enveloped by the larger Indian Ocean warm pool and is believed to affect the intraseasonal variations of summer monsoon rainfall. In this study, we have investigated the mechanisms responsible for the formation of the cold pool using a combination of both satellite data sets and a general circulation model of the Indian Ocean. Sea surface temperature (SST) within the cold pool, after the steady increase during the February-April period, decreases first during a pre-monsoon spell in April and then with the monsoon onset during May. The onset cooling is stronger (~1.8° C) than the pre-monsoon cooling (~0.8° C) and culminates in the formation of the cold pool. Analysis of the model temperature equation shows that SST decrease during both events is primarily due to a decrease in incoming solar radiation and an increase in latent heat loss. These changes in the net heat flux are brought about by the arrival of cloud bands above the cold pool during both periods. During the pre-monsoon period, a cloud band originates in the western equatorial Indian Ocean and subsequently arrives above the cold pool. Similarly, during the monsoon onset, a band of clouds originating in the eastern equatorial Indian Ocean comes over the cold pool region. A lead-lag correlation calculation between daily SST and rainfall anomalies suggest that cooling in SST occurs in response to rainfall events with a lag of 5 days. These sequence of events occur every year with certain amount of interannual variability.

  16. The histone deacetylase inhibitor SAHA sensitizes acute myeloid leukemia cells to a combination of nucleoside analogs and the DNA-alkylating agent busulfan.

    PubMed

    Song, Guiyun; Valdez, Benigno C; Li, Yang; Dominguez, Jose R; Corn, Paul; Champlin, Richard E; Andersson, Borje S

    2014-07-01

    Fludarabine (Flu), clofarabine (Clo) and busulfan (Bu) are used in allogeneic hematopoietic stem cell transplant (allo-HSCT). We reported that combining [Flu + Clo + Bu] had a synergistic cytotoxicity in AML cells. We hypothesized that combining [Flu + Clo + Bu] with the histone deacetylase inhibitor SAHA will further enhance cytotoxicity. We exposed the acute myeloid leukemia (AML) cell lines KBM3/Bu250(6) and OCI-AML3 to Flu, Clo, Bu and SAHA alone and in various combinations. [Flu + Clo + Bu + SAHA] resulted in synergistic cytotoxicity, which can be attributed to (1) activated DNA-damage response and cell cycle checkpoint activation through the ATM-CHK2-P53 (or P73) pathway or ATM-CHK2-cdc25-cdc2 pathway, (2) histone modifications and (3) activated apoptosis pathway. The [Flu + Clo + Bu + SAHA] combination causes mitochondrial outer membrane permeabilization, leakage of cytochrome c and Smac/Diablo into the cytosol with caspase activation, and release of apoptosis-inducing factor (AIF) into the nucleus resulting in nuclear fragmentation and cell death. These results provide a mechanistic basis for using SAHA in future clinical trials with double nucleoside analog-busulfan combinations in pretransplant conditioning therapy. PMID:24144307

  17. Combining plasma Epstein-Barr virus DNA and nodal maximal standard uptake values of 18F-fluoro-2-deoxy-D-glucose positron emission tomography improved prognostic stratification to predict distant metastasis for locoregionally advanced nasopharyngeal carcinoma

    PubMed Central

    Chen, Qiu-Yan; Guo, Shan-Shan; Liu, Li-Ting; Fan, Wei; Zhang, Xu; Guo, Ling; Zhao, Chong; Cao, Ka-Jia; Qian, Chao-Nan; Guo, Xiang; Xie, Dan; Zeng, Mu-Sheng; Mai, Hai-Qiang

    2015-01-01

    Background This study aimed to evaluate the value of combining the nodal maximal standard uptake values (SUVmax) of 18 F-fluoro-2-deoxy-D-glucose positron emission tomography with Epstein-Barr virus DNA(EBV DNA) levels to predict distant metastasis for nasopharyngeal carcinoma (NPC) patients Patients and Methods Eight hundred seventy-four patients with stage III-IVa-b NPC were evaluated for the effects of combining SUVmax and EBV DNA levels on distant metastasis-free survival (DMFS), disease-free survival (DFS) and overall survival (OS). Results The optimal cutoff value was 6,220 copies/mL for EBV DNA and 7.5 for SUVmax-N. Patients with lower EBV DNA levels or SUVmax-N had a significantly better 3-year DMFS, DFS, and OS. Patients were divided into four groups based on EBV DNA and SUVmax-N, as follows: low EBV DNA and low SUVmax-N (LL), low EBV DNA and high SUVmax-N (LH), high EBV DNA and low SUVmax-N (HL), and high EBV DNA and high SUVmax-N (HH). There were significant differences between the four mentioned groups in 3-year DMFS: 95.7%, 92.2%, 92.3%, and 80.1%, respectively (Ptrend < 0.001). When looking at the disease stage, the 3-year DMFS in group LL, LH, HL, HH were 94.2%, 92.9%, 95.0%, and 81.1%, respectively, in stage III patients (Ptrend < 0.001) and 92.7%, 87.2%, 86.3%, and 77.0% in stage IVa–b patients (Ptrend = 0.026). Conclusion Pretreatment EBV DNA and SUVmax of neck lymph nodes were independent prognostic factors for distant metastasis in NPC patients. Combining EBV DNA and SUVmax-N led to an improved risk stratification for distant metastasis in advanced-stage disease. PMID:26512922

  18. Rabies vaccination: comparison of neutralizing antibody responses after priming and boosting with different combinations of DNA, inactivated virus, or recombinant vaccinia virus vaccines.

    PubMed

    Lodmell, D L; Ewalt, L C

    2000-05-01

    Long-term levels of neutralizing antibody were evaluated in mice after a single immunization with experimental DNA or recombinant vaccinia virus (RVV) vaccines encoding the rabies virus glycoprotein (G), or the commercially available inactivated virus human diploid cell vaccine (HDCV). Anamnestic antibody titers were also evaluated after two booster immunizations with vaccines that were identical to or different from the priming vaccine. Five hundred and forty days (1.5 year) after a single immunization with any of the three vaccines, neutralizing antibody titers remained greater than the minimal acceptable human level of antibody titer (0.5 International Units (IU)/ml). In addition, either an HDCV or DNA booster elicited early and elevated anamnestic antibody responses in mice that had been primed with any of the three vaccines. In contrast, RVV boosters failed to elevate titers in mice that had been previously primed with RVV, and elicited slowly rising titers in mice that had been primed with either DNA or HDCV. Thus, a single vaccination with any of the three different vaccines elicited long-term levels of neutralizing antibody that exceeded 0.5 IU/ml. In contrast, different prime-booster vaccine combinations elicited anamnestic neutralizing antibody responses that increased quickly, increased slowly or failed to increase. PMID:10738096

  19. Personal identification of cold case remains through combined contribution from anthropological, mtDNA and bomb–pulse dating analyses*†

    PubMed Central

    Speller, Camilla F.; Spalding, Kirsty L.; Buchholz, Bruce A.; Hildebrand, Dean; Moore, Jason; Mathewes, Rolf; Skinner, Mark F.; Yang, Dongya Y.

    2013-01-01

    In 1968, a child’s cranium was recovered from the banks of a northern Canadian river, and held in trust until the ‘cold case’ was re-opened in 2005. The cranium underwent re-analysis at the Centre for Forensic Research, Simon Fraser University, using recently developed anthropological, ‘bomb-pulse’ radiocarbon analysis and forensic DNA techniques. Craniometrics, skeletal ossification and dental formation indicated an age-at-death of 4.4 ±1 years. Radiocarbon analysis of enamel from two teeth indicated a year of birth between 1958–1962. Forensic DNA analysis indicated the child was male, and the obtained mitochondrial profile matched a living maternal relative of the presumed missing child. These multi-disciplinary analyses resulted in a legal identification 41 years after the discovery of the remains, highlighting the enormous potential of combining radiocarbon analysis with anthropological and mtDNA analyses in producing confident personal identifications for forensic cold cases dating to within the last 60 years. PMID:22804335

  20. Patent pools and diagnostic testing.

    PubMed

    Verbeure, Birgit; van Zimmeren, Esther; Matthijs, Gert; Van Overwalle, Geertrui

    2006-03-01

    There is increasing concern that overlapping patents in the field of genetics will create a costly and legally complex situation known as a patent thicket, which, along with the associated issues of accumulating royalty payments, can act as a disincentive for innovation. One potential means of preventing this is for the patent holders to enter into a so-called patent pool, such as those established in the electronics and telecommunications industries. Precedents for these also exist in the field of genetics, notably with the patents pertaining to the SARS genome. In this review, we initially address the patent pool concept in general and its application in genetics. Following this, we will explore patent pools in the diagnostic field in more detail, and examine some existing and novel examples of patent pools in genetics. PMID:16443296

  1. Reproductive morphology and DNA sequences of the brown alga Platysiphon verticillatus support the new combination Platysiphon glacialis.

    PubMed

    Kawai, Hiroshi; Hanyuda, Takeaki; Yamagishi, Takahiro; Kai, Atsushi; Lane, Chris E; McDevit, Dan; Küpper, Frithjof C; Saunders, Gary W

    2015-10-01

    Platysiphon verticillatus, a brown alga endemic to the Arctic, was described based on vegetative specimens collected at Inglefield Bay, West Greenland. The species is distinctive in having a lanceolate blade-like thallus terminated by a terete portion, both covered with hair-like assimilatory filaments. Punctaria glacialis was described from Eastern Greenland, and the species differs from other Punctaria species in lacking hairs and plurilocular zoidangia. Unilocular zoidangia were reported, but instead of zoids being released they formed cell walls in situ developing the appearance of plurilocular zoidangia. However, the fate of the zoids, as well as the walled cells was not traced, and the life history of the alga has remained unclear. By comparing DNA sequences (cox1, cox3, and rDNA ITS2) of specimens morphologically referable to Platysiphon verticillatus and Punctaria glacialis collected at Baffin Island, as well as re-examining morphology and studying crude cultures, we concluded that they are the same taxonomic entity. Furthermore, their cox3 sequence and vegetative morphology agreed with those of the type specimen of Punctaria glacialis. Consequently, we propose Platysiphon glacialis comb. nov. The life cycle could not be completed in culture, but we hypothesize that in situ germination of the unizoids produces reduced gametophytes housed in peripheral tissue of erect sporophytic thalli. PMID:26986887

  2. Protection against H1N1 influenza challenge by a DNA vaccine expressing H3/H1 subtype hemagglutinin combined with MHC class II-restricted epitopes

    PubMed Central

    2010-01-01

    Background Multiple subtypes of avian influenza viruses have crossed the species barrier to infect humans and have the potential to cause a pandemic. Therefore, new influenza vaccines to prevent the co-existence of multiple subtypes within a host and cross-species transmission of influenza are urgently needed. Methods Here we report a multi-epitope DNA vaccine targeted towards multiple subtypes of the influenza virus. The protective hemagglutinin (HA) antigens from H5/H7/H9 subtypes were screened for MHC II class-restricted epitopes overlapping with predicted B cell epitopes. We then constructed a DNA plasmid vaccine, pV-H3-EHA-H1, based on HA antigens from human influenza H3/H1 subtypes combined with the H5/H7/H9 subtype Th/B epitope box. Results Epitope-specific IFN-γ ELISpot responses were significantly higher in the multi-epitope DNA group than in other vaccine and control groups (P < 0.05). The multi-epitope group significantly enhanced Th2 cell responses as determined by cytokine assays. The survival rate of mice given the multi-epitope vaccine was the highest among the vaccine groups, but it was not significantly different compared to those given single antigen expressing pV-H1HA1 vaccine and dual antigen expressing pV-H3-H1 vaccine (P > 0.05). No measurable virus titers were detected in the lungs of the multi-epitope immunized group. The unique multi-epitope DNA vaccine enhanced virus-specific antibody and cellular immunity as well as conferred complete protection against lethal challenge with A/New Caledonia/20/99 (H1N1) influenza strain in mice. Conclusions This approach may be a promising strategy for developing a universal influenza vaccine to prevent multiple subtypes of influenza virus and to induce long-term protective immune against cross-species transmission. PMID:21134292

  3. Sensitive Electrochemiluminescence Immunosensor for Detection of N-Acetyl-β-d-glucosaminidase Based on a "Light-Switch" Molecule Combined with DNA Dendrimer.

    PubMed

    Wang, Haijun; Yuan, Yali; Zhuo, Ying; Chai, Yaqin; Yuan, Ruo

    2016-06-01

    Here, a novel "light-switch" molecule of Ru (II) complex ([Ru(dcbpy)2dppz](2+)-DPEA) with self-enhanced electrochemiluminescence (ECL) property is proposed, which is almost nonemissive in aqueous solution but is brightly luminescent when it intercalates into DNA duplex. Owing to less energy loss and shorter electron-transfer distance, the intramolecular ECL reaction between the luminescent [Ru(dcbpy)2dppz](2+) and coreactive tertiary amine group in N,N-diisopropylethylenediamine (DPEA) makes the obtained "light-switch" molecule possess much higher light-switch efficiency compared with the traditional "light-switch" molecule. For increasing the loading amount and further enhancing the luminous efficiency of the "light-switch" molecule, biotin labeled DNA dendrimer (the fourth generation, G4) is prepared from Y-shape DNA by a step-by-step assembly strategy, which provides abundant intercalated sites for [Ru(dcbpy)2dppz](2+)-DPEA. Meanwhile, the obtained nanocomposite (G4-[Ru(dcbpy)2dppz](2+)-DPEA) could well bind with streptavidin labeled detection antibody (SA-Ab2) due to the existence of abundant biotin. Through sandwiched immunoreaction, an ECL immunosensor was fabricated for sensitive determination of N-acetyl-β-d-glucosaminidase (NAG), a typical biomarker for diabetic nephropathy (DN). The detemination linear range was 0.1 pg mL(-1) to 1 ng mL(-1), and the detection limit was 0.028 pg mL(-1). The developed strategy combining the ECL self-enhanced "light-switch" molecular and DNA nanotechnology offers an effective signal amplification mean and provides ample potential for further bioanalysis and clinical study. PMID:27185239

  4. Pool impacts of Leidenfrost drop

    NASA Astrophysics Data System (ADS)

    Darbois Texier, Baptiste; Maquet, Laurent; Dorbolo, Stephane; Dehandschoewercker, Eline; Pan, Zhao; Truscott, Tadd

    2015-11-01

    This work concerns the impact of a droplet made of a volatile liquid (typically HFE) on a pool of an other liquid (typically silicone oil) which temperature is above the boiling point of the drop. Depending on the properties of the two liquids and the impacting conditions, four different regimes are observed. For low impacting speeds, the droplet bounces on the surface of the bath and finally levitates above it in a Leidenfrost state. Such a regime occurs as soon as the pool temperature exceeds the boiling point of the drop. This observation means that there is no threshold in temperature for a Leidenfrost effect on a liquid surface contrary to the case of a solid substrate. For intermediate impacting velocities, the pinch-off of the surface of the pool entraps the drop in the liquid bulk. The entrapped drop is separated from the pool by a layer of its own vapour in a similar way of antibulles. For increasing impacting speeds, the vapour layer between the drop and the pool does not hold during the pinch-off event. The contact of the drop with the hot liquid provokes a sudden and intense evaporation. At very large impacting speeds, the drop rapidely contacts the pool, spreads and finally induces a hemi-spherical cavity. In the end, these four different regimes are summarized in a Froud-Weber diagram which boundaries are discussed.

  5. Highly efficient quantum-dot light-emitting diodes with DNA-CTMA as a combined hole-transporting and electron-blocking layer.

    PubMed

    Sun, Qingjiang; Subramanyam, Guru; Dai, Liming; Check, Michael; Campbell, Angela; Naik, Rajesh; Grote, James; Wang, Yongqiang

    2009-03-24

    Owing to their narrow bright emission band, broad size-tunable emission wavelength, superior photostability, and excellent flexible-substrate compatibility, light-emitting diodes based on quantum dots (QD-LEDs) are currently under intensive research and development for multiple consumer applications including flat-panel displays and flat lighting. However, their commercialization is still precluded by the slow development to date of efficient QD-LEDs as even the highest reported efficiency of 2.0% cannot favorably compete with their organic counterparts. Here, we report QD-LEDs with a record high efficiency (approximately 4%), high brightness (approximately 6580 cd/m(2)), low turn-on voltage (approximately 2.6 V), and significantly improved color purity by simply using deoxyribonucleic acid (DNA) complexed with cetyltrimetylammonium (CTMA) (DNA-CTMA) as a combined hole transporting and electron-blocking layer (HTL/EBL). This, together with controlled thermal decomposition of ligand molecules from the QD shell, represents a novel combined, but simple and very effective, approach toward the development of highly efficient QD-LEDs with a high color purity. PMID:19309174

  6. Baculovirus-expressed virus-like particle vaccine in combination with DNA encoding the fusion protein confers protection against respiratory syncytial virus.

    PubMed

    Lee, Jong Seok; Kwon, Young-Man; Hwang, Hye Suk; Lee, Yu-Na; Ko, Eun-Ju; Yoo, Si-Eun; Kim, Min-Chul; Kim, Ki-Hye; Cho, Min Kyoung; Lee, Young-Tae; Lee, You Ri; Quan, Fu-Shi; Kang, Sang-Moo

    2014-10-01

    Respiratory syncytial virus (RSV) is a major viral agent causing significant morbidity and mortality in young infants and the elderly. There is no licensed vaccine against RSV and it is a high priority to develop a safe RSV vaccine. We determined the immunogenicity and protective efficacy of combined virus-like particle and DNA vaccines presenting RSV glycoproteins (Fd.VLP) in comparison with formalin inactivated RSV (FI-RSV). Immunization of mice with Fd.VLP induced higher ratios of IgG2a/IgG1 antibody responses compared to those with FI-RSV. Upon live RSV challenge, Fd.VLP and FI-RSV vaccines were similarly effective in clearing lung viral loads. However, FI-RSV immunized mice showed a substantial weight loss and high levels of T helper type 2 (Th2) cytokines as well as extensive lung histopathology and eosinophil infiltration. In contrast, Fd.VLP immunized mice did not exhibit Th2 type cytokines locally and systemically, which might contribute to preventing vaccine-associated RSV lung disease. These results indicate that virus-like particles in combination with DNA vaccines represent a potential approach for developing a safe and effective RSV vaccine. PMID:25173478

  7. DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops.

    PubMed

    van der Woning, Bas; De Boeck, Gitte; Blanchetot, Christophe; Bobkov, Vladimir; Klarenbeek, Alex; Saunders, Michael; Waelbroeck, Magali; Laeremans, Toon; Steyaert, Jan; Hultberg, Anna; De Haard, Hans

    2016-01-01

    The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins. PMID:27211075

  8. 3D finite element simulation of TIG weld pool

    NASA Astrophysics Data System (ADS)

    Kong, X.; Asserin, O.; Gounand, S.; Gilles, P.; Bergheau, J. M.; Medale, M.

    2012-07-01

    The aim of this paper is to propose a three-dimensional weld pool model for the moving gas tungsten arc welding (GTAW) process, in order to understand the main factors that limit the weld quality and improve the productivity, especially with respect to the welding speed. Simulation is a very powerful tool to help in understanding the physical phenomena in the weld process. A 3D finite element model of heat and fluid flow in weld pool considering free surface of the pool and traveling speed has been developed for the GTAW process. Cast3M software is used to compute all the governing equations. The free surface of the weld pool is calculated by minimizing the total surface energy. The combined effects of surface tension gradient, buoyancy force, arc pressure, arc drag force to drive the fluid flow is included in our model. The deformation of the weld pool surface and the welding speed affect fluid flow, heat flow and thus temperature gradients and molten pool dimensions. Welding trials study is presented to compare our numerical results with macrograph of the molten pool.

  9. Vesicle Pools: Lessons from Adrenal Chromaffin Cells

    PubMed Central

    Stevens, David R.; Schirra, Claudia; Becherer, Ute; Rettig, Jens

    2011-01-01

    The adrenal chromaffin cell serves as a model system to study fast Ca2+-dependent exocytosis. Membrane capacitance measurements in combination with Ca2+ uncaging offers a temporal resolution in the millisecond range and reveals that catecholamine release occurs in three distinct phases. Release of a readily releasable (RRP) and a slowly releasable (SRP) pool are followed by sustained release, due to maturation, and release of vesicles which were not release-ready at the start of the stimulus. Trains of depolarizations, a more physiological stimulus, induce release from a small immediately releasable pool of vesicles residing adjacent to calcium channels, as well as from the RRP. The SRP is poorly activated by depolarization. A sequential model, in which non-releasable docked vesicles are primed to a slowly releasable state, and then further mature to the readily releasable state, has been proposed. The docked state, dependent on membrane proximity, requires SNAP-25, synaptotagmin, and syntaxin. The ablation or modification of SNAP-25 and syntaxin, components of the SNARE complex, as well as of synaptotagmin, the calcium sensor, and modulators such complexins and Snapin alter the properties and/or magnitudes of different phases of release, and in particular can ablate the RRP. These results indicate that the composition of the SNARE complex and its interaction with modulatory molecules drives priming and provides a molecular basis for different pools of releasable vesicles. PMID:21423410

  10. Where is DNA preserved in soil organic matter?

    NASA Astrophysics Data System (ADS)

    Zaccone, Claudio; Beneduce, Luciano; Plaza, César

    2015-04-01

    Deoxyribonucleic acid (DNA) consists of long chains of alternating sugar and phosphate residues twisted in the form of a helix. Upon decomposition of plant and animal debris, this nucleic acid is released into the soil, where its fate is still not completely understood. In fact, although DNA is one of the organic compounds from living cells that is apparently broken down rapidly in soils, it is also potentially capable of being incorporated in (or interact with) the precursors of humic molecules. In order to track DNA occurrence in soil organic matter (SOM) fractions, an experiment was set up as a randomized complete block design with two factors, namely biochar addition and organic amendment. In particular, biochar (BC), applied at a rate of 20 t/ha, was combined with municipal solid waste compost (BC+MC) at a rate equivalent to 75 kg/ha of potentially available N, and with sewage sludge (BC+SS) at a rate equivalent to 75 kg/ha of potentially available N. Using a physical fractionation method, free SOM located between aggregates (unprotected C pool; FR), SOM occluded within macroaggregates (C pool weakly protected by physical mechanisms; MA), SOM occluded within microaggregates (C pool strongly protected by physical mechanisms; MI), and SOM associated with the mineral fractions (chemically-protected C pool; MIN) were separated from soil samples. DNA was then isolated from each fraction of the two series, as well as from the unamended soil (C) and from the bulk soils (WS), using Powersoil DNA isolation kit (MoBio, CA, USA) with a modified protocol. Data clearly show that the DNA survived the SOM fractionation, thus suggesting that physical fractionation methods create less artifacts compared to the chemical ones. Moreover, in both BC+MC and BC+SS series, most of the isolated DNA was present in the FR fraction, followed by the MA and the MI fractions. No DNA was recovered from the MIN fraction. This finding supports the idea that most of the DNA occurring in the SOM

  11. Co-administration of certain DNA vaccine combinations expressing different H5N1 influenza virus antigens can be beneficial or detrimental to immune protection.

    PubMed

    Patel, Ami; Gray, Michael; Li, Yan; Kobasa, Darwyn; Yao, Xiaojian; Kobinger, Gary P

    2012-01-11

    Achieving broad-spectrum immunity against emerging zoonotic viruses such as avian influenza H5N1 and other possible pandemic viruses will require generation of cross-protective immune responses. Strong antibody responses generated against the H5HA protein are protective, however, antigenic variation between diverging isolates can interfere with virus neutralization. The current study investigates co-administration of an H5 HA DNA vaccine with other variable and conserved influenza antigens (NA, NP, and M2). All antigens were derived from the A/Hanoi/30408/2005 (H5N1) virus and the contribution towards overall protection and immune activation was assessed against lethal homologous and heterologous challenges. An (HA+NA) combination afforded the best protection against homologous challenge and (HA+NP) was comparable to HA alone against heterologous A/Hong Kong/483/1997 challenge. Interestingly, combining all four H5 antigens at a single site did not improve protection against matched challenge and unexpectedly reduced survival by 30% against a heterologous challenge. Survival was also significantly decreased against heterologous challenge following combination of (HA+NP) with an unrelated antigen. Although there were no significant changes in antibody titres, significantly lower T-cell responses were detected against all antigens except HA in each combination. Co-administration of the vaccines at different injection sites restored T-cell responses but did not improve overall protection. Similar observations were also recorded following combination of HA and NP antigens using two different adenovirus-based backbones. Overall, the data suggest that co-administering certain H5N1 antigens offer better or comparable protection to HA alone, however, combining extra antigens may be unnecessary and lead to unfavourable immune responses. PMID:22119588

  12. Sediment transport through self-adjusting, bedrock-walled waterfall plunge pools

    NASA Astrophysics Data System (ADS)

    Scheingross, Joel S.; Lamb, Michael P.

    2016-05-01

    Many waterfalls have deep plunge pools that are often partially or fully filled with sediment. Sediment fill may control plunge-pool bedrock erosion rates, partially determine habitat availability for aquatic organisms, and affect sediment routing and debris flow initiation. Currently, there exists no mechanistic model to describe sediment transport through waterfall plunge pools. Here we develop an analytical model to predict steady-state plunge-pool depth and sediment-transport capacity by combining existing jet theory with sediment transport mechanics. Our model predicts plunge-pool sediment-transport capacity increases with increasing river discharge, flow velocity, and waterfall drop height and decreases with increasing plunge-pool depth, radius, and grain size. We tested the model using flume experiments under varying waterfall and plunge-pool geometries, flow hydraulics, and sediment size. The model and experiments show that through morphodynamic feedbacks, plunge pools aggrade to reach shallower equilibrium pool depths in response to increases in imposed sediment supply. Our theory for steady-state pool depth matches the experiments with an R2 value of 0.8, with discrepancies likely due to model simplifications of the hydraulics and sediment transport. Analysis of 75 waterfalls suggests that the water depths in natural plunge pools are strongly influenced by upstream sediment supply, and our model provides a mass-conserving framework to predict sediment and water storage in waterfall plunge pools for sediment routing, habitat assessment, and bedrock erosion modeling.

  13. MGME1 processes flaps into ligatable nicks in concert with DNA polymerase γ during mtDNA replication.

    PubMed

    Uhler, Jay P; Thörn, Christian; Nicholls, Thomas J; Matic, Stanka; Milenkovic, Dusanka; Gustafsson, Claes M; Falkenberg, Maria

    2016-07-01

    Recently, MGME1 was identified as a mitochondrial DNA nuclease with preference for single-stranded DNA (ssDNA) substrates. Loss-of-function mutations in patients lead to mitochondrial disease with DNA depletion, deletions, duplications and rearrangements. Here, we assess the biochemical role of MGME1 in the processing of flap intermediates during mitochondrial DNA replication using reconstituted systems. We show that MGME1 can cleave flaps to enable efficient ligation of newly replicated DNA strands in combination with POLγ. MGME1 generates a pool of imprecisely cut products (short flaps, nicks and gaps) that are converted to ligatable nicks by POLγ through extension or excision of the 3'-end strand. This is dependent on the 3'-5' exonuclease activity of POLγ which limits strand displacement activity and enables POLγ to back up to the nick by 3'-5' degradation. We also demonstrate that POLγ-driven strand displacement is sufficient to generate DNA- but not RNA-flap substrates suitable for MGME1 cleavage and ligation during replication. Our findings have implications for RNA primer removal models, the 5'-end processing of nascent DNA at OriH, and DNA repair. PMID:27220468

  14. MGME1 processes flaps into ligatable nicks in concert with DNA polymerase γ during mtDNA replication

    PubMed Central

    Uhler, Jay P.; Thörn, Christian; Nicholls, Thomas J.; Matic, Stanka; Milenkovic, Dusanka; Gustafsson, Claes M.; Falkenberg, Maria

    2016-01-01

    Recently, MGME1 was identified as a mitochondrial DNA nuclease with preference for single-stranded DNA (ssDNA) substrates. Loss-of-function mutations in patients lead to mitochondrial disease with DNA depletion, deletions, duplications and rearrangements. Here, we assess the biochemical role of MGME1 in the processing of flap intermediates during mitochondrial DNA replication using reconstituted systems. We show that MGME1 can cleave flaps to enable efficient ligation of newly replicated DNA strands in combination with POLγ. MGME1 generates a pool of imprecisely cut products (short flaps, nicks and gaps) that are converted to ligatable nicks by POLγ through extension or excision of the 3′-end strand. This is dependent on the 3′-5′ exonuclease activity of POLγ which limits strand displacement activity and enables POLγ to back up to the nick by 3′-5′ degradation. We also demonstrate that POLγ-driven strand displacement is sufficient to generate DNA- but not RNA-flap substrates suitable for MGME1 cleavage and ligation during replication. Our findings have implications for RNA primer removal models, the 5′-end processing of nascent DNA at OriH, and DNA repair. PMID:27220468

  15. Priming with a Simplified Intradermal HIV-1 DNA Vaccine Regimen followed by Boosting with Recombinant HIV-1 MVA Vaccine Is Safe and Immunogenic: A Phase IIa Randomized Clinical Trial

    PubMed Central

    Nilsson, Charlotta; Joachim, Agricola; Geldmacher, Christof; Mann, Philipp; Moshiro, Candida; Aboud, Said; Lyamuya, Eligius; Maboko, Leonard; Missanga, Marco; Kaluwa, Bahati; Mfinanga, Sayoki; Podola, Lilly; Bauer, Asli; Godoy-Ramirez, Karina; Marovich, Mary; Moss, Bernard; Hoelscher, Michael; Gotch, Frances; Stöhr, Wolfgang; Stout, Richard; McCormack, Sheena; Wahren, Britta; Mhalu, Fred; Robb, Merlin L.; Biberfeld, Gunnel; Sandström, Eric; Bakari, Muhammad

    2015-01-01

    Background Intradermal priming with HIV-1 DNA plasmids followed by HIV-1MVA boosting induces strong and broad cellular and humoral immune responses. In our previous HIVIS-03 trial, we used 5 injections with 2 pools of HIV-DNA at separate sites for each priming immunization. The present study explores whether HIV-DNA priming can be simplified by reducing the number of DNA injections and administration of combined versus separated plasmid pools. Methods In this phase IIa, randomized trial, priming was performed using 5 injections of HIV-DNA, 1000 μg total dose, (3 Env and 2 Gag encoding plasmids) compared to two “simplified” regimens of 2 injections of HIV-DNA, 600 μg total dose, of Env- and Gag-encoding plasmid pools with each pool either administered separately or combined. HIV-DNA immunizations were given intradermally at weeks 0, 4, and 12. Boosting was performed intramuscularly with 108 pfu HIV-MVA at weeks 30 and 46. Results 129 healthy Tanzanian participants were enrolled. There were no differences in adverse events between the groups. The proportion of IFN-γ ELISpot responders to Gag and/or Env peptides after the second HIV-MVA boost did not differ significantly between the groups primed with 2 injections of combined HIV-DNA pools, 2 injections with separated pools, and 5 injections with separated pools (90%, 97% and 97%). There were no significant differences in the magnitude of Gag and/or Env IFN-γ ELISpot responses, in CD4+ and CD8+ T cell responses measured as IFN-γ/IL-2 production by intracellular cytokine staining (ICS) or in response rates and median titers for binding antibodies to Env gp160 between study groups. Conclusions A simplified intradermal vaccination regimen with 2 injections of a total of 600 μg with combined HIV-DNA plasmids primed cellular responses as efficiently as the standard regimen of 5 injections of a total of 1000 μg with separated plasmid pools after boosting twice with HIV-MVA. Trial Registration World Health

  16. No Evidence of Neandertal mtDNA Contribution to Early Modern Humans

    PubMed Central

    2004-01-01

    The retrieval of mitochondrial DNA (mtDNA) sequences from four Neandertal fossils from Germany, Russia, and Croatia has demonstrated that these individuals carried closely related mtDNAs that are not found among current humans. However, these results do not definitively resolve the question of a possible Neandertal contribution to the gene pool of modern humans since such a contribution might have been erased by genetic drift or by the continuous influx of modern human DNA into the Neandertal gene pool. A further concern is that if some Neandertals carried mtDNA sequences similar to contemporaneous humans, such sequences may be erroneously regarded as modern contaminations when retrieved from fossils. Here we address these issues by the analysis of 24 Neandertal and 40 early modern human remains. The biomolecular preservation of four Neandertals and of five early modern humans was good enough to suggest the preservation of DNA. All four Neandertals yielded mtDNA sequences similar to those previously determined from Neandertal individuals, whereas none of the five early modern humans contained such mtDNA sequences. In combination with current mtDNA data, this excludes any large genetic contribution by Neandertals to early modern humans, but does not rule out the possibility of a smaller contribution. PMID:15024415

  17. 47 CFR 13.215 - Question pools.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 1 2011-10-01 2011-10-01 false Question pools. 13.215 Section 13.215... Question pools. The question pool for each written examination element will be composed of questions acceptable to the FCC. Each question pool must contain at least five (5) times the number of...

  18. 47 CFR 13.215 - Question pools.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 1 2010-10-01 2010-10-01 false Question pools. 13.215 Section 13.215... Question pools. The question pool for each written examination element will be composed of questions acceptable to the FCC. Each question pool must contain at least five (5) times the number of...

  19. HYDROLOGY AND LANDSCAPE CONNECTIVITY OF VERNAL POOLS

    EPA Science Inventory

    Vernal pools are shaped by hydrologic processes which influence many aspects of pool function. The hydrologic budget of a pool can be summarized by a water balance equation that relates changes in the amount of water in the pool to precipitation, ground- and surface-water flows, ...

  20. 21 CFR 1250.89 - Swimming pools.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Swimming pools. 1250.89 Section 1250.89 Food and... SANITATION Sanitation Facilities and Conditions on Vessels § 1250.89 Swimming pools. (a) Fill and draw swimming pools shall not be installed or used. (b) Swimming pools of the recirculation type shall...

  1. Swimming Pools. Managing School Facilities, Guide 2.

    ERIC Educational Resources Information Center

    Department for Education and Employment, London (England). Architects and Building Branch.

    This guide for schools with swimming pools offers advice concerning appropriate training for pool managers, the importance of water quality and testing, safety in the handling of chemicals, maintenance and cleaning requirements, pool security, and health concerns. The guide covers both indoor and outdoor pools, explains some technical terms,…

  2. Combining real-time PCR and next-generation DNA sequencing to provide quantitative comparisons of fungal aerosol populations

    NASA Astrophysics Data System (ADS)

    Dannemiller, Karen C.; Lang-Yona, Naama; Yamamoto, Naomichi; Rudich, Yinon; Peccia, Jordan

    2014-02-01

    We examined fungal communities associated with the PM10 mass of Rehovot, Israel outdoor air samples collected in the spring and fall seasons. Fungal communities were described by 454 pyrosequencing of the internal transcribed spacer (ITS) region of the fungal ribosomal RNA encoding gene. To allow for a more quantitative comparison of fungal exposure in humans, the relative abundance values of specific taxa were transformed to absolute concentrations through multiplying these values by the sample's total fungal spore concentration (derived from universal fungal qPCR). Next, the sequencing-based absolute concentrations for Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, and Penicillium/Aspergillus spp. were compared to taxon-specific qPCR concentrations for A. alternata, C. cladosporioides, E. nigrum, and Penicillium/Aspergillus spp. derived from the same spring and fall aerosol samples. Results of these comparisons showed that the absolute concentration values generated from pyrosequencing were strongly associated with the concentration values derived from taxon-specific qPCR (for all four species, p < 0.005, all R > 0.70). The correlation coefficients were greater for species present in higher concentrations. Our microbial aerosol population analyses demonstrated that fungal diversity (number of fungal operational taxonomic units) was higher in the spring compared to the fall (p = 0.02), and principal coordinate analysis showed distinct seasonal differences in taxa distribution (ANOSIM p = 0.004). Among genera containing allergenic and/or pathogenic species, the absolute concentrations of Alternaria, Aspergillus, Fusarium, and Cladosporium were greater in the fall, while Cryptococcus, Penicillium, and Ulocladium concentrations were greater in the spring. The transformation of pyrosequencing fungal population relative abundance data to absolute concentrations can improve next-generation DNA sequencing-based quantitative aerosol exposure

  3. Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors

    PubMed Central

    2013-01-01

    Background The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer. Results We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein. Conclusions Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer. PMID:24257477

  4. Characterisation of cisplatin coordination sites in cellular Escherichia coli DNA-binding proteins by combined biphasic liquid chromatography and ESI tandem mass spectrometry.

    PubMed

    Will, Joanna; Sheldrick, William S; Wolters, Dirk

    2008-03-01

    Combined multidimensional liquid chromatography and electrospray ionisation tandem mass spectrometry was employed to analyse platinated tryptic peptides from Escherichia coli cells treated with the anticancer drug cis-[PtCl2(NH3)2] at pH 7.0. Prerequisites for the LC/LC/MS/MS analysis of protein targets that are fulfilled by cisplatin are (a) that the original protein binding sites have a high kinetic stability over the range 2.3 < pH < 8.5, and (b) that the metal fragment remains coordinated to a significant number of b+ and y+ peptide ions under MS/MS fragmentation conditions. Matching the MS/MS spectra of the platinated tryptic peptides to sequences of proteins in the E. coli database enabled the identification of 31 protein targets for cisplatin. Whereas six of these are high-abundance enzymes and ribosomal proteins in E. coli cells, five low-abundance DNA-binding proteins were also identified as specific targets. These include the DNA mismatch repair protein mutS, the DNA helicase II (uvrD) and topoisomerase I (top1). Two efflux proteins (acrD, mdtA), the redox regulator thioredoxin 1 (thiO) and the external filament-like type-1 fimbrial protein A chain (fimA1) were also characterised as specific cisplatin-binding proteins. Kinetically favoured carboxylate (D, E) and hydroxy (S, T, Y) O atoms were identified as the Pt coordination sites in 18 proteins and methionyl S atoms in 9 proteins. PMID:18157731

  5. Structural of the class II enzyme of human liver alcohol dehydrogenase: combined cDNA and protein sequence determination of the. pi. subunit

    SciTech Connect

    Hoeoeg, J.O.; von Bahr-Lindstroem, H.; Heden, L.O.; Holmquist, B.; Larsson, K.; Hempel, J.; Vallee, B.L.; Joernvall, H.

    1987-04-07

    The class II enzyme of human liver alcohol dehydrogenase was isolated, carboxymethylated, and cleaved with CNBr and proteolytic enzymes. Sequence analysis of peptides established structures corresponding to the ..pi.. subunit. Two segments from the C-terminal region unique to ..pi.. were selected for synthesis of oligodeoxyribonucleotide probes to screen a human liver cDNA library constructed in plasmid pT4. Sequence analysis of two identical hybridization-positive clones with cDNA inserts of about 2000 nucleotides gave the entire coding region of the ..pi.. subunit, a 61-nucleotide 5' noncoding region and a 741-nucleotide 3' noncoding region containing four possible polyadenylation sites. Translation of the coding region yields a 391-residue polypeptide, which in all regions except the C-terminal segment corresponds to the protein structure as determined directly by peptide analysis. With the class I numbering system, the exception concerns a residue exchange at position 368, the actual C-terminus which is Phe-374 by peptide data but a 12 residue extension by cDNA data, and possibly two further residue exchanges at positions 303 and 312. The size difference might indicate the existence of posttranslational modifications of the mature protein or, in combination with the residue exchanges, the existence of polymorphism at the locus for class II subunits. The ..pi.. subunit analyzed directly results in a 379-residue polypeptide and is the only class II size thus far known to occur in the mature protein. Comparison of the ..pi.. structure with those of the class I subunits (..cap alpha.., ..beta.., and ..gamma..) reveals a homology with extensive differences. Large variations in segments affecting relationships at the active site and the area of subunit interactions account for the significant alterations of enzymatic specificities and other properties that differentiate class II from class I enzymes.

  6. Characterization of Nanoscale Transformations in Polyelectrolyte Multilayers Fabricated from Plasmid DNA Using Laser Scanning Confocal Microscopy in Combination with Atomic Force Microscopy

    PubMed Central

    Fredin, Nathaniel J.; Flessner, Ryan M.; Jewell, Christopher M.; Bechler, Shane L.; Buck, Maren E.; Lynn, David M.

    2010-01-01

    Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) were used to characterize changes in nanoscale structure that occur when ultrathin polyelectrolyte multilayers (PEMs) are incubated in aqueous media. The PEMs investigated here were fabricated by the deposition of alternating layers of plasmid DNA and a hydrolytically degradable polyamine onto a precursor film composed of alternating layers of linear poly(ethylene imine) (LPEI) and sodium poly(styrene sulfonate) (SPS). Past studies of these materials in the context of gene delivery revealed transformations from a morphology that is smooth and uniform to one characterized by the formation of nanometer-scale particulate structures. We demonstrate that in-plane registration of LSCM and AFM images acquired from the same locations of films fabricated using fluorescently labeled polyelectrolytes allows the spatial distribution of individual polyelectrolyte species to be determined relative to the locations of topographic features that form during this transformation. Our results suggest that this physical transformation leads to a morphology consisting of a relatively less disturbed portion of film composed of polyamine and DNA juxtaposed over an array of particulate structures composed predominantly of LPEI and SPS. Characterization by scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) microanalysis provides additional support for this interpretation. The combination of these different microscopy techniques provides insight into the structures and dynamics of these multicomponent thin films that cannot be achieved using any one method alone, and that could prove useful for the further development of these assemblies as platforms for the surface-mediated delivery of DNA. PMID:20155860

  7. Mutations in recombinational repair and in checkpoint control genes suppress the lethal combination of srs2Delta with other DNA repair genes in Saccharomyces cerevisiae.

    PubMed Central

    Klein, H L

    2001-01-01

    The SRS2 gene of Saccharomyces cerevisiae encodes a DNA helicase that is active in the postreplication repair pathway and homologous recombination. srs2 mutations are lethal in a rad54Delta background and cause poor growth or lethality in rdh54Delta, rad50Delta, mre11Delta, xrs2Delta, rad27Delta, sgs1Delta, and top3Delta backgrounds. Some of these genotypes are known to be defective in double-strand break repair. Many of these lethalities or poor growth can be suppressed by mutations in other genes in the DSB repair pathway, namely rad51, rad52, rad55, and rad57, suggesting that inhibition of recombination at a prior step prevents formation of a lethal intermediate. Lethality of the srs2Delta rad54Delta and srs2Delta rdh54Delta double mutants can also be rescued by mutations in the DNA damage checkpoint functions RAD9, RAD17, RAD24, and MEC3, indicating that the srs2 rad54 and srs2 rdh54 mutant combinations lead to an intermediate that is sensed by these checkpoint functions. When the checkpoints are intact the cells never reverse from the arrest, but loss of the checkpoints releases the arrest. However, cells do not achieve wild-type growth rates, suggesting that unrepaired damage is still present and may lead to chromosome loss. PMID:11156978

  8. Combination of treatment with death receptor 5-specific antibody with therapeutic HPV DNA vaccination generates enhanced therapeutic anti-tumor effects.

    PubMed

    Tseng, Chih Wen; Monie, Archana; Trimble, Cornelia; Alvarez, Ronald D; Huh, Warner K; Buchsbaum, Donald J; Straughn, J Michael; Wang, Mei-Cheng; Yagita, Hideo; Hung, Chien-Fu; Wu, T-C

    2008-08-12

    There is currently a vital need for the development of novel therapeutic strategies for the control of advanced stage cancers. Antigen-specific immunotherapy and the employment of antibodies against the death receptor 5 (DR5) have emerged as two potentially promising strategies for cancer treatment. In the current study, we hypothesize that the combination of treatment with the anti-DR5 monoclonal antibody, MD5-1 with a DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 (HPV-16) E7 antigen (CRT/E7(detox)) administered via gene gun would lead to further enhancement of E7-specific immune responses as well as anti-tumor effects. Our results indicated that mice bearing the E7-expressing tumor, TC-1 treated with MD5-1 monoclonal antibody followed by CRT/E7(detox) DNA vaccination generated the most potent therapeutic anti-tumor effects as well as highest levels of E7-specific CD8+ T cells among all the groups tested. In addition, treatment with MD5-1 monoclonal antibody was capable of rendering the TC-1 tumor cells more susceptible to lysis by E7-specific cytotoxic T lymphocytes. Our findings serve as an important foundation for future clinical translation. PMID:18598733

  9. Possible effect of biotechnology on plant gene pools in Turkey

    PubMed Central

    Demir, Aynur

    2015-01-01

    The recent rapid developments in biotechnology have made great contributions to the study of plant gene pools. The application of in vitro methods in freeze storage and DNA protection techniques in fast production studies has made major advances. From that aspect, biotechnology is an indispensable means for the protection of plant gene pools, which includes the insurance of sustainable agriculture and development of species. Besides all the positive developments, one of the primary risks posed by the uncontrolled spreading of genetically modified organisms is the possibility for other non-target organisms to be negatively affected. Genes of plant origin should be given priority in this type of studies by taking into consideration such negative effects that may result in disruption of ecological balance and damage to plant genetic pools. Turkey, due to its ecological conditions and history, has a very important position in terms of plant gene pools. This richness ought to be protected without corrupting its natural quality and natural evolution process in order to provide the sources of species that will be required for future sustainable agricultural applications. Thus, attention should be paid to the use of biotechnological methods, which play an important role especially in the protection and use of local and original plant gene pools. PMID:26019612

  10. Response of soil carbon matter pools to elevated CO2 and warming in a semi-arid grassland

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Warming and elevated atmospheric CO2 (eCO2) can elicit contrasting responses on different SOM pools, thus to understand the effects of combined factors it is necessary to evaluate individual pools. Over two years, we assessed responses to eCO2 and warming of SOM pools, their susceptibility to decomp...

  11. Comparison of Exposure Controls, Item Pool Characteristics, and Population Distributions for CAT Using the Partial Credit Model

    ERIC Educational Resources Information Center

    Lee, HwaYoung; Dodd, Barbara G.

    2012-01-01

    This study investigated item exposure control procedures under various combinations of item pool characteristics and ability distributions in computerized adaptive testing based on the partial credit model. Three variables were manipulated: item pool characteristics (120 items for each of easy, medium, and hard item pools), two ability…

  12. An Evaluation of Individualized and Pool Slot Development for Public Service Employment: The Vermont Experience. Final Report.

    ERIC Educational Resources Information Center

    Mattson, Robert E.

    Public Service Employment slots can be developed in three ways: (1) by establishing a pool of jobs into which trainees can be placed; (2) by developing individualized slots for each trainee; (3) by combining the pool and individualized approaches. In terms of administrative and cost efficiency, there is little difference between the pool and the…

  13. Method of measuring a liquid pool volume

    DOEpatents

    Garcia, Gabe V.; Carlson, Nancy M.; Donaldson, Alan D.

    1991-01-01

    A method of measuring a molten metal liquid pool volume and in particular molten titanium liquid pools, including the steps of (a) generating an ultrasonic wave at the surface of the molten metal liquid pool, (b) shining a light on the surface of a molten metal liquid pool, (c) detecting a change in the frequency of light, (d) detecting an ultrasonic wave echo at the surface of the molten metal liquid pool, and (e) computing the volume of the molten metal liquid.

  14. Protective vaccination against experimental canine visceral leishmaniasis using a combination of DNA and protein immunization with cysteine proteinases type I and II of L. infantum.

    PubMed

    Rafati, Sima; Nakhaee, Alireza; Taheri, Tahere; Taslimi, Yasaman; Darabi, Haideh; Eravani, Davood; Sanos, Stephanie; Kaye, Paul; Taghikhani, Mohammad; Jamshidi, Shahram; Rad, Mohammad Ali

    2005-05-25

    Leishmania infantum is known to be associated with visceral leishmaniasis in Iran and canids are natural reservoirs. Control of disease in dogs appears to be one of the most effective approaches for interrupting the domestic cycle of the disease. In search for successful vaccine strategies, we evaluated the cysteine proteinases (CPs) type I and II using a heterologous prime-boost regime for vaccination against experimental visceral leishmaniasis in dogs. Following vaccination and challenge, dogs were followed for 12 months. Ten dogs vaccinated by prime/boost with DNA/recombinant CPs (in combination with CpG ODN and Montanide 720) remained free of infection in their bone morrow. In contrast, three out of four dogs in the control groups had infection in their bone marrow. The peripheral lymphocytes from protected animals had generally higher proliferation responses to F/T antigen, recombinant CPA (rCPA) and recombinant CPB (rCPB) than controls. During post-challenge period, the difference in stimulation index is significant (p<0.05) on months 11 and 12 to F/T antigens, all months for rCPA and 5, 7, 9, 11 and 12 months for rCPB. Analysis of cytokine mRNA level suggested that vaccinated dogs had elevated IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC), whereas there was a consistent increase in the level of IL-10 in the control groups and some vaccinated dogs. The level of total IgG and IgG2, but not IgG1, to rCPA and rCPB was significantly higher in the vaccinated group (p<0.05) than the control groups. We also showed that with the exception of one dog, all dogs in the vaccinated group in comparison to control dogs had strong DTH responses. We propose that the combination of DNA and recombinant protein vaccination using CPs could be instrumental to control (VL) in dogs. PMID:15882533

  15. Triploblastic relationships with emphasis on the acoelomates and the position of Gnathostomulida, Cycliophora, Plathelminthes, and Chaetognatha: a combined approach of 18S rDNA sequences and morphology.

    PubMed

    Giribet, G; Distel, D L; Polz, M; Sterrer, W; Wheeler, W C

    2000-09-01

    Triploblastic relationships were examined in the light of molecular and morphological evidence. Representatives for all triploblastic "phyla" (except Loricifera) were represented by both sources of phylogenetic data. The 18S ribosomal (rDNA) sequence data for 145 terminal taxa and 276 morphological characters coded for 36 supraspecific taxa were combined in a total evidence regime to determine the most consistent picture of triploblastic relationships for these data. Only triploblastic taxa are used to avoid rooting with distant outgroups, which seems to happen because of the extreme distance that separates diploblastic from triploblastic taxa according to the 18S rDNA data. Multiple phylogenetic analyses performed with variable analysis parameters yield largely inconsistent results for certain groups such as Chaetognatha, Acoela, and Nemertodermatida. A normalized incongruence length metric is used to assay the relative merit of the multiple analyses. The combined analysis having the least character incongruence yields the following scheme of relationships of four main clades: (1) Deuterostomia [((Echinodermata + Enteropneusta) (Cephalochordata (Urochordata + Vertebrata)))]; (2) Ecdysozoa [(((Priapulida + Kinorhyncha) (Nematoda + Nematomorpha)) ((Onychophora + Tardigrada) Arthropoda))]; (3) Trochozoa [((Phoronida + Brachiopoda) (Entoprocta (Nemertea (Sipuncula (Mollusca (Pogonophora (Echiura + Annelida)))))))]; and (4) Platyzoa [((Gnathostomulida (Cycliophora + Syndermata)) (Gastrotricha + Plathelminthes))]. Chaetognatha, Nemertodermatida, and Bryozoa cannot be assigned to any one of these four groups. For the first time, a data analysis recognizes a clade of acoelomates, the Platyzoa (sensu Cavalier-Smith, Biol. Rev. 73:203-266, 1998). Other relationships that corroborate some morphological analyses are the existence of a clade that groups Gnathostomulida + Syndermata (= Gnathifera), which is expanded to include the enigmatic phylum Cycliophora, as sister group

  16. Combined Quantification of Pulmonary Pneumocystis jirovecii DNA and Serum (1→3)-β-d-Glucan for Differential Diagnosis of Pneumocystis Pneumonia and Pneumocystis Colonization

    PubMed Central

    Le Gal, Solène; Da Costa, Cécilia; Virmaux, Michèle; Nevez, Gilles; Totet, Anne

    2013-01-01

    This study assessed a quantitative PCR (qPCR) assay for Pneumocystis jirovecii quantification in bronchoalveolar lavage (BAL) fluid samples combined with serum (1→3)-β-d-glucan (BG) level detection to distinguish Pneumocystis pneumonia (PCP) from pulmonary colonization with P. jirovecii. Forty-six patients for whom P. jirovecii was initially detected in BAL fluid samples were retrospectively enrolled. Based on clinical data and results of P. jirovecii detection, 17 and 29 patients were diagnosed with PCP and colonization, respectively. BAL fluid samples were reassayed using a qPCR assay targeting the mitochondrial large subunit rRNA gene. qPCR results and serum BG levels (from a Fungitell kit) were analyzed conjointly. P. jirovecii DNA copy numbers were significantly higher in the PCP group than in the colonization group (1.3 × 107 versus 3.4 × 103 copies/μl, P < 0.05). A lower cutoff value (1.6 × 103 copies/μl) achieving 100% sensitivity for PCP diagnosis and an upper cutoff value (2 × 104 copies/μl) achieving 100% specificity were determined. Applying these two values, 13/17 PCP patients and 19/29 colonized patients were correctly assigned to their patient groups. For the remaining 14 patients with P. jirovecii DNA copy numbers between the cutoff values, PCP and colonization could not be distinguished on the basis of qPCR results. Four of these patients who were initially assigned to the PCP group presented BG levels of ≥100 pg/ml. The other 10 patients, who were initially assigned to the colonization group, presented BG levels of <100 pg/ml. These results suggest that the combination of the qPCR assay, applying cutoff values of 1.6 × 103 and 2 × 104 copies/μl, and serum BG detection, applying a 100 pg/ml threshold, can differentiate PCP and colonization diagnoses. PMID:23903553

  17. UV induced ubiquitination of the yeast Rad4-Rad23 complex promotes survival by regulating cellular dNTP pools.

    PubMed

    Zhou, Zheng; Humphryes, Neil; van Eijk, Patrick; Waters, Raymond; Yu, Shirong; Kraehenbuehl, Rolf; Hartsuiker, Edgar; Reed, Simon H

    2015-09-01

    Regulating gene expression programmes is a central facet of the DNA damage response. The Dun1 kinase protein controls expression of many DNA damage induced genes, including the ribonucleotide reductase genes, which regulate cellular dNTP pools. Using a combination of gene expression profiling and chromatin immunoprecipitation, we demonstrate that in the absence of DNA damage the yeast Rad4-Rad23 nucleotide excision repair complex binds to the promoters of certain DNA damage response genes including DUN1, inhibiting their expression. UV radiation promotes the loss of occupancy of the Rad4-Rad23 complex from the regulatory regions of these genes, enabling their induction and thereby controlling the production of dNTPs. We demonstrate that this regulatory mechanism, which is dependent on the ubiquitination of Rad4 by the GG-NER E3 ligase, promotes UV survival in yeast cells. These results support an unanticipated regulatory mechanism that integrates ubiquitination of NER DNA repair factors with the regulation of the transcriptional response controlling dNTP production and cellular survival after UV damage. PMID:26150418

  18. Flame spread across liquid pools

    NASA Technical Reports Server (NTRS)

    Ross, Howard; Miller, Fletcher; Schiller, David; Sirignano, William A.

    1993-01-01

    For flame spread over liquid fuel pools, the existing literature suggests three gravitational influences: (1) liquid phase buoyant convection, delaying ignition and assisting flame spread; (2) hydrostatic pressure variation, due to variation in the liquid pool height caused by thermocapillary-induced convection; and (3) gas-phase buoyant convection in the opposite direction to the liquid phase motion. No current model accounts for all three influences. In fact, prior to this work, there was no ability to determine whether ignition delay times and flame spread rates would be greater or lesser in low gravity. Flame spread over liquid fuel pools is most commonly characterized by the relationship of the initial pool temperature to the fuel's idealized flash point temperature, with four or five separate characteristic regimes having been identified. In the uniform spread regime, control has been attributed to: (1) gas-phase conduction and radiation; (2) gas-phase conduction only; (3) gas-phase convection and liquid conduction, and most recently (4) liquid convection ahead of the flame. Suggestions were made that the liquid convection was owed to both vuoyancy and thermocapillarity. Of special interest to this work is the determination of whether, and under what conditions, pulsating spread can and will occur in microgravity in the absence of buoyant flows in both phases. The approach we have taken to resolving the importance of buoyancy for these flames is: (1) normal gravity experiments and advanced diagnostics; (2) microgravity experiments; and (3) numerical modelling at arbitrary gravitational level.

  19. Evolution of the assassin's arms: insights from a phylogeny of combined transcriptomic and ribosomal DNA data (Heteroptera: Reduvioidea).

    PubMed

    Zhang, Junxia; Gordon, Eric R L; Forthman, Michael; Hwang, Wei Song; Walden, Kim; Swanson, Daniel R; Johnson, Kevin P; Meier, Rudolf; Weirauch, Christiane

    2016-01-01

    Assassin bugs (Reduvioidea) are one of the most diverse (>7,000 spp.) lineages of predatory animals and have evolved an astounding diversity of raptorial leg modifications for handling prey. The evolution of these modifications is not well understood due to the lack of a robust phylogeny, especially at deeper nodes. We here utilize refined data from transcriptomes (370 loci) to stabilize the backbone phylogeny of Reduvioidea, revealing the position of major clades (e.g., the Chagas disease vectors Triatominae). Analyses combining transcriptomic and Sanger-sequencing datasets result in the first well-resolved phylogeny of Reduvioidea. Despite amounts of missing data, the transcriptomic loci resolve deeper nodes while the targeted ribosomal genes anchor taxa at shallower nodes, both with high support. This phylogeny reveals patterns of raptorial leg evolution across major leg types. Hairy attachment structures (fossula spongiosa), present in the ancestor of Reduvioidea, were lost multiple times within the clade. In contrast to prior hypotheses, this loss is not directly correlated with the evolution of alternative raptorial leg types. Our results suggest that prey type, predatory behavior, salivary toxicity, and morphological adaptations pose intricate and interrelated factors influencing the evolution of this diverse group of predators. PMID:26916580

  20. Evolution of the assassin’s arms: insights from a phylogeny of combined transcriptomic and ribosomal DNA data (Heteroptera: Reduvioidea)

    PubMed Central

    Zhang, Junxia; Gordon, Eric R. L.; Forthman, Michael; Hwang, Wei Song; Walden, Kim; Swanson, Daniel R.; Johnson, Kevin P.; Meier, Rudolf; Weirauch, Christiane

    2016-01-01

    Assassin bugs (Reduvioidea) are one of the most diverse (>7,000 spp.) lineages of predatory animals and have evolved an astounding diversity of raptorial leg modifications for handling prey. The evolution of these modifications is not well understood due to the lack of a robust phylogeny, especially at deeper nodes. We here utilize refined data from transcriptomes (370 loci) to stabilize the backbone phylogeny of Reduvioidea, revealing the position of major clades (e.g., the Chagas disease vectors Triatominae). Analyses combining transcriptomic and Sanger-sequencing datasets result in the first well-resolved phylogeny of Reduvioidea. Despite amounts of missing data, the transcriptomic loci resolve deeper nodes while the targeted ribosomal genes anchor taxa at shallower nodes, both with high support. This phylogeny reveals patterns of raptorial leg evolution across major leg types. Hairy attachment structures (fossula spongiosa), present in the ancestor of Reduvioidea, were lost multiple times within the clade. In contrast to prior hypotheses, this loss is not directly correlated with the evolution of alternative raptorial leg types. Our results suggest that prey type, predatory behavior, salivary toxicity, and morphological adaptations pose intricate and interrelated factors influencing the evolution of this diverse group of predators. PMID:26916580

  1. Monitoring pool-tail fines

    NASA Astrophysics Data System (ADS)

    Bunte, K.; Potyondy, J. P.; Abt, S. R.; Swingle, K. W.

    2010-12-01

    Fine sediment < 2 and < 6 mm deposited in pool-tail areas of mountain streams is often measured to monitor changes in the supply of fines (e.g., by dam removal, bank erosion, or watershed effects including fires and road building) or to assess the status and trend of aquatic ecosystems. Grid counts, pebble counts, and volumetric bedmaterial samples are typically used to quantify pool-tail fines. Grid-count results exhibit a high degree of variability not only among streams and among operators, but also among crews performing a nearly identical procedure (Roper et al. 2010). Variability is even larger when diverse methods are employed, each of which quantifies fines in a different way: grid counts visually count surface fines on small patches within the pool-tail area, pebble counts pick up and tally surface particles along (riffle) transects, and volumetric samples sieve out fines from small-scale bulk samples; and even when delimited to pool-tail areas, individual methods focus on different sampling locales. Two main questions were analyzed: 1) Do pool-tail fines exhibit patterns of spatial variability and are some grid count schemes more likely to provide accurate results than others. 2) How and why does the percentage of fines vary among grid counts, pebble counts, and volumetric samples. In a field study, grids were placed at 7 locales in two rows across the wetted width of 10 pool tails in a 14-m wide 3rd order coarse gravel-bed mountain stream with <4% sand and <8% < 6 mm. Several pebble count transects were placed across each pool-tail area, and three volumetric samples were collected in each of three pool tails. Pebble and grid counts both indicated a fining trend towards one or both banks, sometimes interrupted by a secondary peak of fines within the central half of the wetted width. Among the five sampling schemes tested, grid counts covering the wetted width with 7 locales produced the highest accuracy and the least variability among the pools of the

  2. Interactions between pool geometry and hydraulics

    USGS Publications Warehouse

    Thompson, D.M.; Nelson, J.M.; Wohl, E.E.

    1998-01-01

    An experimental and computational research approach was used to determine interactions between pool geometry and hydraulics. A 20-m-long, 1.8-m-wide flume was used to investigate the effect of four different geometric aspects of pool shape on flow velocity. Plywood sections were used to systematically alter constriction width, pool depth, pool length, and pool exit-slope gradient, each at two separate levels. Using the resulting 16 unique geometries with measured pool velocities in four-way factorial analyses produced an empirical assessment of the role of the four geometric aspects on the pool flow patterns and hence the stability of the pool. To complement the conclusions of these analyses, a two-dimensional computational flow model was used to investigate the relationships between pool geometry and flow patterns over a wider range of conditions. Both experimental and computational results show that constriction and depth effects dominate in the jet section of the pool and that pool length exhibits an increasing effect within the recirculating-eddy system. The pool exit slope appears to force flow reattachment. Pool length controls recirculating-eddy length and vena contracta strength. In turn, the vena contracta and recirculating eddy control velocities throughout the pool.

  3. Statistical methodologies to pool across multiple intervention studies.

    PubMed

    Bangdiwala, Shrikant I; Bhargava, Alok; O'Connor, Daniel P; Robinson, Thomas N; Michie, Susan; Murray, David M; Stevens, June; Belle, Steven H; Templin, Thomas N; Pratt, Charlotte A

    2016-06-01

    Combining and analyzing data from heterogeneous randomized controlled trials of complex multiple-component intervention studies, or discussing them in a systematic review, is not straightforward. The present article describes certain issues to be considered when combining data across studies, based on discussions in an NIH-sponsored workshop on pooling issues across studies in consortia (see Belle et al. in Psychol Aging, 18(3):396-405, 2003). Several statistical methodologies are described and their advantages and limitations are explored. Whether weighting the different studies data differently, or via employing random effects, one must recognize that different pooling methodologies may yield different results. Pooling can be used for comprehensive exploratory analyses of data from RCTs and should not be viewed as replacing the standard analysis plan for each study. Pooling may help to identify intervention components that may be more effective especially for subsets of participants with certain behavioral characteristics. Pooling, when supported by statistical tests, can allow exploratory investigation of potential hypotheses and for the design of future interventions. PMID:27356993

  4. Predicted sub-populations in a marine shrimp proteome as revealed by combined EST and cDNA data from multiple Penaeus species

    PubMed Central

    2010-01-01

    Background Many species of marine shrimp in the Family Penaeidae, viz. Penaeus (Litopenaeus) vannamei, Penaeus monodon, Penaeus (Fenneropenaeus) chinensis, and Penaeus (Marsupenaeus) japonicus, are animals of economic importance in the aquaculture industry. Yet information about their DNA and protein sequences is lacking. In order to predict their collective proteome, we combined over 270,000 available EST and cDNA sequences from the 4 shrimp species with all protein sequences of Drosophila melanogaster and Caenorhabditis elegans. EST data from 4 other crustaceans, the crab Carcinus maenas, the lobster Homarus americanus (Decapoda), the water flea Daphnia pulex, and the brine shrimp Artemia franciscana were also used. Findings Similarity searches from EST collections of the 4 shrimp species matched 64% of the protein sequences of the fruit fly, but only 45% of nematode proteins, indicating that the shrimp proteome content is more similar to that of an insect than a nematode. Combined results with 4 additional non-shrimp crustaceans increased matching to 78% of fruit fly and 56% of nematode proteins, suggesting that present shrimp EST collections still lack sequences for many conserved crustacean proteins. Analysis of matching data revealed the presence of 4 EST groups from shrimp, namely sequences for proteins that are both fruit fly-like and nematode-like, fruit fly-like only, nematode-like only, and non-matching. Gene ontology profiles of proteins for the 3 matching EST groups were analyzed. For non-matching ESTs, a small fraction matched protein sequences from other species in the UniProt database, including other crustacean-specific proteins. Conclusions Shrimp ESTs indicated that the shrimp proteome is comprised of sub-populations of proteins similar to those common to both insect and nematode models, those present specifically in either model, or neither. Combining small EST collections from related species to compensate for their small size allowed

  5. Monte Carlo Test Assembly for Item Pool Analysis and Extension

    ERIC Educational Resources Information Center

    Belov, Dmitry I.; Armstrong, Ronald D.

    2005-01-01

    A new test assembly algorithm based on a Monte Carlo random search is presented in this article. A major advantage of the Monte Carlo test assembly over other approaches (integer programming or enumerative heuristics) is that it performs a uniform sampling from the item pool, which provides every feasible item combination (test) with an equal…

  6. Genomics of crop wild relatives: expanding the gene pool for crop improvement.

    PubMed

    Brozynska, Marta; Furtado, Agnelo; Henry, Robert J

    2016-04-01

    Plant breeders require access to new genetic diversity to satisfy the demands of a growing human population for more food that can be produced in a variable or changing climate and to deliver the high-quality food with nutritional and health benefits demanded by consumers. The close relatives of domesticated plants, crop wild relatives (CWRs), represent a practical gene pool for use by plant breeders. Genomics of CWR generates data that support the use of CWR to expand the genetic diversity of crop plants. Advances in DNA sequencing technology are enabling the efficient sequencing of CWR and their increased use in crop improvement. As the sequencing of genomes of major crop species is completed, attention has shifted to analysis of the wider gene pool of major crops including CWR. A combination of de novo sequencing and resequencing is required to efficiently explore useful genetic variation in CWR. Analysis of the nuclear genome, transcriptome and maternal (chloroplast and mitochondrial) genome of CWR is facilitating their use in crop improvement. Genome analysis results in discovery of useful alleles in CWR and identification of regions of the genome in which diversity has been lost in domestication bottlenecks. Targeting of high priority CWR for sequencing will maximize the contribution of genome sequencing of CWR. Coordination of global efforts to apply genomics has the potential to accelerate access to and conservation of the biodiversity essential to the sustainability of agriculture and food production. PMID:26311018

  7. Bile acids in combination with low pH induce oxidative stress and oxidative DNA damage: relevance to the pathogenesis of Barrett's oesophagus

    PubMed Central

    Dvorak, Katerina; Payne, Claire M; Chavarria, Melissa; Ramsey, Lois; Dvorakova, Barbora; Bernstein, Harris; Holubec, Hana; Sampliner, Richard E; Guy, Naihsuan; Condon, Amanda; Bernstein, Carol; Green, Sylvan B; Prasad, Anil; Garewal, Harinder S

    2007-01-01

    Background Barrett's oesophagus is a premalignant condition associated with an increased risk for the development of oesophageal adenocarcinoma (ADCA). Previous studies indicated that oxidative damage contributes to the development of ADCA. Objective To test the hypothesis that bile acids and gastric acid, two components of refluxate, can induce oxidative stress and oxidative DNA damage. Methods Oxidative stress was evaluated by staining Barrett's oesophagus tissues with different degrees of dysplasia with 8‐hydroxy‐deoxyguanosine (8‐OH‐dG) antibody. The levels of 8‐OH‐dG were also evaluated ex vivo in Barrett's oesophagus tissues incubated for 10 min with control medium and medium acidified to pH 4 and supplemented with 0.5 mM bile acid cocktail. Furthermore, three oesophageal cell lines (Seg‐1 cells, Barrett's oesophagus cells and HET‐1A cells) were exposed to control media, media containing 0.1 mM bile acid cocktail, media acidified to pH 4, and media at pH 4 supplemented with 0.1 mM bile acid cocktail, and evaluated for induction of reactive oxygen species (ROS). Results Immunohistochemical analysis showed that 8‐OH‐dG is formed mainly in the epithelial cells in dysplastic Barrett's oesophagus. Importantly, incubation of Barrett's oesophagus tissues with the combination of bile acid cocktail and acid leads to increased formation of 8‐OH‐dG. An increase in ROS in oesophageal cells was detected after exposure to pH 4 and bile acid cocktail. Conclusions Oxidative stress and oxidative DNA damage can be induced in oesophageal tissues and cells by short exposures to bile acids and low pH. These alterations may underlie the development of Barrett's oesophagus and tumour progression. PMID:17145738

  8. γ-H2AX responds to DNA damage induced by long-term exposure to combined low-dose-rate neutron and γ-ray radiation.

    PubMed

    Zhang, Junlin; He, Ying; Shen, Xianrong; Jiang, Dingwen; Wang, Qingrong; Liu, Qiong; Fang, Wen

    2016-01-01

    Risk estimates for low-dose radiation (LDR) remain controversial. The possible involvement of DNA repair-related genes in long-term low-dose-rate neutron-gamma radiation exposure is poorly understood. In this study, 60 rats were divided into control groups and irradiated groups, which were exposed to low-dose-rate n-γ combined radiation (LDCR) for 15, 30, or 60 days. The effects of different cumulative radiation doses on peripheral blood cell (PBC), subsets of T cells of peripheral blood lymphocytes (PBL) and DNA damage repair were investigated. Real-time PCR and immunoblot analyses were used to detect expression of DNA DSB-repair-related genes involved in the NHEJ pathway, such as Ku70 and Ku80, in PBL. The mRNA level of H2AX and the expression level of γ-H2AX were detected by real-time PCR, immunoblot, and flow cytometry. White blood cells (WBC) and platelets (PLT) of all ionizing radiation (IR) groups decreased significantly, while no difference was seen between the 30 day and 60 day exposure groups. The numbers of CD3(+), CD4(+) T cells and CD4(+)/CD8(+) in the PBL of IR groups were lower than in the control group. In the 30 day and 60 day exposure groups, CD8(+) T cells decreased significantly. Real-time PCR and immunoblot results showed no significant difference in the mRNA and protein expression of Ku70 and Ku80 between the control groups and IR groups. However, the mRNA of H2AX increased significantly, and there was a positive correlation with dose. There was no difference in the protein expression of γ-H2AX between 30 day and 60 day groups, which may help to explain the damage to PBL. In conclusion, PBL damage increased with cumulative dose, suggesting that γ-H2AX, but neither Ku70 nor Ku80, plays an important role in PBL impairment induced by LDCR. PMID:26774665

  9. Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes

    PubMed Central

    Arunrut, Narong; Kampeera, Jantana; Sirithammajak, Sarawut; Sanguanrut, Piyachat; Proespraiwong, Porranee; Suebsing, Rungkarn; Kiatpathomchai, Wansika

    2016-01-01

    Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non

  10. Safety profile of statins alone or combined with ezetimibe: a pooled analysis of 27 studies including over 22,000 patients treated for 6-24 weeks.

    PubMed

    Toth, P P; Morrone, D; Weintraub, W S; Hanson, M E; Lowe, R S; Lin, J; Shah, A K; Tershakovec, A M

    2012-08-01

    Aims:  The aim of this analysis was to assess the overall safety and tolerability profiles of various statins + ezetimibe vs. statin monotherapy and to explore tolerability in sub-populations grouped by age, race, and sex. Methods:  Study-level data were combined from 27 double-blind, placebo-controlled or active-comparator trials that randomized adult hypercholesterolemic patients to statin or statin + ezetimibe for 6-24 weeks. In the full cohort, % patients with AEs within treatment groups (statin: N = 10,517; statin + ezetimibe: N = 11,714) was assessed by logistic regression with terms for first-/second-line therapy (first line = drug-naïve or rendered drug-naïve by washout at study entry; second line = ongoing statin at study entry or statin run-in), trial within first-/second-line therapy, and treatment. The same model was fitted for age (< 65, ≥ 65 years), sex, race (white, black, other) and first-/second-line subgroups with additional terms for subgroup and subgroup-by-treatment interaction. Results:  In the full cohort, the only significant difference between treatments was consecutive AST or ALT elevations ≥ 3 × upper limit of normal (ULN) (statin: 0.35%, statin + ezetimibe: 0.56%; p = 0.017). Significantly more subjects reported ≥ 1 AE; drug-related, hepatitis-related and gastrointestinal-related AEs; and CK elevations ≥ 10 × ULN (all p ≤ 0.008) in first-line vs. second-line therapy studies with both treatments. AEs were generally similar between treatments in subgroups, and similar rates of AEs were reported within age and race subgroups; however, women reported generally higher AE rates. Conclusions:  In conclusion, in second-line studies, ongoing statin treatment at study entry likely screened out participants for previous statin-related AEs and tolerability issues. These results describe the safety profiles of widely used lipid-lowering therapies and encourage their

  11. Pool Safety: A Few Simple Rules.

    ERIC Educational Resources Information Center

    PTA Today, 1993

    1993-01-01

    Presents suggestions by the National Swimming Pool Safety Committee on how to keep children safe while swimming. Ideas include maintaining strict adult supervision, pool and spa barriers, and knowledge of cardiopulmonary resuscitation. (SM)

  12. Cold Pools in the Columbia Basin

    SciTech Connect

    Whiteman, Charles D.; Zhong, Shiyuan; Shaw, William J.; Hubbe, John M.; Bian, Xindi; Mittelstadt, J.

    2001-01-01

    Persistent midwinter cold air pools produce multi-day periods of cold, dreary weather in valleys and basins. Persistent stable stratification leads to the buildup of pollutants and moisture in the pool. Because the pool sometimes has temperatures below freezing while the air above is warmer, freezing precipitation often occurs with consequent effects on transportation and safety. Forecasting the buildup and breakdown of these cold pools is difficult because the physical mechanisms leading to their formation, maintenance, and destruction have received little study. This paper provides a succinct meteorological definition of a cold pool, develops a climatology of Columbia Basin cold pools, and analyzes remote and in situ temperature and wind sounding data for two winter cold pool episodes that were accompanied by fog and stratus, illustrating many of the physical mechanisms affecting cold pool evolution.

  13. Pool power control in remelting systems

    SciTech Connect

    Williamson, Rodney L.; Melgaard, David K.; Beaman, Joseph J.

    2011-12-13

    An apparatus for and method of controlling a remelting furnace comprising adjusting current supplied to an electrode based upon a predetermined pool power reference value and adjusting the electrode drive speed based upon the predetermined pool power reference value.

  14. CDC Study Finds Fecal Contamination in Pools

    MedlinePlus

    ... Communication (404) 639-3286 CDC study finds fecal contamination in pools A study of public pools done ... The E. coli is a marker for fecal contamination. Finding a high percentage of E. coli-positive ...

  15. Improved PCR Amplification of Broad Spectrum GC DNA Templates

    PubMed Central

    Guido, Nicholas; Starostina, Elena; Leake, Devin; Saaem, Ishtiaq

    2016-01-01

    Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10–90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content. PMID:27271574

  16. Combined quantification of pulmonary Pneumocystis jirovecii DNA and serum (1->3)-β-D-glucan for differential diagnosis of pneumocystis pneumonia and Pneumocystis colonization.

    PubMed

    Damiani, Céline; Le Gal, Solène; Da Costa, Cécilia; Virmaux, Michèle; Nevez, Gilles; Totet, Anne

    2013-10-01

    This study assessed a quantitative PCR (qPCR) assay for Pneumocystis jirovecii quantification in bronchoalveolar lavage (BAL) fluid samples combined with serum (1→3)-β-d-glucan (BG) level detection to distinguish Pneumocystis pneumonia (PCP) from pulmonary colonization with P. jirovecii. Forty-six patients for whom P. jirovecii was initially detected in BAL fluid samples were retrospectively enrolled. Based on clinical data and results of P. jirovecii detection, 17 and 29 patients were diagnosed with PCP and colonization, respectively. BAL fluid samples were reassayed using a qPCR assay targeting the mitochondrial large subunit rRNA gene. qPCR results and serum BG levels (from a Fungitell kit) were analyzed conjointly. P. jirovecii DNA copy numbers were significantly higher in the PCP group than in the colonization group (1.3 × 10(7) versus 3.4 × 10(3) copies/μl, P < 0.05). A lower cutoff value (1.6 × 10(3) copies/μl) achieving 100% sensitivity for PCP diagnosis and an upper cutoff value (2 × 10(4) copies/μl) achieving 100% specificity were determined. Applying these two values, 13/17 PCP patients and 19/29 colonized patients were correctly assigned to their patient groups. For the remaining 14 patients with P. jirovecii DNA copy numbers between the cutoff values, PCP and colonization could not be distinguished on the basis of qPCR results. Four of these patients who were initially assigned to the PCP group presented BG levels of ≥100 pg/ml. The other 10 patients, who were initially assigned to the colonization group, presented BG levels of <100 pg/ml. These results suggest that the combination of the qPCR assay, applying cutoff values of 1.6 × 10(3) and 2 × 10(4) copies/μl, and serum BG detection, applying a 100 pg/ml threshold, can differentiate PCP and colonization diagnoses. PMID:23903553

  17. Swimming pools soak up the sun

    SciTech Connect

    Cuoghi, D.; Hesse, P.; Schiller, T.

    1996-05-01

    Solar pool heaters survived the boom and bust solar years of the 1970s and 1980s. Today they are even popular and cost-effective in parts of the country where many people think solar is impractical. This article discusses the following topics: how solar pool heaters work; types of solar pool heater collectors; collector and pump sizing; collector siting and mounting; systems costs and economics; pool covers. 3 figs.

  18. 1968 Listing of Swimming Pool Equipment.

    ERIC Educational Resources Information Center

    National Sanitation Foundation, Ann Arbor, MI. Testing Lab.

    An up-to-date listing of swimming pool equipment including--(1) companies authorized to display the National Sanitation Foundation seal of approval, (2) equipment listed as meeting NSF swimming pool equipment standards relating to diatomite type filters, (3) equipment listed as meeting NSF swimming pool equipment standard relating to sand type…

  19. Apparatus for heating a swimming pool

    SciTech Connect

    Kremen, R.D.

    1983-09-06

    This disclosure relates to a solar heater apparatus for a swimming pool which incorporates a submersible suspendible black body sheet to serve as a device to absorb solar radiation and transfer the collected energy to the pool water so that the pool water can be efficiently heated.

  20. 7 CFR 1131.7 - Pool plant.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1131.7 Section 1131.7 Agriculture... Handling Definitions § 1131.7 Pool plant. Pool Plant means a plant or unit of plants specified in paragraphs (a) through (e) of this section, but excluding a plant specified in paragraph (g) of this...

  1. 7 CFR 1001.7 - Pool plant.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1001.7 Section 1001.7 Agriculture... Handling Definitions § 1001.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant described in paragraph (h)...

  2. 7 CFR 1126.7 - Pool plant.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1126.7 Section 1126.7 Agriculture... Handling Definitions § 1126.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  3. 7 CFR 1005.7 - Pool plant.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1005.7 Section 1005.7 Agriculture... Handling Definitions § 1005.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  4. 7 CFR 1007.7 - Pool plant.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1007.7 Section 1007.7 Agriculture... Handling Definitions § 1007.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  5. 7 CFR 1001.7 - Pool plant.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1001.7 Section 1001.7 Agriculture... Handling Definitions § 1001.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant described in paragraph (h)...

  6. 7 CFR 1030.7 - Pool plant.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1030.7 Section 1030.7 Agriculture... Handling Definitions § 1030.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified in paragraph (h)...

  7. 7 CFR 1006.7 - Pool plant.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1006.7 Section 1006.7 Agriculture... Handling Definitions § 1006.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  8. 7 CFR 1124.7 - Pool plant.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1124.7 Section 1124.7 Agriculture... Regulating Handling Definitions § 1124.7 Pool plant. Pool plant means a plant, unit of plants, or a system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified...

  9. 7 CFR 1032.7 - Pool plant.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1032.7 Section 1032.7 Agriculture... Handling Definitions § 1032.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (i) of...

  10. 7 CFR 1033.7 - Pool plant.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1033.7 Section 1033.7 Agriculture... Handling Definitions § 1033.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (j) of...

  11. 7 CFR 1006.7 - Pool plant.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1006.7 Section 1006.7 Agriculture... Handling Definitions § 1006.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  12. 7 CFR 1033.7 - Pool plant.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1033.7 Section 1033.7 Agriculture... Handling Definitions § 1033.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (j) of...

  13. 7 CFR 1007.7 - Pool plant.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1007.7 Section 1007.7 Agriculture... Handling Definitions § 1007.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  14. 7 CFR 1032.7 - Pool plant.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1032.7 Section 1032.7 Agriculture... Handling Definitions § 1032.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (i) of...

  15. 7 CFR 1030.7 - Pool plant.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1030.7 Section 1030.7 Agriculture... Handling Definitions § 1030.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified in paragraph (h)...

  16. 7 CFR 1005.7 - Pool plant.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1005.7 Section 1005.7 Agriculture... Handling Definitions § 1005.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  17. 7 CFR 1126.7 - Pool plant.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1126.7 Section 1126.7 Agriculture... Handling Definitions § 1126.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  18. 7 CFR 1030.7 - Pool plant.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1030.7 Section 1030.7 Agriculture... Handling Definitions § 1030.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified in paragraph (h)...

  19. 7 CFR 1007.7 - Pool plant.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1007.7 Section 1007.7 Agriculture... Handling Definitions § 1007.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  20. 7 CFR 1001.7 - Pool plant.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1001.7 Section 1001.7 Agriculture... Handling Definitions § 1001.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant described in paragraph (h)...

  1. 7 CFR 1131.7 - Pool plant.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1131.7 Section 1131.7 Agriculture... Handling Definitions § 1131.7 Pool plant. Pool Plant means a plant or unit of plants specified in paragraphs (a) through (e) of this section, but excluding a plant specified in paragraph (g) of this...

  2. 7 CFR 1001.7 - Pool plant.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1001.7 Section 1001.7 Agriculture... Handling Definitions § 1001.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant described in paragraph (h)...

  3. 7 CFR 1124.7 - Pool plant.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1124.7 Section 1124.7 Agriculture... Regulating Handling Definitions § 1124.7 Pool plant. Pool plant means a plant, unit of plants, or a system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified...

  4. 7 CFR 1131.7 - Pool plant.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1131.7 Section 1131.7 Agriculture... Handling Definitions § 1131.7 Pool plant. Pool Plant means a plant or unit of plants specified in paragraphs (a) through (e) of this section, but excluding a plant specified in paragraph (g) of this...

  5. 7 CFR 1030.7 - Pool plant.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1030.7 Section 1030.7 Agriculture... Handling Definitions § 1030.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified in paragraph (h)...

  6. 7 CFR 1007.7 - Pool plant.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1007.7 Section 1007.7 Agriculture... Handling Definitions § 1007.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  7. 7 CFR 1032.7 - Pool plant.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1032.7 Section 1032.7 Agriculture... Handling Definitions § 1032.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (i) of...

  8. 7 CFR 1006.7 - Pool plant.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1006.7 Section 1006.7 Agriculture... Handling Definitions § 1006.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  9. 7 CFR 1006.7 - Pool plant.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1006.7 Section 1006.7 Agriculture... Handling Definitions § 1006.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  10. 7 CFR 1126.7 - Pool plant.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1126.7 Section 1126.7 Agriculture... Handling Definitions § 1126.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  11. 7 CFR 1005.7 - Pool plant.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1005.7 Section 1005.7 Agriculture... Handling Definitions § 1005.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  12. 7 CFR 1033.7 - Pool plant.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1033.7 Section 1033.7 Agriculture... Handling Definitions § 1033.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (j) of...

  13. 7 CFR 1005.7 - Pool plant.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1005.7 Section 1005.7 Agriculture... Handling Definitions § 1005.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  14. 7 CFR 1007.7 - Pool plant.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1007.7 Section 1007.7 Agriculture... Handling Definitions § 1007.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  15. 7 CFR 1126.7 - Pool plant.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1126.7 Section 1126.7 Agriculture... Handling Definitions § 1126.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  16. 7 CFR 1006.7 - Pool plant.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1006.7 Section 1006.7 Agriculture... Handling Definitions § 1006.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  17. 7 CFR 1124.7 - Pool plant.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1124.7 Section 1124.7 Agriculture... Regulating Handling Definitions § 1124.7 Pool plant. Pool plant means a plant, unit of plants, or a system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified...

  18. 7 CFR 1033.7 - Pool plant.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1033.7 Section 1033.7 Agriculture... Handling Definitions § 1033.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (j) of...

  19. 7 CFR 1005.7 - Pool plant.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1005.7 Section 1005.7 Agriculture... Handling Definitions § 1005.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant...

  20. 7 CFR 1032.7 - Pool plant.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1032.7 Section 1032.7 Agriculture... Handling Definitions § 1032.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (i) of...

  1. 7 CFR 1124.7 - Pool plant.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1124.7 Section 1124.7 Agriculture... Regulating Handling Definitions § 1124.7 Pool plant. Pool plant means a plant, unit of plants, or a system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified...

  2. 7 CFR 1131.7 - Pool plant.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1131.7 Section 1131.7 Agriculture... Handling Definitions § 1131.7 Pool plant. Pool Plant means a plant or unit of plants specified in paragraphs (a) through (e) of this section, but excluding a plant specified in paragraph (g) of this...

  3. 7 CFR 1033.7 - Pool plant.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... its route distribution in this marketing area for 3 consecutive months or if the plant is required to... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1033.7 Section 1033.7 Agriculture... Handling Definitions § 1033.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants...

  4. 7 CFR 1030.7 - Pool plant.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... its route distribution in this marketing area for 3 consecutive months or if the plant is required to... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1030.7 Section 1030.7 Agriculture... Handling Definitions § 1030.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants...

  5. 7 CFR 1131.7 - Pool plant.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... its route distribution in this marketing area for 3 consecutive months or if the plant is required to... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1131.7 Section 1131.7 Agriculture... Handling Definitions § 1131.7 Pool plant. Pool Plant means a plant or unit of plants specified...

  6. 7 CFR 1001.7 - Pool plant.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1001.7 Section 1001.7 Agriculture... Handling Definitions § 1001.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant described in paragraph (h)...

  7. 7 CFR 1124.7 - Pool plant.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1124.7 Section 1124.7 Agriculture... Regulating Handling Definitions § 1124.7 Pool plant. Pool plant means a plant, unit of plants, or a system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified...

  8. 7 CFR 1032.7 - Pool plant.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... a majority of its route distribution in this marketing area for 3 consecutive months or if the plant... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1032.7 Section 1032.7 Agriculture... Handling Definitions § 1032.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants...

  9. Genotyping common and rare variation using overlapping pool sequencing

    PubMed Central

    2011-01-01

    Background Recent advances in sequencing technologies set the stage for large, population based studies, in which the ANA or RNA of thousands of individuals will be sequenced. Currently, however, such studies are still infeasible using a straightforward sequencing approach; as a result, recently a few multiplexing schemes have been suggested, in which a small number of ANA pools are sequenced, and the results are then deconvoluted using compressed sensing or similar approaches. These methods, however, are limited to the detection of rare variants. Results In this paper we provide a new algorithm for the deconvolution of DNA pools multiplexing schemes. The presented algorithm utilizes a likelihood model and linear programming. The approach allows for the addition of external data, particularly imputation data, resulting in a flexible environment that is suitable for different applications. Conclusions Particularly, we demonstrate that both low and high allele frequency SNPs can be accurately genotyped when the DNA pooling scheme is performed in conjunction with microarray genotyping and imputation. Additionally, we demonstrate the use of our framework for the detection of cancer fusion genes from RNA sequences. PMID:21989232

  10. Use of vibrational spectroscopy to study protein and DNA structure, hydration, and binding of biomolecules: A combined theoretical and experimental approach

    NASA Astrophysics Data System (ADS)

    Jalkanen, K. J.; Jürgensen, V. Würtz; Claussen, A.; Rahim, A.; Jensen, G. M.; Wade, R. C.; Nardi, F.; Jung, C.; Degtyarenko, I. M.; Nieminen, R. M.; Herrmann, F.; Knapp-Mohammady, M.; Niehaus, T. A.; Frimand, K.; Suhai, S.

    We report on our work with vibrational absorption, vibrational circular dichroism, Raman scattering, Raman optical activity, and surface-enhanced Raman spectroscopy to study protein and DNA structure, hydration, and the binding of ligands, drugs, pesticides, or herbicides via a combined theoretical and experimental approach. The systems we have studied systematically are the amino acids (L-alanine, L-tryptophan, and L-histidine), peptides (N-4271 acetyl L-alanine N?-methyl amide, N-acetyl L-tryptophan N?-methyl amide, N-acetyl L-histidine N?-methyl amide, L-alanyl L-alanine, tri-L-serine, N-acetyl L-alanine L-proline L-tyrosine N?-methyl amide, Leu-enkephalin, cyclo-(gly-L-pro)3, N-acetyl (L-alanine)n N?-methyl amide), 3-methyl indole, and a variety of small molecules (dichlobenil and 2,6-dochlorobenzamide) of relevance to the protein systems under study. We have used molecular mechanics, the SCC-DFTB, SCC-DFTB+disp, RHF, MP2, and DFT methodologies for the modeling studies with the goal of interpreting the experimentally measured vibrational spectra for these molecules to the greatest extent possible and to use this combined approach to understand the structure, function, and electronic properties of these molecules in their various environments. The application of these spectroscopies to biophysical and environmental assays is expanding, and therefore a thorough understanding of the phenomenon from a rigorous theoretical basis is required. In addition, we give some exciting and new preliminary results which allow us to extend our methods to even larger and more complex systems. The work presented here is the current state of the art to this ever and fast changing field of theoretical spectroscopic interpretation and use of VA, VCD, Raman, ROA, EA, and ECD spectroscopies.

  11. Regression for Skewed Biomarker Outcomes Subject to Pooling

    PubMed Central

    Mitchell, Emily M.; Lyles, Robert H.; Manatunga, Amita K.; Danaher, Michelle; Perkins, Neil J.; Schisterman, Enrique F.

    2014-01-01

    Summary Epidemiological studies involving biomarkers are often hindered by prohibitively expensive laboratory tests. Strategically pooling specimens prior to performing these lab assays has been shown to effectively reduce cost with minimal information loss in a logistic regression setting. When the goal is to perform regression with a continuous biomarker as the outcome, regression analysis of pooled specimens may not be straightforward, particularly if the outcome is right-skewed. In such cases, we demonstrate that a slight modification of a standard multiple linear regression model for poolwise data can provide valid and precise coefficient estimates when pools are formed by combining biospecimens from subjects with identical covariate values. When these x-homogeneous pools cannot be formed, we propose a Monte Carlo Expectation Maximization (MCEM) algorithm to compute maximum likelihood estimates (MLEs). Simulation studies demonstrate that these analytical methods provide essentially unbiased estimates of coefficient parameters as well as their standard errors when appropriate assumptions are met. Furthermore, we show how one can utilize the fully observed covariate data to inform the pooling strategy, yielding a high level of statistical efficiency at a fraction of the total lab cost. PMID:24521420

  12. Assessment of DNA damage in Ehlrich carcinoma after treatment with doxorubicin encapsulated in nanoscales thermosensitive liposomes in combination with localized hyperthermia.

    PubMed

    Rageh, Monira M; Shafaa, Medhat W; Elhefnawy, Mona R; El-Nagdy, Mohamed S

    2016-07-01

    Nanoscales thermosensitive liposomes (TSL) composed of synthetic lipids (dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine), were used for doxorubicin encapsulation with 70% encapsulated efficiency. The liposomes were characterized by dynamic light scattering, transmission electron microscopy and turbidity method. Additionally, the liposomes exhibited a significant release of doxorubicin (Dox) by 60% within 5 min at 42°C. To assess the therapeutic efficacy of Dox in combination with hyperthermia, Dox free and encapsulated TSL were administered directly to Ehrlich tumor bearing mice at 1 mg/kg dose. Immediately after the drug administration, hyperthermia was applied to mention the temperature inside the tumor site at 42°C either for 5 min and 30 min. The results indicate a significant increase in the percent of apoptotic and necrotic cells in the treated group. Moreover, disrupts the integrity and the amount of intact DNA in tumor cells. In conclusion, Dox and hyperthermia may serve as a useful targeted drug delivery system for management of Ehrlich carcinoma. PMID:27174899

  13. Combined virus-like particle and fusion protein-encoding DNA vaccination of cotton rats induces protection against respiratory syncytial virus without causing vaccine-enhanced disease.

    PubMed

    Hwang, Hye Suk; Lee, Young-Tae; Kim, Ki-Hye; Park, Soojin; Kwon, Young-Man; Lee, Youri; Ko, Eun-Ju; Jung, Yu-Jin; Lee, Jong Seok; Kim, Yu-Jin; Lee, Yu-Na; Kim, Min-Chul; Cho, Minkyoung; Kang, Sang-Moo

    2016-07-01

    A safe and effective vaccine against respiratory syncytial virus (RSV) should confer protection without causing vaccine-enhanced disease. Here, using a cotton rat model, we investigated the protective efficacy and safety of an RSV combination vaccine composed of F-encoding plasmid DNA and virus-like particles containing RSV fusion (F) and attachment (G) glycoproteins (FFG-VLP). Cotton rats with FFG-VLP vaccination controlled lung viral replication below the detection limit, and effectively induced neutralizing activity and antibody-secreting cell responses. In comparison with formalin inactivated RSV (FI-RSV) causing severe RSV disease after challenge, FFG-VLP vaccination did not cause weight loss, airway hyper-responsiveness, IL-4 cytokines, histopathology, and infiltrates of proinflammatory cells such as eosinophils. FFG-VLP was even more effective in preventing RSV-induced pulmonary inflammation than live RSV infections. This study provides evidence that FFG-VLP can be developed into a safe and effective RSV vaccine candidate. PMID:27123586

  14. Metformin synergizes 5-fluorouracil, epirubicin, and cyclophosphamide (FEC) combination therapy through impairing intracellular ATP production and DNA repair in breast cancer stem cells.

    PubMed

    Soo, Jaslyn Sian-Siu; Ng, Char-Hong; Tan, Si Hoey; Malik, Rozita Abdul; Teh, Yew-Ching; Tan, Boon-Shing; Ho, Gwo-Fuang; See, Mee-Hoong; Taib, Nur Aishah Mohd; Yip, Cheng-Har; Chung, Felicia Fei-Lei; Hii, Ling-Wei; Teo, Soo-Hwang; Leong, Chee-Onn

    2015-10-01

    Metformin, an AMPK activator, has been reported to improve pathological response to chemotherapy in diabetic breast cancer patients. To date, its mechanism of action in cancer, especially in cancer stem cells (CSCs) have not been fully elucidated. In this study, we demonstrated that metformin, but not other AMPK activators (e.g. AICAR and A-769662), synergizes 5-fluouracil, epirubicin, and cyclophosphamide (FEC) combination chemotherapy in non-stem breast cancer cells and breast cancer stem cells. We show that this occurs through an AMPK-dependent mechanism in parental breast cancer cell lines. In contrast, the synergistic effects of metformin and FEC occurred in an AMPK-independent mechanism in breast CSCs. Further analyses revealed that metformin accelerated glucose consumption and lactate production more severely in the breast CSCs but the production of intracellular ATP was severely hampered, leading to a severe energy crisis and impairs the ability of CSCs to repair FEC-induced DNA damage. Indeed, addition of extracellular ATP completely abrogated the synergistic effects of metformin on FEC sensitivity in breast CSCs. In conclusion, our results suggest that metformin synergizes FEC sensitivity through distinct mechanism in parental breast cancer cell lines and CSCs, thus providing further evidence for the clinical relevance of metformin for the treatment of cancers. PMID:26276035

  15. Patent pools: intellectual property rights and competition.

    PubMed

    Rodriguez, Victor

    2010-01-01

    Patent pools do not correct all problems associated with patent thickets. In this respect, patent pools might not stop the outsider problem from striking pools. Moreover, patent pools can be expensive to negotiate, can exclude patent holders with smaller numbers of patents or enable a group of major players to form a cartel that excludes new competitors. For all the above reasons, patent pools are subject to regulatory clearance because they could result in a monopoly. The aim of this article is to present the relationship between patents and competition in a broad context. PMID:20200607

  16. Patent Pools: Intellectual Property Rights and Competition

    PubMed Central

    Rodriguez, Victor

    2010-01-01

    Patent pools do not correct all problems associated with patent thickets. In this respect, patent pools might not stop the outsider problem from striking pools. Moreover, patent pools can be expensive to negotiate, can exclude patent holders with smaller numbers of patents or enable a group of major players to form a cartel that excludes new competitors. For all the above reasons, patent pools are subject to regulatory clearance because they could result in a monopoly. The aim of this article is to present the relationship between patents and competition in a broad context. PMID:20200607

  17. Method of measuring a liquid pool volume

    DOEpatents

    Garcia, G.V.; Carlson, N.M.; Donaldson, A.D.

    1991-03-19

    A method of measuring a molten metal liquid pool volume and in particular molten titanium liquid pools is disclosed, including the steps of (a) generating an ultrasonic wave at the surface of the molten metal liquid pool, (b) shining a light on the surface of a molten metal liquid pool, (c) detecting a change in the frequency of light, (d) detecting an ultrasonic wave echo at the surface of the molten metal liquid pool, and (e) computing the volume of the molten metal liquid. 3 figures.

  18. A new genomic tool, ultra-frequently cleaving TaqII/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse DNA libraries

    PubMed Central

    2013-01-01

    Background Genomics and metagenomics are currently leading research areas, with DNA sequences accumulating at an exponential rate. Although enormous advances in DNA sequencing technologies are taking place, progress is frequently limited by factors such as genomic contig assembly and generation of representative libraries. A number of DNA fragmentation methods, such as hydrodynamic sharing, sonication or DNase I fragmentation, have various drawbacks, including DNA damage, poor fragmentation control, irreproducibility and non-overlapping DNA segment representation. Improvements in these limited DNA scission methods are consequently needed. An alternative method for obtaining higher quality DNA fragments involves partial digestion with restriction endonucleases (REases). We have shown previously that class-IIS/IIC/IIG TspGWI REase, the prototype member of the Thermus sp. enzyme family, can be chemically relaxed by a cofactor analogue, allowing it to recognize very short DNA sequences of 3-bp combined frequency. Such frequently cleaving REases are extremely rare, with CviJI/CviJI*, SetI and FaiI the only other ones found in nature. Their unusual features make them very useful molecular tools for the development of representative DNA libraries. Results We constructed a horse genomic library and a deletion derivative library of the butyrylcholinesterase cDNA coding region using a novel method, based on TaqII, Thermus sp. family bifunctional enzyme exhibiting cofactor analogue specificity relaxation. We used sinefungin (SIN) – an S-adenosylmethionine (SAM) analogue with reversed charge pattern, and dimethylsulfoxide (DMSO), to convert the 6-bp recognition site TaqII (5′-GACCGA-3′ [11/9]) into a theoretical 2.9-bp REase, with 70 shortened variants of the canonical recognition sequence detected. Because partial DNA cleavage is an inherent feature of the Thermus sp. enzyme family, this modified TaqII is uniquely suited to quasi-random library generation. Conclusions

  19. Transient pool boiling in microgravity

    NASA Technical Reports Server (NTRS)

    Ervin, J. S.; Merte, H., Jr.; Keller, R. B.; Kirk, K.

    1992-01-01

    Transient nucleate pool boiling experiments using R113 are conducted for short times in microgravity and in earth gravity with different heater surface orientations and subcoolings. The heating surface is a transparent gold film sputtered on a quartz substrate, which simultaneously provides surface temperature measurements and permits viewing of the boiling process from beneath. For the microgravity experiments, which have uniform initial temperatures and no fluid motion, the temperature distribution in the R 113 at the moment of boiling inception is known. High speed cameras with views both across and through the heating surface record the boiling spread across the heater surface, which is classified into six distinct categories.

  20. Nonoverlapping clone pooling for high-throughput sequencing.

    PubMed

    Kuroshu, Reginaldo M

    2013-01-01

    Simultaneously sequencing multiple clones using second-generation sequencers can speed up many essential clone-based sequencing methods. However, in applications such as fosmid clone sequencing and full-length cDNA sequencing, it is important to create pools of clones that do not overlap on the genome for the identification of structural variations and alternatively spliced transcripts, respectively. We define the nonoverlapping clone pooling problem and provide practical solutions based on optimal graph coloring and bin-packing algorithms with constant absolute worst-case ratios, and further extend them to cope with repetitive mappings. Using theoretical analysis and experiments, we also show that the proposed methods are applicable. PMID:24384700

  1. Microbiological Analysis in Three Diverse Natural Geothermal Bathing Pools in Iceland

    PubMed Central

    Thorolfsdottir, Berglind Osk Th.; Marteinsson, Viggo Thor

    2013-01-01

    Natural thermal bathing pools contain geothermal water that is very popular to bathe in but the water is not sterilized, irradiated or treated in any way. Increasing tourism in Iceland will lead to increasing numbers of bath guests, which can in turn affect the microbial flora in the pools and therefore user safety. Today, there is no legislation that applies to natural geothermal pools in Iceland, as the water is not used for consumption and the pools are not defined as public swimming pools. In this study, we conducted a microbiological analysis on three popular but different natural pools in Iceland, located at Lýsuhóll, Hveravellir and Landmannalaugar. Total bacterial counts were performed by flow cytometry, and with plate count at 22 °C, 37 °C and 50 °C. The presence of viable coliforms, Enterococcus spp. and pseudomonads were investigated by growth experiments on selective media. All samples were screened for noroviruses by real time PCR. The results indicate higher fecal contamination in the geothermal pools where the geothermal water flow was low and bathing guest count was high during the day. The number of cultivated Pseudomonas spp. was high (13,000–40,000 cfu/100 mL) in the natural pools, and several strains were isolated and classified as opportunistic pathogens. Norovirus was not detected in the three pools. DNA was extracted from one-liter samples in each pool and analyzed by partial 16S rRNA gene sequencing. Microbial diversity analysis revealed different microbial communities between the pools and they were primarily composed of alpha-, beta- and gammaproteobacteria. PMID:23493033

  2. Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

    PubMed Central

    Makarova, Alena V.; Grabow, Corinn; Gening, Leonid V.; Tarantul, Vyacheslav Z.; Tahirov, Tahir H.; Bessho, Tadayoshi; Pavlov, Youri I.

    2011-01-01

    Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts. PMID:21304950

  3. A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA.

    PubMed

    Diot, Alan; Hinks-Roberts, Alex; Lodge, Tiffany; Liao, Chunyan; Dombi, Eszter; Morten, Karl; Brady, Stefen; Fratter, Carl; Carver, Janet; Muir, Rebecca; Davis, Ryan; Green, Charlotte J; Johnston, Iain; Hilton-Jones, David; Sue, Carolyn; Mortiboys, Heather; Poulton, Joanna

    2015-10-01

    Mitophagy is a cellular mechanism for the recycling of mitochondrial fragments. This process is able to improve mitochondrial DNA (mtDNA) quality in heteroplasmic mtDNA disease, in which mutant mtDNA co-exists with normal mtDNA. In disorders where the load of mutant mtDNA determines disease severity it is likely to be an important determinant of disease progression. Measuring mitophagy is technically demanding. We used pharmacological modulators of autophagy to validate two techniques for quantifying mitophagy. First we used the IN Cell 1000 analyzer to quantify mitochondrial co-localisation with LC3-II positive autophagosomes. Unlike conventional fluorescence and electron microscopy, this high-throughput system is sufficiently sensitive to detect transient low frequency autophagosomes. Secondly, because mitophagy preferentially removes pathogenic heteroplasmic mtDNA mutants, we developed a heteroplasmy assay based on loss of m.3243A>G mtDNA, during culture conditions requiring oxidative metabolism ("energetic stress"). The effects of the pharmacological modulators on these two measures were consistent, confirming that the high throughput imaging output (autophagosomes co-localising with mitochondria) reflects mitochondrial quality control. To further validate these methods, we performed a more detailed study using metformin, the most commonly prescribed antidiabetic drug that is still sometimes used in Maternally Inherited Diabetes and Deafness (MIDD). This confirmed our initial findings and revealed that metformin inhibits mitophagy at clinically relevant concentrations, suggesting that it may have novel therapeutic uses. PMID:26196248

  4. DNA and RNA "traffic lights": synthetic wavelength-shifting fluorescent probes based on nucleic acid base substitutes for molecular imaging.

    PubMed

    Holzhauser, Carolin; Wagenknecht, Hans-Achim

    2013-08-01

    The DNA base substitute approach by the (S)-3-amino-1,2-propanediol linker allows placing two fluorophores in a precise way inside a given DNA framework. The double helical architecture around the fluorophores, especially the DNA-induced twist, is crucial for the desired photophysical interactions. Excitonic, excimer, and energy transfer interactions yield fluorescent DNA and RNA probes with dual emission color readout. Especially, our DNA and RNA "traffic light" that combines the green emission of TO with the red emission of TR represents an important tool for molecular imaging and can be applied as aptasensors and as probes to monitor the siRNA delivery into cells. The concept can be extended to the synthetically easier to access postsynthetic 2'-modifications and the NIR range. Thereby, the pool of tailor-made fluorescent nucleic acid conjugates can be extended. PMID:23796243

  5. Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.

    PubMed

    Watthanapanpituck, Khanistha; Kiatpathomchai, Wansika; Chu, Eric; Panvisavas, Nathinee

    2014-11-01

    A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments. PMID:24827529

  6. Combined effects of DNA methyltransferase 1 and 3A polymorphisms and urinary total arsenic levels on the risk for clear cell renal cell carcinoma.

    PubMed

    Yang, Shu-Mei; Huang, Chao-Yuan; Shiue, Horng-Sheng; Pu, Yeong-Shiau; Hsieh, Yi-Hsun; Chen, Wei-Jen; Lin, Ying-Chin; Hsueh, Yu-Mei

    2016-08-15

    Our previous study showed that high urinary total arsenic levels were associated with higher odds ratio (OR) for renal cell carcinoma (RCC). Single nucleotide polymorphisms (SNPs) of DNA methyltransferases (DNMTs) might influence DNMT enzyme activity associated with tumorigenesis. In this study, we investigated the association of five SNPs from DNMT1 (rs8101626 and rs2228611), DNMT3A (rs34048824 and rs1550117), and DNMT3B (rs1569686) with the risk of clear cell renal cell carcinoma (ccRCC). We also examined the combined effects of DNMT genotypes and urinary arsenic levels on ccRCC risk. We conducted a hospital-based case-control study, which included 293 subjects with ccRCC and 293 age- and gender-matched controls. The urinary arsenic species were determined by a high performance liquid chromatography-linked hydride generator and atomic absorption spectrometry. Genotypes were investigated using polymerase chain reaction and restriction fragment length polymorphism analyses. We observed that the DNMT1 rs8101626 G/G genotype was significantly associated with reduced odds ratio (OR) of ccRCC [OR=0.38, 95% confidence interval (CI) 0.14-0.99]. Subjects with concurrent DNMT1 rs8101626 A/A+A/G and DNMT3A rs34048824 T/T+T/C genotypes had significantly higher OR for ccRCC [OR=2.88, 95% CI 1.44-5.77]. Participants with the high-risk genotype of DNMT1 rs8101626 and DNMT3A rs34048824 with concurrently high urinary total arsenic levels had even higher OR of ccRCC in a dose-response manner. This is the first study to evaluate variant DNMT1 rs8101626 and DNMT3A rs34048824 genotypes that modify the arsenic-related ccRCC risk in a geographic area without significant arsenic exposure in Taiwan. PMID:27292127

  7. Combining pseudo dinucleotide composition with the Z curve method to improve the accuracy of predicting DNA elements: a case study in recombination spots.

    PubMed

    Dong, Chuan; Yuan, Ya-Zhou; Zhang, Fa-Zhan; Hua, Hong-Li; Ye, Yuan-Nong; Labena, Abraham Alemayehu; Lin, Hao; Chen, Wei; Guo, Feng-Biao

    2016-08-16

    Pseudo dinucleotide composition (PseDNC) and Z curve showed excellent performance in the classification issues of nucleotide sequences in bioinformatics. Inspired by the principle of Z curve theory, we improved PseDNC to give the phase-specific PseDNC (psPseDNC). In this study, we used the prediction of recombination spots as a case to illustrate the capability of psPseDNC and also PseDNC fused with Z curve theory based on a novel machine learning method named large margin distribution machine (LDM). We verified that combining the two widely used approaches could generate better performance compared to only using PseDNC with a support vector machine based (SVM-based) model. The best Mathew's correlation coefficient (MCC) achieved by our LDM-based model was 0.7037 through the rigorous jackknife test and improved by ∼6.6%, ∼3.2%, and ∼2.4% compared with three previous studies. Similarly, the accuracy was improved by 3.2% compared with our previous iRSpot-PseDNC web server through an independent data test. These results demonstrate that the joint use of PseDNC and Z curve enhances performance and can extract more information from a biological sequence. To facilitate research in this area, we constructed a user-friendly web server for predicting hot/cold spots, HcsPredictor, which can be freely accessed from . In summary, we provided a united algorithm by integrating Z curve with PseDNC. We hope this united algorithm could be extended to other classification issues in DNA elements. PMID:27410247

  8. Drowning in disinfection byproducts? Assessing swimming pool water.

    PubMed

    Zwiener, Christian; Richardson, Susan D; DeMarini, David M; De Marini, David M; Grummt, Tamara; Glauner, Thomas; Frimmel, Fritz H

    2007-01-15

    Disinfection is mandatory for swimming pools: public pools are usually disinfected by gaseous chlorine or sodium hypochlorite and cartridge filters; home pools typically use stabilized chlorine. These methods produce a variety of disinfection byproducts (DBPs), such as trihalomethanes (THMs), which are regulated carcinogenic DBPs in drinking water that have been detected in the blood and breath of swimmers and of nonswimmers at indoor pools. Also produced are halogenated acetic acids (HAAs) and haloketones, which irritate the eyes, skin, and mucous membranes; trichloramine, which is linked with swimming-pool-associated asthma; and halogenated derivatives of UV sun screens, some of which show endocrine effects. Precursors of DBPs include human body substances, chemicals used in cosmetics and sun screens, and natural organic matter. Analytical research has focused also on the identification of an additional portion of unknown DBPs using gas chromatography (GC)/mass spectrometry (MS) and liquid chromatography (LC)/MS/MS with derivatization. Children swimmers have an increased risk of developing asthma and infections of the respiratory tract and ear. A 1.6-2.0-fold increased risk for bladder cancer has been associated with swimming or showering/bathing with chlorinated water. Bladder cancer risk from THM exposure (all routes combined) was greatest among those with the GSTT1-1 gene. This suggests a mechanism involving distribution of THMs to the bladder by dermal/inhalation exposure and activation there by GSTT1-1 to mutagens. DBPs may be reduced by engineering and behavioral means, such as applying new oxidation and filtration methods, reducing bromide and iodide in the source water, increasing air circulation in indoor pools, and assuring the cleanliness of swimmers. The positive health effects gained by swimming can be increased by reducing the potential adverse health risks. PMID:17310693

  9. Radioisotope Power System Pool Concept

    NASA Technical Reports Server (NTRS)

    Rusick, Jeffrey J.; Bolotin, Gary S.

    2015-01-01

    Advanced Radioisotope Power Systems (RPS) for NASA deep space science missions have historically used static thermoelectric-based designs because they are highly reliable, and their radioisotope heat sources can be passively cooled throughout the mission life cycle. Recently, a significant effort to develop a dynamic RPS, the Advanced Stirling Radioisotope Generator (ASRG), was conducted by NASA and the Department of Energy, because Stirling based designs offer energy conversion efficiencies four times higher than heritage thermoelectric designs; and the efficiency would proportionately reduce the amount of radioisotope fuel needed for the same power output. However, the long term reliability of a Stirling based design is a concern compared to thermoelectric designs, because for certain Stirling system architectures the radioisotope heat sources must be actively cooled via the dynamic operation of Stirling converters throughout the mission life cycle. To address this reliability concern, a new dynamic Stirling cycle RPS architecture is proposed called the RPS Pool Concept.

  10. A combination of computational and experimental approaches identifies DNA sequence constraints associated with target site binding specificity of the transcription factor CSL.

    PubMed

    Torella, Rubben; Li, Jinghua; Kinrade, Eddie; Cerda-Moya, Gustavo; Contreras, Ashley N; Foy, Robert; Stojnic, Robert; Glen, Robert C; Kovall, Rhett A; Adryan, Boris; Bray, Sarah J

    2014-01-01

    Regulation of transcription is fundamental to development and physiology, and occurs through binding of transcription factors to specific DNA sequences in the genome. CSL (CBF1/Suppressor of Hairless/LAG-1), a core component of the Notch signaling pathway, is one such transcription factor that acts in concert with co-activators or co-repressors to control the activity of associated target genes. One fundamental question is how CSL can recognize and select among different DNA sequences available in vivo and whether variations between selected sequences can influence its function. We have therefore investigated CSL-DNA recognition using computational approaches to analyze the energetics of CSL bound to different DNAs and tested the in silico predictions with in vitro and in vivo assays. Our results reveal novel aspects of CSL binding that may help explain the range of binding observed in vivo. In addition, using molecular dynamics simulations, we show that domain-domain correlations within CSL differ significantly depending on the DNA sequence bound, suggesting that different DNA sequences may directly influence CSL function. Taken together, our results, based on computational chemistry approaches, provide valuable insights into transcription factor-DNA binding, in this particular case increasing our understanding of CSL-DNA interactions and how these may impact on its transcriptional control. PMID:25114055

  11. A Combination DNA and Attenuated Simian Immunodeficiency Virus Vaccine Strategy Provides Enhanced Protection from Simian/Human Immunodeficiency Virus-Induced Disease†

    PubMed Central

    Amara, Rama Rao; Patel, Kalpana; Niedziela, Genevieve; Nigam, Pragati; Sharma, Sunita; Staprans, Silvija I.; Montefiori, David C.; Chenareddi, Lakshmi; Herndon, James G.; Robinson, Harriet L.; McClure, Harold M.; Novembre, Francis J.

    2005-01-01

    Among the most effective vaccine candidates tested in the simian immunodeficiency virus (SIV)/macaque system, live attenuated viruses have been shown to provide the best protection from challenge. To investigate if preimmunization would increase the level of protection afforded by live attenuated SIVmac239Δnef (Δnef), macaques were given two priming immunizations of DNA encoding SIV Gag and Pol proteins, with control macaques receiving vector DNA immunizations. In macaques receiving the SIV DNA inoculation, SIV-specific cellular but not humoral responses were readily detectable 2 weeks after the second DNA inoculation. Following boosting with live attenuated virus, control of Δnef replication was superior in SIV-DNA-primed macaques versus vector-DNA-primed macaques and was correlated with higher levels of CD8+/gamma-interferon-positive and/or interleukin-2-positive cells. Challenge with an intravenous inoculation of simian/human immunodeficiency virus (SHIV) strain SHIV89.6p resulted in infection of all animals. However, macaques receiving SIV DNA as the priming immunizations had statistically lower viral loads than control animals and did not develop signs of disease, whereas three of seven macaques receiving vector DNA showed severe CD4+ T-cell decline, with development of AIDS in one of these animals. No correlation of immune responses to protection from disease could be derived from our analyses. These results demonstrate that addition of a DNA prime to a live attenuated virus provided better protection from disease following challenge than live attenuated virus alone. PMID:16306607

  12. National decline in invasive prenatal diagnostic procedures in association with uptake of combined first trimester and cell-free DNA aneuploidy screening.

    PubMed

    Robson, Stephen J; Hui, Lisa

    2015-10-01

    In late 2012, a new screening test for fetal aneuploidy based on circulating cell-free DNA (cfDNA) became available to Australian women. The introduction of this technology in the United States has led to a reduction in invasive diagnostic procedures. Analysis of the number of amniocentesis and chorionic villus sampling (CVS) procedures performed in Australia from 1994 to 2014 shows that the introduction of cfDNA testing has been associated with the most rapid decline in invasive procedures in the last 20 years. This change has important implications for training in, and maintenance of, the procedural skills of amniocentesis and CVS. PMID:26259499

  13. Designing ARVs Patent Pool Up to Trade & Policy Evolutionary Dynamics

    PubMed Central

    Dionisio, Daniele; Racalbuto, Vincenzo; Messeri, Daniela

    2010-01-01

    Patent pools for second and third-line Fixed Dose Combination (FDC) antiretroviral drugs (ARVs) should not be delayed as they are instrumental to urgent public health needs in the under-served markets. Nonetheless, multinational originator companies still seem to perceive patent pooling for ARVs as a minefield that would offer the generic competitors lots of deeply exploitable opportunities, to the detriment of patent owner’s rights. This paper analyses the brand industry concerns, while looking for a strategy up to a really equitable and free world market, without any discrimination between end-users in wealthy and resource-limited countries. This strategy would urge partnerships between originator companies first to make newer FDC ARVs quickly available and allow patent pool agreements with generic counterparts to be negotiated straight afterwards. The patent pool strategy highlighted in this paper would assert the primacy of health over for-profit policies, while aligning with the 61st WHO’s Assembly recommendations and G7, G8 and World Trade Organisation’s warnings and pledges against trade protectionism. PMID:20200604

  14. A NOVEL APPROACH TO SPENT FUEL POOL DECOMMISSIONING

    SciTech Connect

    R. L. Demmer

    2011-04-01

    The Idaho National Laboratory (INL) has been at the forefront of developing methods to reduce the cost and schedule of deactivating spent fuel pools (SFP). Several pools have been deactivated at the INL using an underwater approach with divers. These projects provided a basis for the INL cooperation with the Dresden Nuclear Power Station Unit 1 SFP (Exelon Generation Company) deactivation. It represents the first time that a commercial nuclear power plant (NPP) SFP was decommissioned using this underwater coating process. This approach has advantages in many aspects, particularly in reducing airborne contamination and allowing safer, more cost effective deactivation. The INL pioneered underwater coating process was used to decommission three SFPs with a total combined pool volume of over 900,000 gallons. INL provided engineering support and shared project plans to successfully initiate the Dresden project. This report outlines the steps taken by INL and Exelon to decommission SFPs using the underwater coating process. The rationale used to select the underwater coating process and the advantages and disadvantages are described. Special circumstances are also discussed, such as the use of a remotely-operated underwater vehicle to visually and radiologically map the pool areas that were not readily accessible. A larger project, the INTEC-603 SFP in-situ (grouting) deactivation, is reviewed. Several specific areas where special equipment was employed are discussed and a Lessons Learned evaluation is included.

  15. Recombinant protein production from stable mammalian cell lines and pools.

    PubMed

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. PMID:27322762

  16. Inferring parental genomic ancestries using pooled semi-Markov processes

    PubMed Central

    Zou, James Y.; Halperin, Eran; Burchard, Esteban; Sankararaman, Sriram

    2015-01-01

    Motivation: A basic problem of broad public and scientific interest is to use the DNA of an individual to infer the genomic ancestries of the parents. In particular, we are often interested in the fraction of each parent’s genome that comes from specific ancestries (e.g. European, African, Native American, etc). This has many applications ranging from understanding the inheritance of ancestry-related risks and traits to quantifying human assortative mating patterns. Results: We model the problem of parental genomic ancestry inference as a pooled semi-Markov process. We develop a general mathematical framework for pooled semi-Markov processes and construct efficient inference algorithms for these models. Applying our inference algorithm to genotype data from 231 Mexican trios and 258 Puerto Rican trios where we have the true genomic ancestry of each parent, we demonstrate that our method accurately infers parameters of the semi-Markov processes and parents’ genomic ancestries. We additionally validated the method on simulations. Our model of pooled semi-Markov process and inference algorithms may be of independent interest in other settings in genomics and machine learning. Contact: jazo@microsoft.com PMID:26072482

  17. Experimental characterization of the weld pool flow in a TIG configuration

    NASA Astrophysics Data System (ADS)

    Stadler, M.; Masquère, M.; Freton, P.; Franceries, X.; Gonzalez, J. J.

    2014-11-01

    Tungsten Inert Gas (TIG) welding process relies on heat transfer between plasma and work piece leading to a metallic weld pool. Combination of different forces produces movements on the molten pool surface. One of our aims is to determine the velocity on the weld pool surface. This provides a set of data that leads to a deeper comprehension of the flow behavior and allows us to validate numerical models used to study TIG parameters. In this paper, two diagnostic methods developed with high speed imaging for the determination of velocity of an AISI 304L stainless steel molten pool are presented. Application of the two methods to a metallic weld pool under helium with a current intensity of 100 A provides velocity values around 0.70 m/s which are in good agreement with literature works.

  18. Promising combination therapies with gemcitabine.

    PubMed

    Robinson, Blaine W; Ostruszka, Leo; Im, Michael M; Shewach, Donna S

    2004-04-01

    Because treatment regimens for breast cancer commonly include gemcitabine, we evaluated two promising combinations in preclinical studies: gemcitabine (Gemzar; Eli Lilly and Company, Indianapolis, IN) with either ionizing radiation or docetaxel (Taxotere; Aventis Pharmaceuticals, Inc, Parsippany, NJ). In breast cancer cell lines that expressed either wild-type p53 (MCF-7) or mutant p53 (MCF-7/Adr), sensitivity to the cytotoxic effects of gemcitabine during a 24-hour incubation was similar (IC(50) values 80 and 60 nmol/L in MCF-7 and MCF-7/Adr, respectively). Both cell lines were well radiosensitized by gemcitabine at the corresponding IC(50), with radiation enhancement ratios of 1.6 to 1.7. Although the MCF-7 cells accumulated nearly twice as much gemcitabine triphosphate compared with the MCF-7/Adr cells, a similar reduction in 2'-deoxyadenosine 5'-triphosphate pools was observed. While the number of dying cells, as measured by sub-G1 DNA content or S-phase cells unable to replicate DNA, differed between the wild-type p53 or mutant p53-expressing cell lines, neither parameter correlated with radiosensitization. Docetaxel was a more potent cytotoxic agent than gemcitabine in MCF-7 cells (IC(50) = 1 nmol/L). Strong synergistic cytotoxicity was observed in cells treated with gemcitabine (24 hours) followed by docetaxel (24 hours) or the reverse sequence. However, simultaneous addition of the two drugs was antagonistic. To determine whether synergy with radiation or docetaxel was mediated by increased DNA damage, DNA double-strand breaks (double-strand breaks) were measured by immunostaining for phosphorylated H2AX. Ionizing radiation produced more double-strand breaks than gemcitabine alone, while no significant double-strand breaks formed with docetaxel alone. The addition of docetaxel or ionizing radiation to gemcitabine-treated cells did not increase H2AX foci formation. These results show that the combination of gemcitabine with ionizing radiation or docetaxel

  19. A randomised, phase II trial of the DNA-hypomethylating agent 5-aza-2′-deoxycytidine (decitabine) in combination with carboplatin vs carboplatin alone in patients with recurrent, partially platinum-sensitive ovarian cancer

    PubMed Central

    Glasspool, R M; Brown, R; Gore, M E; Rustin, G J S; McNeish, I A; Wilson, R H; Pledge, S; Paul, J; Mackean, M; Hall, G D; Gabra, H; Halford, S E R; Walker, J; Appleton, K; Ullah, R; Kaye, S

    2014-01-01

    Background: Our previous laboratory and clinical data suggested that one mechanism underlying the development of platinum resistance in ovarian cancer is the acquisition of DNA methylation. We therefore tested the hypothesis that the DNA hypomethylating agent 5-aza-2′-deoxycytodine (decitabine) can reverse resistance to carboplatin in women with relapsed ovarian cancer. Methods: Patients progressing 6–12 months after previous platinum therapy were randomised to decitabine on day 1 and carboplatin (AUC 6) on day 8, every 28 days or carboplatin alone. The primary objective was response rate in patients with methylated hMLH1 tumour DNA in plasma. Results: After a pre-defined interim analysis, the study closed due to lack of efficacy and poor treatment deliverability in 15 patients treated with the combination. Responses by GCIG criteria were 9 out of 14 vs 3 out of 15 and by RECIST were 6 out of 13 vs 1 out of 12 for carboplatin and carboplatin/decitabine, respectively. Grade 3/4 neutropenia was more common with the combination (60% vs 15.4%) as was G2/3 carboplatin hypersensitivity (47% vs 21%). Conclusions: With this schedule, the addition of decitabine appears to reduce rather than increase the efficacy of carboplatin in partially platinum-sensitive ovarian cancer and is difficult to deliver. Patient-selection strategies, different schedules and other demethylating agents should be considered in future combination studies. PMID:24642620

  20. Single and Combined Effects of Deoxynivalenol Mycotoxin and a Microbial Feed Additive on Lymphocyte DNA Damage and Oxidative Stress in Broiler Chickens

    PubMed Central

    Awad, Wageha A.; Ghareeb, Khaled; Dadak, Agnes; Hess, Michael; Böhm, Josef

    2014-01-01

    The immune and intestinal epithelial cells are particularly sensitive to the toxic effects of deoxynivalenol (DON). The aim of this experiment was to study the effects of DON and/or a microbial feed additive on the DNA damage of blood lymphocytes and on the level of thiobarbituric acid reactive substance (TBARS) as an indicator of lipid peroxidation and oxidative stress in broilers. A total of forty 1-d-old broiler chicks were randomly assigned to 1 of 4 dietary treatments (10 birds per group) for 5 wk. The dietary treatments were 1) basal diet; 2) basal diet contaminated with 10 mg DON/kg feed; 3) basal diet contaminated with 10 mg DON/kg feed and supplemented with 2.5 kg/ton of feed of Mycofix Select; 4) basal diet supplemented with Mycofix Select (2.5 kg/ton of feed). At the end of the feeding trial, blood were collected for measuring the level of lymphocyte DNA damage of blood and the TBARS level was measured in plasma, heart, kidney, duodenum and jejunum. The dietary exposure of DON caused a significant increase (P = 0.001) of DNA damage in blood lymphocytes (31.99±0.89%) as indicated in the tail of comet assay. Interestingly addition of Mycofix Select to DON contaminated diet decreased (P = 0.001) the DNA damage (19.82±1.75%) induced by DON. In order to clarify the involvement of lipid peroxidation in the DNA damage of DON, TBARS levels was measured. A significant increase (P = 0.001) in the level of TBARS (23±2 nmol/mg) was observed in the jejunal tissue suggesting that the lipid peroxidation might be involved in the DNA damage. The results indicate that DON is cytotoxic and genotoxic to the chicken intestinal and immune cells and the feed additive have potential ability to prevent DNA damage induced by DON. PMID:24498242

  1. Virtual manipulation of topography to test potential pool-riffle maintenance mechanisms

    NASA Astrophysics Data System (ADS)

    Jackson, J. R.; Pasternack, G. B.; Wheaton, J. M.

    2015-01-01

    In this study, numerical experimentation with two-dimensional hydraulic modeling of pool-riffle river topography drawing on the testbed data from the classic Keller (1971) study was used to investigate the effect of synthetically manipulating topography on the occurrence and magnitude of velocity and Shields stress reversals in a pool-riffle sequence. Reversals in velocity and shear stress have been used to explore mechanisms of pool-riffle maintenance, while Shields stress (a combined measure of transport capacity and substrate erodibility) is emerging in importance. The original site topography was modeled alongside six altered ones to evaluate the sensitivity of hydraulic reversals to subtle morphology - five incrementally wider pools and a filled pool. The Caamaño (2009) criterion, a simplified geometric threshold for predicting velocity reversals, was applied to each terrain to evaluate its utility. The original pool-riffle topography was just over the threshold for a velocity reversal and well over the threshold for a strong Shields stress reversal. Overall, pool widening caused a predominantly local response, with change to pool hydraulics and no change in section-averaged velocity in the riffle beyond the initial widening of 10%. Filling in the pool significantly increased the magnitude of reversals, whereas expanding it eliminated the occurrence of a reversal in mean velocity, though the Shields stress reversal persisted because of strong differentiation in bed material texture. Using Shields stress as a reversal parameter enabled the quantification of pool modification effects on pool-riffle resiliency. The Caamaño (2009) criterion accurately predicted reversal occurrence for the altered terrains with exaggerated effects, but failed to predict the weak reversal for the original topography. Two-dimensional modeling coupled with previously accepted hydrologic, geomorphic, and engineering analyses is vital in project design and evaluation prior to

  2. 21 CFR 1250.89 - Swimming pools.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... types of salt water pools shall be so operated that complete circulation and replacement of the water in... equipped so as to provide complete circulation, replacement, and filtration of the water in the pool every six hours or less. Suitable means of chlorination and, if necessary, other treatment of the...

  3. 47 CFR 97.523 - Question pools.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 5 2012-10-01 2012-10-01 false Question pools. 97.523 Section 97.523 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES AMATEUR RADIO SERVICE Qualifying Examination Systems § 97.523 Question pools. All VECs must cooperate in maintaining...

  4. 47 CFR 97.523 - Question pools.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 5 2013-10-01 2013-10-01 false Question pools. 97.523 Section 97.523 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES AMATEUR RADIO SERVICE Qualifying Examination Systems § 97.523 Question pools. All VECs must cooperate in maintaining...

  5. 47 CFR 97.523 - Question pools.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 5 2014-10-01 2014-10-01 false Question pools. 97.523 Section 97.523 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES AMATEUR RADIO SERVICE Qualifying Examination Systems § 97.523 Question pools. All VECs must cooperate in maintaining...

  6. 47 CFR 97.523 - Question pools.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 5 2011-10-01 2011-10-01 false Question pools. 97.523 Section 97.523 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES AMATEUR RADIO SERVICE Qualifying Examination Systems § 97.523 Question pools. All VECs must cooperate in maintaining...

  7. 47 CFR 97.523 - Question pools.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 5 2010-10-01 2010-10-01 false Question pools. 97.523 Section 97.523 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES AMATEUR RADIO SERVICE Qualifying Examination Systems § 97.523 Question pools. All VECs must cooperate in maintaining...

  8. The Chemistry of Swimming Pool Maintenance

    ERIC Educational Resources Information Center

    Salter, Carl; Langhus, David L.

    2007-01-01

    The study of chemistry involved in the maintenance of a swimming pool provides a lot of chemical education to the students, including the demonstration of the importance of pH in water chemistry. The various chemical aspects hidden in the maintenance of the pool are being described.

  9. 10 CFR 36.33 - Irradiator pools.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... purification system designed to be capable of maintaining the water during normal operation at a conductivity..., irradiator pools must either: (1) Have a water-tight stainless steel liner or a liner metallurgically... water level that could allow water to drain out of the pool. Pipes that have intakes more than 0.5...

  10. 10 CFR 36.33 - Irradiator pools.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... purification system designed to be capable of maintaining the water during normal operation at a conductivity..., irradiator pools must either: (1) Have a water-tight stainless steel liner or a liner metallurgically... water level that could allow water to drain out of the pool. Pipes that have intakes more than 0.5...

  11. Camera Would Monitor Weld-Pool Contours

    NASA Technical Reports Server (NTRS)

    Gordon, Stephen S.; Gutow, David A.

    1990-01-01

    Weld pool illuminated and viewed coaxially along welding torch. Proposed monitoring subsystem for arc welder provides image in which horizontal portions of surface of weld pool highlighted. Monitoring and analyzing subsystems integrated into overall control system of robotic welder. Control system sets welding parameters to adapt to changing conditions, maintaining surface contour giving desired pattern of reflections.

  12. 10 CFR 36.33 - Irradiator pools.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 1 2014-01-01 2014-01-01 false Irradiator pools. 36.33 Section 36.33 Energy NUCLEAR....02 millisievert (2 millirems) per hour. ... issued after July 1, 1993, irradiator pools must have no outlets more than 0.5 meter below the normal...

  13. 21 CFR 1250.89 - Swimming pools.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... types of salt water pools shall be so operated that complete circulation and replacement of the water in... equipped so as to provide complete circulation, replacement, and filtration of the water in the pool every six hours or less. Suitable means of chlorination and, if necessary, other treatment of the...

  14. 21 CFR 1250.89 - Swimming pools.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... equipped so as to provide complete circulation, replacement, and filtration of the water in the pool every six hours or less. Suitable means of chlorination and, if necessary, other treatment of the water... types of salt water pools shall be so operated that complete circulation and replacement of the water...

  15. 21 CFR 1250.89 - Swimming pools.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... equipped so as to provide complete circulation, replacement, and filtration of the water in the pool every six hours or less. Suitable means of chlorination and, if necessary, other treatment of the water... types of salt water pools shall be so operated that complete circulation and replacement of the water...

  16. A Training Program for Swimming Pool Operators.

    ERIC Educational Resources Information Center

    Pope, James R., Jr.; Mihalik, Brian J.

    1985-01-01

    In the United States today, there is a dramatic shortage of qualified public swimming pool operators. This article describes a training program initiated in South Carolina to serve the needs of everyone responsible for and involved in the safe operation and management of a public swimming pool. (MT)

  17. A homogenous nature of native Chinese duck matrilineal pool

    PubMed Central

    2008-01-01

    Background China, with around 30 unique breeds, has a diverse duck genetic pool. Currently, there is no systematic report which investigates the genetic diversity, phylogenetic relationship, and matrilineal genetic structure of these domestic breeds and wild mallards (Anas platyrhynchos). Results In this study, we sequenced the mitochondrial DNA (mtDNA) control region segments in 278 domestic ducks (Anas platyrhynchos domestica) from 19 indigenous breeds/populations and 70 wild mallard samples and analyzed them together with the 101 control region sequences from published sources. Fifty-two samples were then sequenced for a cytochrome b (Cyt b) gene fragment to solidify the pattern emerged from the control region sequences. All domestic duck and wild mallard haplotypes were essentially indistinguishable and were clustered together in the phylogenetic tree. There was no geographic differentiation and breed/population-specific distribution of duck lineages. Conclusion Our results showed that unlike other domesticated farm animals in China such as chicken, cattle, goat, and yak with multiple matrilineal components, the matrilineal pool of Chinese ducks was homogenous. PMID:18957137

  18. An Enhanced Synthetic Multiclade DNA Prime Induces Improved Cross-Clade-Reactive Functional Antibodies when Combined with an Adjuvanted Protein Boost in Nonhuman Primates

    PubMed Central

    Wise, Megan C.; Hutnick, Natalie A.; Pollara, Justin; Myles, Devin J. F.; Williams, Constance; Yan, Jian; LaBranche, Celia C.; Khan, Amir S.; Sardesai, Niranjan Y.; Montefiori, David; Barnett, Susan W.; Zolla-Pazner, Susan; Ferrari, Guido

    2015-01-01

    ABSTRACT The search for an efficacious human immunodeficiency virus type 1 (HIV-1) vaccine remains a pressing need. The moderate success of the RV144 Thai clinical vaccine trial suggested that vaccine-induced HIV-1-specific antibodies can reduce the risk of HIV-1 infection. We have made several improvements to the DNA platform and have previously shown that improved DNA vaccines alone are capable of inducing both binding and neutralizing antibodies in small-animal models. In this study, we explored how an improved DNA prime and recombinant protein boost would impact HIV-specific vaccine immunogenicity in rhesus macaques (RhM). After DNA immunization with either a single HIV Env consensus sequence or multiple constructs expressing HIV subtype-specific Env consensus sequences, we detected both CD4+ and CD8+ T-cell responses to all vaccine immunogens. These T-cell responses were further increased after protein boosting to levels exceeding those of DNA-only or protein-only immunization. In addition, we observed antibodies that exhibited robust cross-clade binding and neutralizing and antibody-dependent cellular cytotoxicity (ADCC) activity after immunization with the DNA prime-protein boost regimen, with the multiple-Env formulation inducing a more robust and broader response than the single-Env formulation. The magnitude and functionality of these responses emphasize the strong priming effect improved DNA immunogens can induce, which are further expanded upon protein boost. These results support further study of an improved synthetic DNA prime together with a protein boost for enhancing anti-HIV immune responses. IMPORTANCE Even with effective antiretroviral drugs, HIV remains an enormous global health burden. Vaccine development has been problematic in part due to the high degree of diversity and poor immunogenicity of the HIV Env protein. Studies suggest that a relevant HIV vaccine will likely need to induce broad cellular and humoral responses from a simple vaccine

  19. Combined experimental and computational analysis of DNA damage signaling reveals context-dependent roles for Erk in apoptosis and G1/S arrest after genotoxic stress.

    PubMed

    Tentner, Andrea R; Lee, Michael J; Ostheimer, Gerry J; Samson, Leona D; Lauffenburger, Douglas A; Yaffe, Michael B

    2012-01-01

    Following DNA damage, cells display complex multi-pathway signaling dynamics that connect cell-cycle arrest and DNA repair in G1, S, or G2/M phase with phenotypic fate decisions made between survival, cell-cycle re-entry and proliferation, permanent cell-cycle arrest, or cell death. How these phenotypic fate decisions are determined remains poorly understood, but must derive from integrating genotoxic stress signals together with inputs from the local microenvironment. To investigate this in a systematic manner, we undertook a quantitative time-resolved cell signaling and phenotypic response study in U2OS cells receiving doxorubicin-induced DNA damage in the presence or absence of TNFα co-treatment; we measured key nodes in a broad set of DNA damage signal transduction pathways along with apoptotic death and cell-cycle regulatory responses. Two relational modeling approaches were then used to identify network-level relationships between signals and cell phenotypic events: a partial least squares regression approach and a complementary new technique which we term 'time-interval stepwise regression.' Taken together, the results from these analysis methods revealed complex, cytokine-modulated inter-relationships among multiple signaling pathways following DNA damage, and identified an unexpected context-dependent role for Erk in both G1/S arrest and apoptotic cell death following treatment with this commonly used clinical chemotherapeutic drug. PMID:22294094

  20. Hydrology of the Floral City Pool of Tsala Apopka Lake, west-central Florida

    USGS Publications Warehouse

    Bradner, L.A.

    1988-01-01

    Tsala Apopka Lake, in west-central Florida, has an area of about 19,000 acres and is divided into three water-management pools, with the Floral City Pool, the most upgradient. The Floral City Pool, which has a surface area of approximately 4,750 acres, contains an extensive combination of lakes, wetlands, and connecting canals. The Pool receives inflow from the Withlacoochee River through two canals. Outflow is through one manmade canal and one natural slough. Canal flow is partially controlled by manmade structures. A cumulative deficit of 19.4 inches of rainfall from August 1984 through May 1985 reduced surface-water inflow to the Floral City Pool to about 0.5 cu ft/sec by May 1985. During May 1985, pool levels declined approximately 0.04 ft/day. By the end of May, there was no observable outflow. From June 1985 through September 1985, 39.8 inches of rainfall caused above-average inflow to the Floral City Pool and a pool-level increase of 6.2 ft. The inflow of 340 CFS nearly equaled the outflow of 338 CFS by the end of September. (USGS)

  1. A biosignature suite from cave pool precipitates, Cottonwood Cave, New Mexico.

    PubMed

    Melim, L A; Liescheidt, R; Northup, D E; Spilde, M N; Boston, P J; Queen, J M

    2009-11-01

    Calcite cave pool precipitates often display a variety of potential biosignatures from the macroscopic to the submicroscopic. A fossil cave pool in Cottonwood Cave, New Mexico, exhibits older stalactites and stalagmites that are completely coated in brown, laminated calcitic crust that extends down as pool fingers and u-loops. The pool fingers and u-loops are mainly micrite to clotted micrite, some recrystallized to microspar, with some isopachous spar layers. Micrite, particularly clotted micrite, is usually interpreted by carbonate workers as microbial in origin. Scanning electron microscopy examination of etched pool fingers, u-loops, and the brown crust revealed abundant calcified microbial filaments and biofilm. Energy dispersive X-ray analysis showed that these features have excess carbon, above that found in pure calcite. Independent carbon analysis indicated that these same samples contain up to 0.2% organic carbon. Since pool fingers hang down but form underwater, we hypothesize they are biogenic with hanging microbial filaments or biofilm acting as nuclei for calcite precipitation. Because of the abundance of micrite and fossil filaments, we further hypothesize that these pendant features formed during a period of plentiful nutrients and active hydrological activity when the pool was literally dripping with microbial slime. Although each of these lines of evidence could be interpreted in other ways, their combined weight strongly suggests the cave pool precipitates in Cottonwood Cave are biogenic. These investigations can be used to help inform extraterrestrial life-detection studies. PMID:19968466

  2. A magnetotelluric model of the Mana Pools basin, northern Zimbabwe

    NASA Astrophysics Data System (ADS)

    Bailey, D.; Whaler, K. A.; Zengeni, T.; Jones, P. C.; Gwavava, O.

    2000-05-01

    The Mana Pools sedimentary basin lies within the Zambezi mobile belt in northern Zimbabwe. New and preexisting magnetotelluric data and the available seismic reflection data are used to constrain the basin structure and the depth to the electrical basement. Long-period magnetotelluric (LMT) data were collected at five stations along a 60 km north-south profile across the Mana Pools basin and onto the southern escarpment. These data augment an existing audiofrequency (AMT) data set from 11 sites in the same area. The subsurface apparent resistivities measured at periods sampling the basin are very low (a few Ωm). After processing both data sets, the estimated impedance tensor is decomposed, showing that the resistivity structure of the Mana Pools basin can be modeled two dimensionally. The ρ+ algorithm is used to show that there is no systematic offset in magnitude between the AMT and LMT data sets before they are combined. Minimum structure resistivity models of the Mana Pools basin compare well with the information from reflection seismic data and support its previous description as a half graben basin of ˜7 km depth. The excellent conductor in the Mana Pools basin is quite different to those seen elsewhere in the orogenic belt in that it is a feature of the sedimentary fill rather than the basement. The resistivity of the basement is low but no localized good conductor is observed; these low resistivities may result from a high degree of either chemical or tectonic alteration to the underlying rocks due to metamorphic processes and tectonic disruption during rift formation.

  3. The Tropical Warm Pool International Cloud Experiment

    SciTech Connect

    May, Peter T.; Mather, James H.; Vaughan, Geraint; Jakob, Christian; McFarquhar, Greg; Bower, Keith; Mace, Gerald G.

    2008-05-01

    One of the most complete data sets describing tropical convection ever collected will result from the upcoming Tropical Warm Pool International Cloud Experiment (TWP-ICE) in the area around Darwin, Northern Australia in January and February 2006. The aims of the experiment, which will be operated in conjunction with the DOE Atmospheric Radiation Measurement (ARM) site in Darwin, will be to examine convective cloud systems from their initial stages through to the decay of the cirrus generated and to measure their impact on the environment. The experiment will include an unprecedented network of ground-based observations (soundings, active and passive remote sensors) combined with low, mid and high altitude aircraft for in-situ and remote sensing measurements. A crucial outcome of the experiment will be a data set suitable to provide the forcing and evaluation data required by cloud resolving and single column models as well as global climate models (GCMs) with the aim to contribute to parameterization development. This data set will provide the necessary link between the observed cloud properties and the models that are attempting to simulate them. The experiment is a large multi-agency experiment including substantial contributions from the United States DOE ARM program, ARM-UAV program, NASA, the Australian Bureau of Meteorology, CSIRO, EU programs and many universities.

  4. Assessment of the role of DNA repair in damaged forensic samples.

    PubMed

    Ambers, Angie; Turnbough, Meredith; Benjamin, Robert; King, Jonathan; Budowle, Bruce

    2014-11-01

    Previous studies on DNA damage and repair have involved in vitro laboratory procedures that induce a single type of lesion in naked templates. Although repair of singular, sequestered types of DNA damage has shown some success, forensic and ancient specimens likely contain a number of different types of lesions. This study sought to (1) develop protocols to damage DNA in its native state, (2) generate a pool of candidate samples for repair that more likely emulate authentic forensic samples, and (3) assess the ability of the PreCR(TM) Repair Mix to repair the resultant lesions. Complexed, native DNA is more difficult to damage than naked DNA. Modified procedures included the use of higher concentrations and longer exposure times. Three types of samples, those that demonstrated damage based on short tandem repeat (STR) profile signals, were selected for repair experiments: environmentally damaged bloodstains, bleach-damaged whole blood, and human skeletal remains. Results showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR(TM) assay. The data suggest that the use of PreCR in casework should be considered with caution due to the assay's varied results. PMID:24792635

  5. BAC sequencing using pooled methods.

    PubMed

    Saski, Christopher A; Feltus, F Alex; Parida, Laxmi; Haiminen, Niina

    2015-01-01

    Shotgun sequencing and assembly of a large, complex genome can be both expensive and challenging to accurately reconstruct the true genome sequence. Repetitive DNA arrays, paralogous sequences, polyploidy, and heterozygosity are main factors that plague de novo genome sequencing projects that typically result in highly fragmented assemblies and are difficult to extract biological meaning. Targeted, sub-genomic sequencing offers complexity reduction by removing distal segments of the genome and a systematic mechanism for exploring prioritized genomic content through BAC sequencing. If one isolates and sequences the genome fraction that encodes the relevant biological information, then it is possible to reduce overall sequencing costs and efforts that target a genomic segment. This chapter describes the sub-genome assembly protocol for an organism based upon a BAC tiling path derived from a genome-scale physical map or from fine mapping using BACs to target sub-genomic regions. Methods that are described include BAC isolation and mapping, DNA sequencing, and sequence assembly. PMID:25239741

  6. Design report on SCDAP/RELAP5 model improvements - debris bed and molten pool behavior

    SciTech Connect

    Allison, C.M.; Rempe, J.L.; Chavez, S.A.

    1994-11-01

    the SCDAP/RELAP5/MOD3 computer code is designed to describe the overall reactor coolant system thermal-hydraulic response, core damage progression, and in combination with VICTORIA, fission product release and transport during severe accidents. Improvements for existing debris bed and molten pool models in the SCDAP/RELAP5/MOD3.1 code are described in this report. Model improvements to address (a) debris bed formation, heating, and melting; (b) molten pool formation and growth; and (c) molten pool crust failure are discussed. Relevant data, existing models, proposed modeling changes, and the anticipated impact of the changes are discussed. Recommendations for the assessment of improved models are provided.

  7. Core-concrete molten pool dynamics and interfacial heat transfer. [PWR; BWR

    SciTech Connect

    Benjamin, A.S.

    1980-01-01

    Theoretical models are derived for the heat transfer from molten oxide pools to an underlying concrete surface and from molten steel pools to a general concrete containment. To accomplish this, two separate effects models are first developed, one emphasizing the vigorous agitation of the molten pool by gases evolving from the concrete and the other considering the insulating effect of a slag layer produced by concrete melting. The resulting algebraic expressions, combined into a general core-concrete heat transfer representation, are shown to provide very good agreement with experiments involving molten steel pours into concrete crucibles.

  8. Apparatus for draining lower drywell pool water into suppresion pool in boiling water reactor

    DOEpatents

    Gluntz, Douglas M.

    1996-01-01

    An apparatus which mitigates temperature stratification in the suppression pool water caused by hot water drained into the suppression pool from the lower drywell pool. The outlet of a spillover hole formed in the inner bounding wall of the suppression pool is connected to and in flow communication with one end of piping. The inlet end of the piping is above the water level in the suppression pool. The piping is routed down the vertical downcomer duct and through a hole formed in the thin wall separating the downcomer duct from the suppression pool water. The piping discharge end preferably has an elevation at or near the bottom of the suppression pool and has a location in the horizontal plane which is removed from the point where the piping first emerges on the suppression pool side of the inner bounding wall of the suppression pool. This enables water at the surface of the lower drywell pool to flow into and be discharged at the bottom of the suppression pool.

  9. 10 CFR 36.63 - Pool water purity.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 1 2011-01-01 2011-01-01 false Pool water purity. 36.63 Section 36.63 Energy NUCLEAR... § 36.63 Pool water purity. (a) Pool water purification system must be run sufficiently to maintain the conductivity of the pool water below 20 microsiemens per centimeter under normal circumstances. If pool...

  10. 10 CFR 36.63 - Pool water purity.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 1 2014-01-01 2014-01-01 false Pool water purity. 36.63 Section 36.63 Energy NUCLEAR... § 36.63 Pool water purity. (a) Pool water purification system must be run sufficiently to maintain the conductivity of the pool water below 20 microsiemens per centimeter under normal circumstances. If pool...

  11. 10 CFR 36.63 - Pool water purity.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 1 2013-01-01 2013-01-01 false Pool water purity. 36.63 Section 36.63 Energy NUCLEAR... § 36.63 Pool water purity. (a) Pool water purification system must be run sufficiently to maintain the conductivity of the pool water below 20 microsiemens per centimeter under normal circumstances. If pool...

  12. 13 CFR 120.1709 - Transfers of Pool Certificates.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Establishment of SBA Secondary Market Guarantee Program for First Lien Position 504 Loan Pools § 120.1709... transfer. The Pool Investor must supply the following information in the letter: (1) Pool number; (2) Pool Certificate number; (3) Name of purchaser of Pool Certificate; (4) Address and tax identification number...

  13. Swimming Pools. A Guide to Their Planning, Design and Operation.

    ERIC Educational Resources Information Center

    Gabrielsen, M. Alexander, Ed.

    Information is presented regarding all phases of swimming pool development and operation from earliest planning considerations to final programing. This comprehensive book covers--(1) the steps involved in planning a pool, (2) designing the pool, (3) water circulation, filtration, and treatment, (4) community pools, school and agency pools, and…

  14. DNA precursor compartmentation in mammalian cells: metabolic and antimetabolic studies of nuclear and mitochondrial DNA synthesis

    SciTech Connect

    Bestwick, R.K.

    1983-01-01

    HeLa cells were used for the quantitation of cellular and mitochondrial deoxyribonucleoside triphosphate (dNTP) and ribonucleoside triphosphate (rNTP) pools and of changes in pools in response to treatment with the antimetabolites methotrexate (mtx) and 5-fluorodeoxyuridine (FUdR). Use of an enzymatic assay of dNTPs and of improved nucleotide extraction methods allowed quantitation of mitochondrial dNTP pools. All four mitochondrial dNTP pools expand following treatment with mtx or FUdR whereas cellular dTTP and dGTP pools are depleted. Mitochrondrial rNTP pools were also found to expand in response to these antimetabolites. Mouse L-cells were used to determine the relative contributions of an exogenously supplied precursor to nuclear and mitochrondrial DNA replication. Cells were labeled to near steady state specific activities with /sup 32/P-orthophosphate and subsequently labeled with (/sup 3/H)uridine, a general pyrimidine precursor, in the continuing presence of /sup 32/P. Deoxyribonucleoside monophosphates derived from these DNAs were separated by HPLC and the /sup 3/H//sup 32/P ratio in each pyrimidine determined. The dCMP residues in mitochondrial DNA (mtDNA) were found to be derived exclusively from the exogenous supplied uridine. The dTMP residues from nuclear and mtDNA and the dCMP residues from nuclear DNA were seen to be synthesized partly from exogenous sources and partly from other sources, presumably de novo pyrimidine synthesis.

  15. Pool-Riffle Formation in Mountain Streams

    NASA Astrophysics Data System (ADS)

    Chartrand, S. M.; Hassan, M. A.

    2014-12-01

    Pool-riffle formation in low-sinuosity mountain streams is thought to occur through at least two different mechanisms: (1) a transient flow obstruction converges flow, drives pool development and results in an upstream and downstream riffle, (2) variations in channel or valley width set up converging and expanding zones of flow, which leads to pool and riffle development, respectively. These two mechanisms require a width gradient which is not always present, or obviously so along all pool-riffle channel reaches. We propose a third formative mechanism to account for nearly uniform channel width profiles which depends on a spatially discrete and strong change in the bed surface grain size distribution that is characterized as coarse and perhaps better sorted than prevailing conditions. We are testing each of these formative mechanisms plus a no forcing alternative with physical experimentation and numerical modeling. We will present initial observations and findings from our work, focused on addressing the following three questions: (a) does each formative mechanism result in pool-riffle pair development? (b) if a pool-riffle pair forms, are the associated perturbations to the flow and sediment transport fields sufficient for development of additional pool-riffle pairs? and (c) are resultant morphologies comparable in terms of geometries?

  16. Using the ubiquitous pH meter combined with a loop mediated isothermal amplification method for facile and sensitive detection of Nosema bombycis genomic DNA PTP1.

    PubMed

    Xie, Shunbi; Yuan, Yali; Song, Yue; Zhuo, Ying; Li, Tian; Chai, Yaqin; Yuan, Ruo

    2014-12-28

    Here we show an amplification-coupled detection method for directly measuring released hydrogen ions during the loop mediated isothermal amplification (LAMP) procedure by using a pH meter. The genomic DNA of Nosema bombycis (N. bombycis) was amplified and detected by employing this LAMP-pH meter platform for the first time. PMID:25381873

  17. Floral and macroecological evolution within Cyrtanthus (Amaryllidaceae) inferences from combined analyses of plastid ndhF and nrDNA ITS sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One of the most diverse members of Amaryllidaceae is Cyrtanthus Aiton, a large, sub-Saharan Africa genus of approximately 55 species found mostly in South Africa. To investigate phylogenetic and biogeographic relationships within Cyrtanthus, sequence data from the plastid ndhF gene and the ITS nrDNA...

  18. Aberrant DNA Methylation of P16, MGMT, and hMLH1 Genes in Combination with MTHFR C677T Genetic Polymorphism in gastric cancer

    PubMed Central

    Song, Binbin; Ai, Jiang; Kong, Xianghong; Liu, Dexin; Li, Jun

    2013-01-01

    Objective: We aimed to explore the association of P16, MGMT and HMLH1 with gastric cancer and their relation with Methylenetetrahydrofolate reductase (MTHFR). Methods: 322 gastric patients who were confirmed with pathological diagnosis were included in our study. Aberrant DNA methylation of P16, MGMT and HMLH1 and polymorphisms of MTHFR C677T and A1298C were detected using PCR-RFLP. Results: The proportions of DNA hypermethylation in P16, MGMT and hMLH1 genes in gastric cancer tissues were 75.2% (242/322), 27.6% (89/322) and 5.3% (17/322), respectively. In the remote normal-appearing tissues, 29.5% (95/322) and 16.1%(52/322) showed hypermethylation in P16 and MGMT genes, respectively. We found a significantly higher proportion of DNA hypermethylation of P16 in patients with N1 TNM stage in cancer tissues and remote normal-appearing tissues (P<0.05). Similarly, we found DNA hypermethylation of MGMT had significantly higher proportion in N1 and M1 TNM stage (P<0.05). Individuals with homozygotes (TT) of MTHFR C677T had significant risk of DNA hypermethylation of MGMT in cancer tissues [OR (95% CI)=4.27(1.76-7.84)], and a significant risk was also found in those carrying MTHFR 677CT/TT genotype [OR (95% CI)= 3.27(1.21-4.77)]. Conclusion: We found the aberrant hypermethylation of cancer-related genes, such as P16, MGMT and HMLH1, could be predictive biomarkers for detection of gastric cancer. PMID:24550949

  19. Forensic DNA Profiling and Database

    PubMed Central

    Panneerchelvam, S.; Norazmi, M.N.

    2003-01-01

    The incredible power of DNA technology as an identification tool had brought a tremendous change in crimnal justice . DNA data base is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. This article discusses the essential steps in compilation of COmbined DNA Index System (CODIS) on validated polymerase chain amplified STRs and their use in crime detection. PMID:23386793

  20. Airways disorders and the swimming pool.

    PubMed

    Bougault, Valérie; Boulet, Louis-Philippe

    2013-08-01

    Concerns have been expressed about the possible detrimental effects of chlorine derivatives in indoor swimming pool environments. Indeed, a controversy has arisen regarding the possibility that chlorine commonly used worldwide as a disinfectant favors the development of asthma and allergic diseases. The effects of swimming in indoor chlorinated pools on the airways in recreational and elite swimmers are presented. Recent studies on the influence of swimming on airway inflammation and remodeling in competitive swimmers, and the phenotypic characteristics of asthma in this population are reviewed. Preventative measures that could potentially reduce the untoward effects of pool environment on airways of swimmers are discussed. PMID:23830132

  1. Genotoxic Effects in Swimmers Exposed to Disinfection By-products in Indoor Swimming Pools

    PubMed Central

    Kogevinas, Manolis; Villanueva, Cristina M.; Font-Ribera, Laia; Liviac, Danae; Bustamante, Mariona; Espinoza, Felicidad; Nieuwenhuijsen, Mark J.; Espinosa, Aina; Fernandez, Pilar; DeMarini, David M.; Grimalt, Joan O.; Grummt, Tamara; Marcos, Ricard

    2010-01-01

    Background Exposure to disinfection by-products (DBPs) in drinking water has been associated with cancer risk. A recent study (Villanueva et al. 2007; Am J Epidemiol 165:148–156) found an increased bladder cancer risk among subjects attending swimming pools relative to those not attending. Objectives We evaluated adults who swam in chlorinated pools to determine whether exposure to DBPs in pool water is associated with biomarkers of genotoxicity. Methods We collected blood, urine, and exhaled air samples from 49 nonsmoking adult volunteers before and after they swam for 40 min in an indoor chlorinated pool. We estimated associations between the concentrations of four trihalomethanes (THMs) in exhaled breath and changes in micronuclei (MN) and DNA damage (comet assay) in peripheral blood lymphocytes before and 1 hr after swimming; urine mutagenicity (Ames assay) before and 2 hr after swimming; and MN in exfoliated urothelial cells before and 2 weeks after swimming. We also estimated associations and interactions with polymorphisms in genes related to DNA repair or to DBP metabolism. Results After swimming, the total concentration of the four THMs in exhaled breath was seven times higher than before swimming. The change in the frequency of micronucleated lymphocytes after swimming increased in association with higher exhaled concentrations of the brominated THMs (p = 0.03 for bromodichloromethane, p = 0.05 for chlorodibromomethane, p = 0.01 for bromoform) but not chloroform. Swimming was not associated with DNA damage detectable by the comet assay. Urine mutagenicity increased significantly after swimming, in association with the higher concentration of exhaled bromoform (p = 0.004). We found no significant associations with changes in micronucleated urothelial cells. Conclusions Our findings support potential genotoxic effects of exposure to DBPs from swimming pools. The positive health effects gained by swimming could be increased by reducing the potential health

  2. DNA Oligonucleotide 3'-Phosphorylation by a DNA Enzyme.

    PubMed

    Camden, Alison J; Walsh, Shannon M; Suk, Sarah H; Silverman, Scott K

    2016-05-10

    T4 polynucleotide kinase is widely used for 5'-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3'-phosphorylation. Here, we report the in vitro selection of deoxyribozymes (DNA enzymes) capable of DNA oligonucleotide 3'-phosphorylation, using a 5'-triphosphorylated RNA transcript (pppRNA) as the phosphoryl donor. The basis of selection was the capture, during each selection round, of the 3'-phosphorylated DNA substrate terminus by 2-methylimidazole activation of the 3'-phosphate (forming 3'-MeImp) and subsequent splint ligation with a 5'-amino DNA oligonucleotide. Competing and precedented DNA-catalyzed reactions were DNA phosphodiester hydrolysis or deglycosylation, each also leading to a 3'-phosphate but at a different nucleotide position within the DNA substrate. One oligonucleotide 3'-kinase deoxyribozyme, obtained from an N40 random pool and named 3'Kin1, can 3'-phosphorylate nearly any DNA oligonucleotide substrate for which the 3'-terminus has the sequence motif 5'-NKR-3', where N denotes any oligonucleotide sequence, K = T or G, and R = A or G. These results establish the viabilty of in vitro selection for identifying DNA enzymes that 3'-phosphorylate DNA oligonucleotides. PMID:27063020

  3. Photolysis of inorganic chloramines and efficiency of trichloramine abatement by UV treatment of swimming pool water.

    PubMed

    Soltermann, Fabian; Widler, Tobias; Canonica, Silvio; von Gunten, Urs

    2014-06-01

    Trichloramine, one of the three inorganic chloramines (mono-, di- and trichloramine), is a problematic disinfection by-product in recreational pool water since it causes skin and eye irritations as well as irritations of the respiratory tract. The most commonly used chloramine mitigation strategy in pool water is UV treatment. Experiments with membrane inlet mass spectrometry (MIMS) confirmed that inorganic chloramines are effectively degraded by UV irradiation with low-pressure (LP) and medium-pressure (MP) mercury lamps (apparent quantum yields (QY): NH2Cl = 0.50 (LP) and 0.31 (MP) mol einstein(-1), NHCl2: 1.06 (LP) and 0.85 (MP) mol einstein(-1)). Trichloramine showed the fastest depletion with a quantum yield slightly above 2 mol einstein(-1) in purified (LP and MP) and pool water (MP). This high quantum yield can partly be explained by reactions involving OH radicals (purified water) and the reaction of trichloramine with moieties formed during UV irradiation of pool water. The presence of free chlorine affects trichloramine degradation (QY: ∼1.5 mol einstein(-1)) since it scavenges OH radicals and competes with trichloramine for reactive species (e.g. organic amines). Measurements in a pool facility revealed that the installed UV reactors degraded trichloramine by 40-50% as expected from laboratory experiments. However, trichloramine reduction in the pools was less pronounced than in the UV reactors. Model calculations combining pool hydraulics with formation/abatement of trichloramine showed that there was a fast trichloramine formation in the pool from the residual chlorine and nitrogenous precursors. The main factors influencing trichloramine concentrations in pool water are the free chlorine concentration and the UV treatment in combination with the recirculation rate through the water treatment system. PMID:24699420

  4. Viruses-to-mobile genetic elements skew in the deep Atlantis II brine pool sediments.

    PubMed

    Adel, Mustafa; Elbehery, Ali H A; Aziz, Sherry K; Aziz, Ramy K; Grossart, Hans-Peter; Siam, Rania

    2016-01-01

    The central rift of the Red Sea has 25 brine pools with different physical and geochemical characteristics. Atlantis II (ATIID), Discovery Deeps (DD) and Chain Deep (CD) are characterized by high salinity, temperature and metal content. Several studies reported microbial communities in these brine pools, but few studies addressed the brine pool sediments. Therefore, sediment cores were collected from ATIID, DD, CD brine pools and an adjacent brine-influenced site. Sixteen different lithologic sediment sections were subjected to shotgun DNA pyrosequencing to generate 1.47 billion base pairs (1.47 × 10(9) bp). We generated sediment-specific reads and attempted to annotate all reads. We report the phylogenetic and biochemical uniqueness of the deepest ATIID sulfur-rich brine pool sediments. In contrary to all other sediment sections, bacteria dominate the deepest ATIID sulfur-rich brine pool sediments. This decrease in virus-to-bacteria ratio in selected sections and depth coincided with an overrepresentation of mobile genetic elements. Skewing in the composition of viruses-to-mobile genetic elements may uniquely contribute to the distinct microbial consortium in sediments in proximity to hydrothermally active vents of the Red Sea and possibly in their surroundings, through differential horizontal gene transfer. PMID:27596223

  5. Viruses-to-mobile genetic elements skew in the deep Atlantis II brine pool sediments

    PubMed Central

    Adel, Mustafa; Elbehery, Ali H. A.; Aziz, Sherry K.; Aziz, Ramy K.; Grossart, Hans-Peter; Siam, Rania

    2016-01-01

    The central rift of the Red Sea has 25 brine pools with different physical and geochemical characteristics. Atlantis II (ATIID), Discovery Deeps (DD) and Chain Deep (CD) are characterized by high salinity, temperature and metal content. Several studies reported microbial communities in these brine pools, but few studies addressed the brine pool sediments. Therefore, sediment cores were collected from ATIID, DD, CD brine pools and an adjacent brine-influenced site. Sixteen different lithologic sediment sections were subjected to shotgun DNA pyrosequencing to generate 1.47 billion base pairs (1.47 × 109 bp). We generated sediment-specific reads and attempted to annotate all reads. We report the phylogenetic and biochemical uniqueness of the deepest ATIID sulfur-rich brine pool sediments. In contrary to all other sediment sections, bacteria dominate the deepest ATIID sulfur-rich brine pool sediments. This decrease in virus-to-bacteria ratio in selected sections and depth coincided with an overrepresentation of mobile genetic elements. Skewing in the composition of viruses-to-mobile genetic elements may uniquely contribute to the distinct microbial consortium in sediments in proximity to hydrothermally active vents of the Red Sea and possibly in their surroundings, through differential horizontal gene transfer. PMID:27596223

  6. Unique Prokaryotic Consortia in Geochemically Distinct Sediments from Red Sea Atlantis II and Discovery Deep Brine Pools

    PubMed Central

    Siam, Rania; Mustafa, Ghada A.; Sharaf, Hazem; Moustafa, Ahmed; Ramadan, Adham R.; Antunes, Andre; Bajic, Vladimir B.; Stingl, Uli; Marsis, Nardine G. R.; Coolen, Marco J. L.; Sogin, Mitchell; Ferreira, Ari J. S.; Dorry, Hamza El

    2012-01-01

    The seafloor is a unique environment, which allows insights into how geochemical processes affect the diversity of biological life. Among its diverse ecosystems are deep-sea brine pools - water bodies characterized by a unique combination of extreme conditions. The ‘polyextremophiles’ that constitute the microbial assemblage of these deep hot brines have not been comprehensively studied. We report a comparative taxonomic analysis of the prokaryotic communities of the sediments directly below the Red Sea brine pools, namely, Atlantis II, Discovery, Chain Deep, and an adjacent brine-influenced site. Analyses of sediment samples and high-throughput pyrosequencing of PCR-amplified environmental 16S ribosomal RNA genes (16S rDNA) revealed that one sulfur (S)-rich Atlantis II and one nitrogen (N)-rich Discovery Deep section contained distinct microbial populations that differed from those found in the other sediment samples examined. Proteobacteria, Actinobacteria, Cyanobacteria, Deferribacteres, and Euryarchaeota were the most abundant bacterial and archaeal phyla in both the S- and N-rich sections. Relative abundance-based hierarchical clustering of the 16S rDNA pyrotags assigned to major taxonomic groups allowed us to categorize the archaeal and bacterial communities into three major and distinct groups; group I was unique to the S-rich Atlantis II section (ATII-1), group II was characteristic for the N-rich Discovery sample (DD-1), and group III reflected the composition of the remaining sediments. Many of the groups detected in the S-rich Atlantis II section are likely to play a dominant role in the cycling of methane and sulfur due to their phylogenetic affiliations with bacteria and archaea involved in anaerobic methane oxidation and sulfate reduction. PMID:22916172

  7. Mitochondrial deoxynucleotide pools in quiescent fibroblasts: a possible model for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).

    PubMed

    Ferraro, Paola; Pontarin, Giovanna; Crocco, Laura; Fabris, Sonia; Reichard, Peter; Bianchi, Vera

    2005-07-01

    Mitochondrial (mt) DNA depletion syndromes can arise from genetic deficiencies for enzymes of dNTP metabolism, operating either inside or outside mitochondria. MNGIE is caused by the deficiency of cytosolic thymidine phosphorylase that degrades thymidine and deoxyuridine. The extracellular fluid of the patients contains 10-20 microM deoxynucleosides leading to changes in dTTP that may disturb mtDNA replication. In earlier work, we suggested that mt dTTP originates from two distinct pathways: (i) the reduction of ribonucleotides in the cytosol (in cycling cells) and (ii) intra-mt salvage of thymidine (in quiescent cells). In MNGIE and most other mtDNA depletion syndromes, quiescent cells are affected. Here, we demonstrate in quiescent fibroblasts (i) the existence of small mt dNTP pools, each usually 3-4% of the corresponding cytosolic pool; (ii) the rapid metabolic equilibrium between mt and cytosolic pools; and (iii) the intra-mt synthesis and rapid turnover of dTTP in the absence of DNA replication. Between 0.1 and 10 microM extracellular thymidine, intracellular thymidine rapidly approaches the extracellular concentration. We mimic the conditions of MNGIE by maintaining quiescent fibroblasts in 10-40 microM thymidine and/or deoxyuridine. Despite a large increase in intracellular thymidine concentration, cytosolic and mt dTTP increase at most 4-fold, maintaining their concentration for 41 days. Other dNTPs are marginally affected. Deoxyuridine does not increase the normal dNTP pools but gives rise to a small dUTP and a large dUMP pool, both turning over rapidly. We discuss these results in relation to MNGIE. PMID:15878850

  8. Postdoctoral Pool Growth Reviewed in Academy Reports.

    ERIC Educational Resources Information Center

    Henig, Robin Marantz

    1979-01-01

    Two National Research Council studies investigate the growing number of postdoctoral trainees, particularly in the biomedical fields, and the implications of this postdoctoral pool growth for federal support of pre- and postdoctoral research training. (BB)

  9. Fire safety distances for open pool fires

    NASA Astrophysics Data System (ADS)

    Sudheer, S.; Kumar, Lokendra; Manjunath, B. S.; Pasi, Amit; Meenakshi, G.; Prabhu, S. V.

    2013-11-01

    Fire accidents that carry huge loss with them have increased in the previous two decades than at any time in the history. Hence, there is a need for understanding the safety distances from different fires with different fuels. Fire safety distances are computed for different open pool fires. Diesel, gasoline and hexane are used as fuels for circular pool diameters of 0.5 m, 0.7 m and 1.0 m. A large square pool fire of 4 m × 4 m is also conducted with diesel as a fuel. All the prescribed distances in this study are purely based on the thermal analysis. IR camera is used to get the thermal images of pool fires and there by the irradiance at different locations is computed. The computed irradiance is presented with the threshold heat flux limits for human beings.

  10. Investigations in Marine Chemistry: Tide Pool Ecology.

    ERIC Educational Resources Information Center

    Schlenker, Richard M.

    Students investigated the salinity of tide pools at different levels in the intertidal zone. Data are analyzed collectively. Students graphed and discussed data. Included are suggestions for evaluation and further study. (Author)

  11. Identification and characterization of new DNA G-quadruplex binders selected by a combination of ligand and structure-based virtual screening approaches.

    PubMed

    Alcaro, Stefano; Musetti, Caterina; Distinto, Simona; Casatti, Margherita; Zagotto, Giuseppe; Artese, Anna; Parrotta, Lucia; Moraca, Federica; Costa, Giosuè; Ortuso, Francesco; Maccioni, Elias; Sissi, Claudia

    2013-02-14

    Nowadays, it has been demonstrated that DNA G-quadruplex arrangements are involved in cellular aging and cancer, thus boosting the discovery of selective binders for these DNA secondary structures. By taking advantage of available structural and biological information on these structures, we performed a high throughput in silico screening of commercially available molecules databases by merging ligand- and structure-based approaches by means of docking experiments. Compounds selected by the virtual screening procedure were then tested for their ability to interact with the human telomeric G-quadruplex folding by circular dichroism, fluorescence spectroscopy, and photodynamic techniques. Interestingly, our screening succeeded in retrieving a new promising scaffold for G-quadruplex binders characterized by a psoralen moiety. PMID:23294188

  12. Welding pool measurement using thermal array sensor

    NASA Astrophysics Data System (ADS)

    Cho, Chia-Hung; Hsieh, Yi-Chen; Chen, Hsin-Yi

    2015-08-01

    Selective laser melting (SLM) is an additive manufacturing (AM) technology that uses a high-power laser beam to melt metal powder in chamber of inert gas. The process starts by slicing the 3D CAD data as a digital information source into layers to create a 2D image of each layer. Melting pool was formed by using laser irradiation on metal powders which then solidified to consolidated structure. In a selective laser melting process, the variation of melt pool affects the yield of a printed three-dimensional product. For three dimensional parts, the border conditions of the conductive heat transport have a very large influence on the melt pool dimensions. Therefore, melting pool is an important behavior that affects the final quality of the 3D object. To meet the temperature and geometry of the melting pool for monitoring in additive manufacturing technology. In this paper, we proposed the temperature sensing system which is composed of infrared photodiode, high speed camera, band-pass filter, dichroic beam splitter and focus lens. Since the infrared photodiode and high speed camera look at the process through the 2D galvanometer scanner and f-theta lens, the temperature sensing system can be used to observe the melting pool at any time, regardless of the movement of the laser spot. In order to obtain a wide temperature detecting range, 500 °C to 2500 °C, the radiation from the melting pool to be measured is filtered into a plurality of radiation portions, and since the intensity ratio distribution of the radiation portions is calculated by using black-body radiation. The experimental result shows that the system is suitable for melting pool to measure temperature.

  13. Electromagnetic Interference in a Private Swimming Pool

    PubMed Central

    Iskandar, Sandia; Lavu, Madhav; Atoui, Moustapha; Lakkireddy, Dhanunjaya

    2016-01-01

    Although current lead design and filtering capabilities have greatly improved, Electromagnetic Interference (EMI) from environmental sources has been increasingly reported in patients with Cardiac Implantable Electronic Device (CIED) [1]. Few cases of inappropriate intracardiac Cardioverter Defibrillator (ICD) associated with swimming pool has been described [2]. Here we present a case of 64 year old male who presented with an interesting EMI signal that was subsequently identified to be related to AC current leak in his swimming pool. PMID:27479205

  14. Performance Study of Swimming Pool Heaters

    SciTech Connect

    McDonald, R.J.

    2009-01-01

    The objective of this report is to perform a controlled laboratory study on the efficiency and emissions of swimming pool heaters based on a limited field investigation into the range of expected variations in operational parameters. Swimming pool heater sales trends have indicated a significant decline in the number of conventional natural gas-fired swimming pool heaters (NGPH). On Long Island the decline has been quite sharp, on the order of 50%, in new installations since 2001. The major portion of the decline has been offset by a significant increase in the sales of electric powered heat pump pool heaters (HPPH) that have been gaining market favor. National Grid contracted with Brookhaven National Laboratory (BNL) to measure performance factors in order to compare the relative energy, environmental and economic consequences of using one technology versus the other. A field study was deemed inappropriate because of the wide range of differences in actual load variations (pool size), geographic orientations, ground plantings and shading variations, number of hours of use, seasonal use variations, occupancy patterns, hour of the day use patterns, temperature selection, etc. A decision was made to perform a controlled laboratory study based on a limited field investigation into the range of expected operational variations in parameters. Critical to this are the frequency of use, temperature selection, and sizing of the heater to the associated pool heating loads. This would be accomplished by installing a limited amount of relatively simple compact field data acquisition units on selected pool installations. This data included gas usage when available and alternately heater power or gas consumption rates were inferred from the manufacturer's specifications when direct metering was not available in the field. Figure 1 illustrates a typical pool heater installation layout.

  15. How to map your industry's profit pool.

    PubMed

    Gadiesh, O; Gilbert, J L

    1998-01-01

    Many managers chart strategy without a full understanding of the sources and distribution of profits in their industry. Sometimes they focus their sights on revenues instead of profits, mistakenly assuming that revenue growth will eventually translate into profit growth. In other cases, they simply lack the data or the analytical tools required to isolate and measure variations in profitability. In this Manager's Tool Kit, the authors present a way to think clearly about where the money's being made in any industry. They describe a framework for analyzing how profits are distributed among the activities that form an industry's value chain. Such an analysis can provide a company's managers with a rich understanding of their industry's profit structure--what the authors call its profit pool--enabling them to identify which activities are generating disproportionately large or small shares of profits. Even more important, a profit-pool map opens a window onto the underlying structure of the industry, helping managers see the various forces that are determining the distribution of profits. As such, a profit-pool map provides a solid basis for strategic thinking. Mapping a profit pool involves four steps: defining the boundaries of the pool, estimating the pool's overall size, estimating the size of each value-chain activity in the pool, and checking and reconciling the calculations. The authors briefly describe each step and then apply the process by providing a detailed example of a hypothetical retail bank. They conclude by looking at ways of organizing the data in chart form as a first step toward plotting a profit-pool strategy. PMID:10179650

  16. Profit pools: a fresh look at strategy.

    PubMed

    Gadiesh, O; Gilbert, J L

    1998-01-01

    In charting strategy, many managers focus on revenue growth, assuming that profits will follow. But that approach is dangerous: today's deep revenue pool may become tomorrow's dry hole. To create strategies that result in profitable growth, managers need to look beyond revenues to see the shape of their industry's profit pool. The authors define an industry's profit pool as the total profits earned at all points along the industry's value chain. Although the concept is simple, the structure of a profit pool is usually quite complex. The pool will be deeper in some segments of the value chain than in others, and depths will vary within an individual segment as well. Segment profitability may, for example, vary widely by customer group, product category, geographic market, and distribution channel. Moreover, the pattern of profit concentration in an industry will often be very different from the pattern of revenue concentration. The authors describe how successful companies have gained competitive advantage by developing sophisticated profit-pool strategies. They explain how U-Haul identified new sources of profit in the consumer-truck-rental industry; how Merck reached beyond its traditional value-chain role to protect its profits in the pharmaceuticals industry; how Dell rebounded from a misguided channel decision by refocusing on its traditional source of profit; and how Anheuser-Busch made a series of astute product, pricing, and operating decisions to dominate the beer industry's profit pool. The companies with the best understanding of their industry's profit pool, the authors argue, will be in the best position to thrive over the long term. PMID:10179649

  17. Phylogenetic Analysis of the Hoplolaiminae Inferred from Combined D2 and D3 Expansion Segments of 28S rDNA.

    PubMed

    Bae, C H; Szalanski, A L; Robbins, R T

    2009-03-01

    DNA sequences of the D2-D3 expansion segments of the 28S gene of ribosomal DNA from 23 taxa of the subfamily Hoplolaiminae were obtained and aligned to infer phylogenetic relationships. The D2 and D3 expansion regions are G-C rich (59.2%), with up to 20.7% genetic divergence between Scutellonema brachyurum and Hoplolaimus concaudajuvencus. Molecular phylogenetic analysis using maximum likelihood and maximum parsimony was conducted using the D2-D3 sequence data. Of 558 characters, 254 characters (45.5%) were variable and 198 characters (35.4%) were parsimony informative. All phylogenetic methods produced a similar topology with two distinct clades: One clade consists of all Hoplolaimus species while the other clade consists of the rest of the studied Hoplolaiminae genera. This result suggests that Hoplolaimus is monophyletic. Another clade consisted of Aorolaimus, Helicotylenchus, Rotylenchus, and Scutellonema species. Phylogenetic analysis using the outgroup species Globodera rostocheinsis suggests that Hoplolaiminae is paraphyletic. In this study, the D2-D3 region had levels of DNA sequence divergence sufficient for phylogenetic analysis and delimiting species of Hoplolaiminae. PMID:22661775

  18. Combined QM/MM investigation on the light-driven electron-induced repair of the (6-4) thymine dimer catalyzed by DNA photolyase.

    PubMed

    Faraji, Shirin; Groenhof, Gerrit; Dreuw, Andreas

    2013-09-01

    The (6-4) photolyases are blue-light-activated enzymes that selectively bind to DNA and initiate splitting of mutagenic thymine (6-4) thymine photoproducts (T(6-4)T-PP) via photoinduced electron transfer from flavin adenine dinucleotide anion (FADH(-)) to the lesion triggering repair. In the present work, the repair mechanism after the initial electron transfer and the effect of the protein/DNA environment are investigated theoretically by means of hybrid quantum mechanical/molecular mechanical (QM/MM) simulations using X-ray structure of the enzyme-DNA complex. By comparison of three previously proposed repair mechanisms, we found that the lowest activation free energy is required for the pathway in which the key step governing the repair photocycle is electron transfer coupled with the proton transfer from the protonated histidine, His365, to the N3' nitrogen of the pyrimidone thymine. The transfer simultaneously occurs with concerted intramolecular OH transfer without formation of an oxetane or isolated water molecule intermediate. In contrast to previously suggested mechanisms, this newly identified pathway requires neither a subsequent two-photon process nor electronic excitation of the photolesion. PMID:23915283

  19. Characterisation of the Permafrost Carbon Pool

    USGS Publications Warehouse

    Kuhry, P.; Grosse, G.; Harden, J.W.; Hugelius, G.; Koven, C.D.; Ping, C.-L.; Schirrmeister, L.; Tarnocai, C.

    2013-01-01

    The current estimate of the soil organic carbon (SOC) pool in the northern permafrost region of 1672 Petagrams (Pg) C is much larger than previously reported and needs to be incorporated in global soil carbon (C) inventories. The Northern Circumpolar Soil Carbon Database (NCSCD), extended to include the range 0–300 cm, is now available online for wider use by the scientific community. An important future aim is to provide quantitative uncertainty ranges for C pool estimates. Recent studies have greatly improved understanding of the regional patterns, landscape distribution and vertical (soil horizon) partitioning of the permafrost C pool in the upper 3 m of soils. However, the deeper C pools in unconsolidated Quaternary deposits need to be better constrained. A general lability classification of the permafrost C pool should be developed to address potential C release upon thaw. The permafrost C pool and its dynamics are beginning to be incorporated into Earth System models, although key periglacial processes such as thermokarst still need to be properly represented to obtain a better quantification of the full permafrost C feedback on global climate change.

  20. Pool Boiling Experiment Has Five Successful Flights

    NASA Technical Reports Server (NTRS)

    Chiaramonte, Fran

    1997-01-01

    The Pool Boiling Experiment (PBE) is designed to improve understanding of the fundamental mechanisms that constitute nucleate pool boiling. Nucleate pool boiling is a process wherein a stagnant pool of liquid is in contact with a surface that can supply heat to the liquid. If the liquid absorbs enough heat, a vapor bubble can be formed. This process occurs when a pot of water boils. On Earth, gravity tends to remove the vapor bubble from the heating surface because it is dominated by buoyant convection. In the orbiting space shuttle, however, buoyant convection has much less of an effect because the forces of gravity are very small. The Pool Boiling Experiment was initiated to provide insight into this nucleate boiling process, which has many earthbound applications in steamgeneration power plants, petroleum plants, and other chemical plants. In addition, by using the test fluid R-113, the Pool Boiling Experiment can provide some basic understanding of the boiling behavior of cryogenic fluids without the large cost of an experiment using an actual cryogen.

  1. Pool Boiling Experiment Has Successful Flights

    NASA Technical Reports Server (NTRS)

    1996-01-01

    The Pool Boiling Experiment (PBE) is designed to improve understanding of the fundamental mechanisms that constitute nucleate pool boiling. Nucleate pool boiling is a process wherein a stagnant pool of liquid is in contact with a surface that can supply heat to the liquid. If the liquid absorbs enough heat, a vapor bubble can be formed. This process occurs when a pot of water boils. On Earth, gravity tends to remove the vapor bubble from the heating surface because it is dominated by buoyant convection. In the orbiting space shuttle, however, buoyant convection has much less of an effect because the forces of gravity are very small. The Pool Boiling Experiment was initiated to provide insight into this nucleate boiling process, which has many Earthbound applications, such as steam-generation power plants, petroleum, and other chemical plants. Also, by using the test fluid R-113, the Pool Boiling Experiment can provide some basic understanding of the boiling behavior of cryogenic fluids without the large cost of an experiment using an actual cryogen.

  2. Occurrence of enteroviruses in community swimming pools.

    PubMed Central

    Keswick, B H; Gerba, C P; Goyal, S M

    1981-01-01

    Municipal swimming pools and wading pools were examined for the presence of human enteric viruses using a portable virus concentrator at the site to concentrate viruses from 100-gallon to 500-gallon samples. Ten of 14 samples contained viruses; three of these were positive for virus in the presence of residual free chlorine. Enteroviruses were isolated from two pools which exceeded the 0.4 ppm free residual chlorine standard. This study appears to be supportive of recent evidence that indicates a higher incidence of enterovirus infection among bathers. All seven wading pool samples contained virus. Coxsackieviruses B3 and B4, poliovirus 1, and echovirus 7 were isolated. Total coliform bacteria were not adequate indicators of the presence of virus, as six of the samples were positive for virus but negative for coliforms. Total plate counts appeared to provide a better indication of the sanitary quality of the pool water, but viruses could still be detected in samples that met currently recommended bacterial levels. It is possible that swimming and wading pools may serve as a means of transmission of enteroviral disease, especially in children, during summer months. PMID:6267950

  3. Molecular identification of potential pathogens in water and air of a hospital therapy pool

    PubMed Central

    Angenent, Largus T.; Kelley, Scott T.; Amand, Allison St.; Pace, Norman R.; Hernandez, Mark T.

    2005-01-01

    Indoor warm-water therapy pool workers in a Midwestern regional hospital were diagnosed with non-tuberculosis pulmonary hypersensitive pneumonitis and Mycobacterium avium infections. In response, we conducted a multiseason survey of microorganisms present in this therapy pool water, in biofilms associated with the pool containment walls, and in air immediately above the pool. The survey used culture, microscopy, and culture-independent molecular phylogenetic analyses. Although outfitted with a state-of-the-art UV-peroxide disinfection system, the numbers of bacteria in the therapy pool water were relatively high compared with the potable water used to fill the pool. Regardless of the source, direct microscopic counts of microbes were routinely ≈1,000 times greater than conventional plate counts. Analysis of clone libraries of small subunit rRNA genes from environmental DNA provided phylogenetic diversity estimates of the microorganisms collected in and above the pool. A survey of >1,300 rRNA genes yielded a total of 628 unique sequences, the most common of which was nearly identical to that of M. avium strains. The high proportion of clones with different Mycobacterium spp. rRNA genes suggested that such organisms comprised a significant fraction of microbes in the pool water (to >30%) and preferentially partition into aerosols (to >80%) relative to other waterborne bacteria present. The results of the study strongly validate aerosol partitioning as a mechanism for disease transfer in these environments. The results also show that culture protocols currently used by public health facilities and agencies are seriously inadequate for the detection and enumeration of potential pathogens. PMID:15769858

  4. Regulation of power pools and system operators: An international comparison

    SciTech Connect

    Barker, J. Jr.; Tenenbaum, B.; Woolf, F.

    1997-12-31

    This paper focuses on the governance and regulation of power pools outside the United States. The current governance and regulatory arrangements for four power pools, as developed in pool documents and government regulations and laws, are compared and contrasted. The power pools analyzed are located in England and Wales, Australia, Canada, and Scandinavia. Topics discussed in relation to these pools are the effects of structure on governance, how each pool has dealt with a number of basic governance decisions, how the pools monitor the markets, ways in which regulators and other institutions control pools, and self-governance issues.

  5. The UNITAID Patent Pool Initiative: Bringing Patents Together for the Common Good.

    PubMed

    Bermudez, Jorge; 't Hoen, Ellen

    2010-01-01

    Developing and delivering appropriate, affordable, well-adapted medicines for HIV/AIDS remains an urgent challenge: as first-line therapies fail, increasing numbers of people require costly second-line therapy; one-third of ARVs are not available in pediatric formulations; and certain key first- and second-line triple fixed-dose combinations do not exist or sufficient suppliers are lacking. UNITAID aims to help solve these problems through an innovative initiative for the collective management of intellectual property (IP) rights - a patent pool for HIV medicines. The idea behind a patent pool is that patent holders - companies, governments, researchers or universities - voluntarily offer, under certain conditions, the IP related to their inventions to the patent pool. Any company that wants to use the IP to produce or develop medicines can seek a license from the pool against the payment of royalties, and may then produce the medicines for use in developing countries (conditional upon meeting agreed quality standards). The patent pool will be a voluntary mechanism, meaning its success will largely depend on the willingness of pharmaceutical companies to participate and commit their IP to the pool. Generic producers must also be willing to cooperate. The pool has the potential to provide benefits to all. PMID:20309404

  6. Stroke Treatment Academic Industry Roundtable Recommendations for Individual Data Pooling Analyses in Stroke.

    PubMed

    Lees, Kennedy R; Khatri, Pooja

    2016-08-01

    Pooled analysis of individual patient data from stroke trials can deliver more precise estimates of treatment effect, enhance power to examine prespecified subgroups, and facilitate exploration of treatment-modifying influences. Analysis plans should be declared, and preferably published, before trial results are known. For pooling trials that used diverse analytic approaches, an ordinal analysis is favored, with justification for considering deaths and severe disability jointly. Because trial pooling is an incremental process, analyses should follow a sequential approach, with statistical adjustment for iterations. Updated analyses should be published when revised conclusions have a clinical implication. However, caution is recommended in declaring pooled findings that may prejudice ongoing trials, unless clinical implications are compelling. All contributing trial teams should contribute to leadership, data verification, and authorship of pooled analyses. Development work is needed to enable reliable inferences to be drawn about individual drug or device effects that contribute to a pooled analysis, versus a class effect, if the treatment strategy combines ≥2 such drugs or devices. Despite the practical challenges, pooled analyses are powerful and essential tools in interpreting clinical trial findings and advancing clinical care. PMID:27406108

  7. Measurement of convectional heat transfer coefficients in a primary containment vessel with outer pool

    SciTech Connect

    Fukui, Toru; Kataoka, Yoshiyuki; Hatamiya, Shigeo

    1990-01-01

    New concepts with passive safety systems that use no active compounds, such as pumps, have been recently developed for next-generation nuclear power plants. In these concepts, several ideas and their combination of passive components were adopted for emergency core cooling and residual heat removal systems. For the residual heat removal system, utilization of natural circulation heat transfer in water pools was proposed as a passive containment cooling system (PCCS), which removes decay heat from the primary containment vessel (PCV) during loss-of-coolant accidents (LOCAs). This system consists of a suppression pool (S/P) and an outer pool (O/P), which are set adjacently inside and outside of the steel PCV wall. The core decay heat during LOCA is released through a break as steam and is led into the S/P. The injected steam condenses there, resulting a pool temperature rise. The adsorbed heat in the S/P is transferred to the O/P by convection in both pools and thermal conduction through the steel PCV wall. The heat transferred to the O/P is finally released to the atmosphere by vaporization of the O/P water. Estimation of the convectional heat transfer coefficients in both pools is necessary to predict the heat removal capability in this system precisely. The heat transfer coefficients measured in this study are useful for the design of the next-generation nuclear reactor as the fundamental thermal-hydraulic data in the primary containment vessel with the outer pool.

  8. Modulation of the equilibrative nucleoside transporter by inhibitors of DNA synthesis.

    PubMed Central

    Pressacco, J.; Wiley, J. S.; Jamieson, G. P.; Erlichman, C.; Hedley, D. W.

    1995-01-01

    Expression of the equilibrative, S-(p-nitrobenzyl)-6-thioinosine (NBMPR)-sensitive nucleoside transporter (es), a component of the nucleoside salvage pathway, was measured during unperturbed growth and following exposure to various antimetabolites at growth-inhibitory concentrations. The probe 5-(SAENTA-x8)-fluorescein is a highly modified form of adenosine incorporating a fluorescein molecule. It binds. with high affinity and specificity to the (es) nucleoside transporter at a 1:1 stoichiometry, allowing reliable estimates of es expression by flow cytometry. Using a dual labelling technique which combined the vital DNA dye Hoechst-33342 and 5-(SAENTA-x8)-fluorescein, we found that surface expression of es approximately doubled between G1 and G2 + M phases of the cell cycle. To address the question of whether es expression could be modulated in cells exposed to drugs which inhibit de novo synthesis of nucleotides, cells were exposed to antimetabolite drugs having different modes of action. Hydroxyurea and 5-fluorouracil (5-FU), which inhibit the de novo synthesis of DNA precursors, produced increases in the expression of es. In contrast, cytosine arabinoside (ara-C) and aphidicolin, which directly inhibit DNA synthesis, produced no significant increase in es expression. Thymidine (TdR), which is an allosteric inhibitor of ribonucleotide reductase that depletes dATP, dCTP and dGTP pools while repleting the dTTP pool, had no significant effect on es expression. These data suggest that surface expression of the es nucleoside transporter is regulated by a mechanism which is sensitive to the supply of deoxynucleotides. Because 5-FU (which specifically depletes dTTP pools) causes a large increase in expression whereas TdR (which depletes all precursors except dTTP) does not, this mechanism might be particularly sensitive to dTTP pools. PMID:7547244

  9. A high-fidelity approach towards simulation of pool boiling

    NASA Astrophysics Data System (ADS)

    Yazdani, Miad; Radcliff, Thomas; Soteriou, Marios; Alahyari, Abbas A.

    2016-01-01

    A novel numerical approach is developed to simulate the multiscale problem of pool-boiling phase change. The particular focus is to develop a simulation technique that is capable of predicting the heat transfer and hydrodynamic characteristics of nucleate boiling and the transition to critical heat flux on surfaces of arbitrary shape and roughness distribution addressing a critical need to design enhanced boiling heat transfer surfaces. The macro-scale of the phase change and bubble dynamics is addressed through employing off-the-shelf Computational Fluid Dynamics (CFD) methods for interface tracking and interphase mass and energy transfer. The micro-scale of the microlayer, which forms at early stage of bubble nucleation near the wall, is resolved through asymptotic approximation of the thin-film theory which provides a closed-form solution for the distribution of the micro-layer and its influence on the evaporation process. In addition, the sub-grid surface roughness is represented stochastically through probabilistic density functions and its role in bubble nucleation and growth is then represented based on the thermodynamics of nucleation process. This combination of deterministic CFD, local approximation, and stochastic representation allows the simulation of pool boiling on any surface with known roughness and enhancement characteristics. The numerical model is validated for dynamics and hydrothermal characteristics of a single nucleated bubble on a flat surface against available literature data. In addition, the prediction of pool-boiling heat transfer coefficient is verified against experimental measurements as well as reputable correlations for various roughness distributions and different surface orientations. Finally, the model is employed to demonstrate pool-boiling phenomenon on enhanced structures with reentrance cavities and to explore the effect of enhancement feature design on thermal and hydrodynamic characteristics of these surfaces.

  10. Alkali metal pool boiler life tests for a 25 kWe advanced Stirling conversion system

    NASA Astrophysics Data System (ADS)

    Anderson, W. G.; Rosenfeld, J. H.; Noble, J.

    The overall operating temperature and efficiency of solar-powered Stirling engines can be improved by adding an alkali metal pool boiler heat transport system to supply heat more uniformly to the heater head tubes. One issue with liquid metal pool boilers is unstable boiling. Stable boiling is obtained with an enhanced boiling surface containing nucleation sites that promote continuous boiling. Over longer time periods, it is possible that the boiling behavior of the system will change. An 800-h life test was conducted to verify that pool boiling with the chosen fluid/surface combination remains stable as the system ages. The apparatus uses NaK boiling on a - 100 + 140 stainless steel sintered porous layer, with the addition of a small amount of xenon. Pool boiling remained stable to the end of life test. The pool boiler life test included a total of 82 cold starts, to simulate startup each morning, and 60 warm restarts, to simulate cloud cover transients. The behavior of the cold and warm starts showed no significant changes during the life test. In the experiments, the fluid/surface combination provided stable, high-performance boiling at the operating temperature of 700 C. Based on these experiments, a pool boiler was designed for a full-scale 25-kWe Stirling system.

  11. Alkali metal pool boiler life tests for a 25 kWe advanced Stirling conversion system

    NASA Technical Reports Server (NTRS)

    Anderson, W. G.; Rosenfeld, J. H.; Noble, J.

    1991-01-01

    The overall operating temperature and efficiency of solar-powered Stirling engines can be improved by adding an alkali metal pool boiler heat transport system to supply heat more uniformly to the heater head tubes. One issue with liquid metal pool boilers is unstable boiling. Stable boiling is obtained with an enhanced boiling surface containing nucleation sites that promote continuous boiling. Over longer time periods, it is possible that the boiling behavior of the system will change. An 800-h life test was conducted to verify that pool boiling with the chosen fluid/surface combination remains stable as the system ages. The apparatus uses NaK boiling on a - 100 + 140 stainless steel sintered porous layer, with the addition of a small amount of xenon. Pool boiling remained stable to the end of life test. The pool boiler life test included a total of 82 cold starts, to simulate startup each morning, and 60 warm restarts, to simulate cloud cover transients. The behavior of the cold and warm starts showed no significant changes during the life test. In the experiments, the fluid/surface combination provided stable, high-performance boiling at the operating temperature of 700 C. Based on these experiments, a pool boiler was designed for a full-scale 25-kWe Stirling system.

  12. Ribonucleotide triggered DNA damage and RNA-DNA damage responses

    PubMed Central

    Wallace, Bret D; Williams, R Scott

    2014-01-01

    Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage. PMID:25692233

  13. How cold pool triggers deep convection?

    NASA Astrophysics Data System (ADS)

    Yano, Jun-Ichi

    2014-05-01

    The cold pool in the boundary layer is often considered a major triggering mechanism of convection. Here, presented are basic theoretical considerations on this issue. Observations suggest that cold pool-generated convective cells is available for shallow maritime convection (Warner et al. 1979; Zuidema et al. 2012), maritime deep convection (Barnes and Garstang 1982; Addis et al. 1984; Young et al. 1995) and continental deep convection (e.g., Lima and Wilson 2008; Flamant 2009; Lothon et al. 2011; Dione et al. 2013). Moreover, numerical studies appear to suggest that cold pools promote the organization of clouds into larger structures and thereby aid the transition from shallow to deep convection (Khairoutdinov and Randall 2006, Boing et al. 2012, Schlemmer and Hohenegger, 2014). Even a cold--pool parameterization coupled with convection is already proposed (Grandpeix and Lafore 2010: but see also Yano 2012). However, the suggested link between the cold pool and deep convection so far is phenomenological at the best. A specific process that the cold pool leads to a trigger of deep convection must still to be pinned down. Naively, one may imagine that a cold pool lifts up the air at the front as it propagates. Such an uplifting leads to a trigger of convection. However, one must realize that a shift of air along with its propagation does not necessarily lead to an uplifting, and even if it may happen, it would not far exceed a depth of the cold pool itself. Thus, the uplifting can never be anything vigorous. Its thermodynamic characteristics do help much either for inducing convection. The cold-pool air is rather under rapid recovering process before it can induce convection under a simple parcel-lifting argument. The most likely reason that the cold pool may induce convection is its gust winds that may encounter an air mass from an opposite direction. This induces a strong convergence, also leading to a strong uplifting. This is an argument essentially developed

  14. Building a Better Applicant Pool--A Case Study of the Use of Predictive Modeling and Market Segmentation to Build and Enroll Better Pools of Students

    ERIC Educational Resources Information Center

    Herridge, Bart; Heil, Robert

    2003-01-01

    Predictive modeling has been a popular topic in higher education for the last few years. This case study shows an example of an effective use of modeling combined with market segmentation to strategically divide large, unmanageable prospect and inquiry pools and convert them into applicants, and eventually, enrolled students. (Contains 6 tables.)

  15. Induction of immune tolerance in asthmatic mice by vaccination with DNA encoding an allergen-cytotoxic T lymphocyte-associated antigen 4 combination.

    PubMed

    Zhang, Fang; Huang, Gang; Hu, Bo; Song, Yong; Shi, Yi

    2011-05-01

    Allergen-specific immunotherapy is a potential treatment for allergic diseases. We constructed an allergen-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)-encoding DNA vaccine, administered it directly to antigen-presenting cells (APCs), and investigated its ability and mechanisms to ameliorate allergic airway inflammation in an asthmatic mouse model. An allergen-CTLA-4 DNA plasmid (OVA-CTLA-4-pcDNA₃.₁) encoding an ovalbumin (OVA) and the mouse CTLA-4 extracellular domain was constructed and transfected into COS-7 cells to obtain the fusion protein OVA-CTLA-4, which was able to bind the B7 ligand on dendritic cells (DCs), and induced CD25⁺ Foxp3⁺ regulatory T (Treg) cells by the coculture of naive CD4⁺ T cells with DCs in vitro. In an animal study, BALB/c mice were sensitized and challenged with OVA to establish the asthmatic model. Vaccination with a high dose of OVA-CTLA-4-pcDNA₃.₁ significantly decreased interleukin-4 (IL-4) and IL-5 levels and eosinophil counts and prevented OVA-induced reduction of the gamma interferon level in the bronchoalveolar lavage fluid. In addition, these mice suffered less severe airway inflammation and had lower levels of OVA-specific IgE and IgG1 titers in serum. Also, high-dose OVA-CTLA-4-pcDNA₃.₁ vaccination inhibited the development of airway hyperreactivity and prevented OVA-induced reduction of the percentages of Foxp3⁺ Treg cells in the spleen. Our results indicate that a high dose of allergen-CTLA-4-encoding DNA vaccine was more effective in preventing an allergen-induced Th2-skewed immune response through the induction of Treg cells and may be a new alternative therapy for asthma. PMID:21346053

  16. Coupling dynamic blow down and pool evaporation model for LNG.

    PubMed

    Woodward, John L

    2007-02-20

    Treating the dynamic effects of accidental discharges of liquefied natural gas (LNG) is important for realistic predictions of pool radius. Two phenomena have important influence on pool spread dynamics, time-varying discharge (blow down) and pool ignition. Time-varying discharge occurs because a punctured LNG tanker or storage tank drains with a decreasing liquid head and decreasing head-space pressure. Pool ignition increases the evaporation rate of a pool and consequently decreases the ultimate pool area. This paper describes an approach to treat these phenomena in a dynamic pool evaporation model. The pool evaporation model developed here has two separate regimes. Early in the spill, momentum forces dominate and the pool spreads independently of pool evaporation rate and the corresponding heat transfer rate. After the average pool depth drops below a minimum value, momentum forces are largely dissipated and the thin edges of the pool completely evaporate, so pool area is established by the heat transfer rate. The maximum extent of a burning pool is predicted to be significantly less than that of an unignited pool because the duration of the first regime is reduced by higher heat transfer rates. The maximum extent of an LNG pool is predicted to be larger upon accounting for blow down compared with using a constant average discharge rate. However, the maximum pool extent occurs only momentarily before retreating. PMID:17184912

  17. Enhancement of Blood–Brain Barrier Permeability and Delivery of Antisense Oligonucleotides or Plasmid DNA to the Brain by the Combination of Bubble Liposomes and High-Intensity Focused Ultrasound

    PubMed Central

    Negishi, Yoichi; Yamane, Masaya; Kurihara, Naho; Endo-Takahashi, Yoko; Sashida, Sanae; Takagi, Norio; Suzuki, Ryo; Maruyama, Kazuo

    2015-01-01

    The blood–brain barrier (BBB) is a major obstacle that prevents therapeutic drugs or genes from being delivered to the central nervous system. Therefore, it is important to develop methods to enhance the permeability of the BBB. We have developed echo-contrast gas (C3F8) entrapping liposomes (Bubble liposomes, BLs) that can work as a gene delivery tool in combination with ultrasound (US) exposure. Here, we studied whether the permeability of the BBB can be enhanced by the combination of BLs and high-intensity focused ultrasound (HIFU). Mice were intravenously injected with Evans blue (EB). BLs were subsequently injected, and the right hemispheres were exposed to HIFU. As a result, the accumulation of EB in the HIFU-exposed brain hemispheres was increased over that observed in the non-HIFU-exposed hemispheres, depending on the intensity and the duration of the HIFU. Similarly, the combination of BLs and HIFU allowed fluorescent-labeled antisense oligonucleotides to be delivered into the HIFU-exposed left hemispheres of the treated mice. Furthermore, a firefly luciferase-expressing plasmid DNA was delivered to the brain by the combination method of BLs and HIFU, which resulted in the increased gene expression in the brain at the focused-US exposure site. These results suggest that the method of combining BLs and HIFU together serves as a useful means for accelerating the permeability of BBB and thereby enabling antisense oligonucleotides or genes to be delivered to the focused brain site. PMID:26402694

  18. Cleaving DNA with DNA

    NASA Astrophysics Data System (ADS)

    Carmi, Nir; Balkhi, Shameelah R.; Breaker, Ronald R.

    1998-03-01

    A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This ``deoxyribozyme'' can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a kobs of 0.2 min-1, whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple ``restriction enzymes'' for the site-specific cleavage of single-stranded DNA.

  19. Intrauterine devices and endometrial cancer risk: a pooled analysis of the Epidemiology of Endometrial Cancer Consortium

    PubMed Central

    Felix, Ashley S.; Gaudet, Mia M.; La Vecchia, Carlo; Nagle, Christina M.; Ou Shu, Xiao; Weiderpass, Elisabete; Olov Adami, Hans; Beresford, Shirley; Bernstein, Leslie; Chen, Chu; Cook, Linda S.; De Vivo, Immaculata; Doherty, Jennifer A.; Friedenreich, Christine M.; Gapstur, Susan M.; Hill, Dierdre; Horn-Ross, Pamela L.; Lacey, James V.; Levi, Fabio; Liang, Xiaolin; Lu, Lingeng; Magliocco, Anthony; McCann, Susan E.; Negri, Eva; Olson, Sara H.; Palmer, Julie R.; Patel, Alpa V.; Petruzella, Stacey; Prescott, Jennifer; Risch, Harvey A.; Rosenberg, Lynn; Sherman, Mark E.; Spurdle, Amanda B.; Webb, Penelope M.; Wise, Lauren A.; Xiang, Yong-Bing; Xu, Wanghong; Yang, Hannah P.; Yu, Herbert; Zeleniuch-Jacquotte, Anne; Brinton, Louise A.

    2014-01-01

    Intrauterine devices (IUDs), long-acting and reversible contraceptives, induce a number of immunological and biochemical changes in the uterine environment that could affect endometrial cancer (EC) risk. We addressed this relationship through a pooled analysis of data collected in the Epidemiology of Endometrial Cancer Consortium. We combined individual-level data from 4 cohort and 14 case-control studies, in total 8,801 EC cases and 15,357 controls. Using multivariable logistic regression, we estimated pooled odds ratios (pooled-ORs) and 95% confidence intervals (CIs) for EC risk associated with ever use, type of device, ages at first and last use, duration of use, and time since last use, stratified by study and adjusted for confounders. Ever use of IUDs was inversely related to EC risk (pooled-OR=0.81, 95% CI=0.74–0.90). Compared with never use, reduced risk of EC was observed for inert IUDs (pooled-OR=0.69, 95% CI=0.58–0.82), older age at first use (≥35 years pooled-OR=0.53, 95% CI=0.43–0.67), older age at last use (≥45 years pooled-OR=0.60, 95% CI=0.50–0.72), longer duration of use (≥10 years pooled-OR=0.61, 95% CI=0.52–0.71), and recent use (within 1 year of study entry pooled-OR=0.39, 95% CI=0.30–0.49). Future studies are needed to assess the respective roles of detection biases and biologic effects related to foreign body responses in the endometrium, heavier bleeding (and increased clearance of carcinogenic cells), and localized hormonal changes. PMID:25242594

  20. Condensation in a two-phase pool

    SciTech Connect

    Duffey, R.B.; Hughes, E.D.

    1991-12-31

    We consider the case of vapor condensation in a liquid pool, when the heat transfer is controlled by heat losses through the walls. The analysis is based on drift flux theory for phase separation in the pool, and determines the two-phase mixture height for the pool. To our knowledge this is the first analytical treatment of this classic problem that gives an explicit result, previous work having established the result for the evaporative case. From conservation of mass and energy in a one-dimensional steady flow, together with a void relation between the liquid and vapor fluxes, we determine the increase in the mixture level from the base level of the pool. It can be seen that the thermal and hydrodynamic influences are separable. Thus, the thermal influence of the wall heat transfer appears through its effect on the condensing length L*, so that at high condensation rates the pool is all liquid, and at low rates overflows (the level swell or foaming effect). Similarly, the phase separation effect hydrodynamically determines the height via the relative velocity of the mixture to the entering flux. We examine some practical applications of this result to level swell in condensing flows, and also examine some limits in ideal cases.