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Sample records for amplicon va derived

  1. Constitutive and Inducible Innate Responses in Cells Infected by HSV-1-Derived Amplicon Vectors

    PubMed Central

    Tsitoura, Eliza; Epstein, Alberto L

    2010-01-01

    Amplicons are helper-dependent herpes simplex virus type 1 (HSV-1)-based vectors that can deliver very large foreign DNA sequences and, as such, are good candidates both for gene delivery and vaccine development. However, many studies have shown that innate constitutive or induced cellular responses, elicited or activated by the entry of HSV-1 particles, can play a significant role in the control of transgenic expression and in the induction of inflammatory responses. Moreover, transgene expression from helper-free amplicon stocks is often weak and transient, depending on the particular type of infected cells, suggesting that cellular responses could be also responsible for the silencing of amplicon-mediated transgene expression. This review summarizes the current experimental evidence underlying these latter concepts, focusing on the impact on transgene expression of very-early interactions between amplicon particles and the infected cells, and speculates on possible ways to counteract the cellular protective mechanisms, thus allowing stable transgene expression without enhancement of vector toxicity. PMID:20811588

  2. Use of Amplicon-6 Vectors Derived from Human Herpesvirus 6 for Efficient Expression of Membrane-Associated and -Secreted Proteins in T Cells

    PubMed Central

    Borenstein, Ronen; Singer, Oded; Moseri, Adi; Frenkel, Niza

    2004-01-01

    The composite amplicon-6 vectors, which are derived from human herpesvirus 6 (HHV-6), can target hematopoietic cells. In the presence of the respective helper viruses, the amplicons are replicated by the rolling circle mechanism, yielding defective genomes of overall size 135 to 150 kb, composed of multiple repeats of units, containing the viral DNA replication origin, packaging signals, and the selected transgene(s). We report the use of amplicon-6 vectors designed for transgene expression in T cells. The selected transgenes included the green fluorescent protein marker, the herpes simplex virus type 1 glycoprotein D (gD), and the gD gene deleted in the transmembrane region (gDsec). The vectors were tested after electroporation and passage in T cells with or without helper HHV-6A superinfections. The results were as follows. (i)The vectors could be passaged both as cell-associated and as cell-free secreted virions infectious to new cells. (ii)The intact gD accumulated at the cell surface, whereas the gDsec was dispersed at internal locations of the cells or was secreted into the medium. (iii)Analyses of amplicon-6-gD expression by flow cytometry have shown significant expression in cultures with reiterated amplicons and helper viruses. The vector has spread to >60% of the cells, and the efficiency of expression per cell increased 15-fold, most likely due to the presence of concatemeric amplicon repeats. Current studies are designed to test whether amplicon-6 vectors can be used for gene therapy in lymphocytes and whether amplicon-6 vectors expressed in T cells and dendritic cells can induce strong cellular and humoral immune responses. PMID:15078955

  3. Functional Overexpression of Vomeronasal Receptors Using a Herpes Simplex Virus Type 1 (HSV-1)-Derived Amplicon.

    PubMed

    Stein, Benjamin; Alonso, María Teresa; Zufall, Frank; Leinders-Zufall, Trese; Chamero, Pablo

    2016-01-01

    In mice, social behaviors such as mating and aggression are mediated by pheromones and related chemosignals. The vomeronasal organ (VNO) detects olfactory information from other individuals by sensory neurons tuned to respond to specific chemical cues. Receptors expressed by vomeronasal neurons are implicated in selective detection of these cues. Nearly 400 receptor genes have been identified in the mouse VNO, but the tuning properties of individual receptors remain poorly understood, in part due to the lack of a robust heterologous expression system. Here we develop a herpes virus-based amplicon delivery system to overexpress three types of vomeronasal receptor genes and to characterize cell responses to their proposed ligands. Through Ca2+ imaging in native VNO cells we show that virus-induced overexpression of V1rj2, V2r1b or Fpr3 caused a pronounced increase of responsivity to sulfated steroids, MHC-binding peptide or the synthetic hexapeptide W-peptide, respectively. Other related ligands were not recognized by infected individual neurons, indicating a high degree of selectivity by the overexpressed receptor. Removal of G-protein signaling eliminates Ca2+ responses, indicating that the endogenous second messenger system is essential for observing receptor activation. Our results provide a novel expression system for vomeronasal receptors that should be useful for understanding the molecular logic of VNO ligand detection. Functional expression of vomeronasal receptors and their deorphanization provides an essential requirement for deciphering the neural mechanisms controlling behavior. PMID:27195771

  4. Functional Overexpression of Vomeronasal Receptors Using a Herpes Simplex Virus Type 1 (HSV-1)-Derived Amplicon

    PubMed Central

    Stein, Benjamin; Alonso, María Teresa; Zufall, Frank; Leinders-Zufall, Trese; Chamero, Pablo

    2016-01-01

    In mice, social behaviors such as mating and aggression are mediated by pheromones and related chemosignals. The vomeronasal organ (VNO) detects olfactory information from other individuals by sensory neurons tuned to respond to specific chemical cues. Receptors expressed by vomeronasal neurons are implicated in selective detection of these cues. Nearly 400 receptor genes have been identified in the mouse VNO, but the tuning properties of individual receptors remain poorly understood, in part due to the lack of a robust heterologous expression system. Here we develop a herpes virus-based amplicon delivery system to overexpress three types of vomeronasal receptor genes and to characterize cell responses to their proposed ligands. Through Ca2+ imaging in native VNO cells we show that virus-induced overexpression of V1rj2, V2r1b or Fpr3 caused a pronounced increase of responsivity to sulfated steroids, MHC-binding peptide or the synthetic hexapeptide W-peptide, respectively. Other related ligands were not recognized by infected individual neurons, indicating a high degree of selectivity by the overexpressed receptor. Removal of G-protein signaling eliminates Ca2+ responses, indicating that the endogenous second messenger system is essential for observing receptor activation. Our results provide a novel expression system for vomeronasal receptors that should be useful for understanding the molecular logic of VNO ligand detection. Functional expression of vomeronasal receptors and their deorphanization provides an essential requirement for deciphering the neural mechanisms controlling behavior. PMID:27195771

  5. Murine Bone Marrow-Derived Dendritic Cells Transduced by Light-Helper-Dependent Herpes Simplex Virus-1 Amplicon Vector Acquire a Mature Dendritic Cell Phenotype.

    PubMed

    Oz-Arslan, Devrim; Tsitoura, Eliza; Kazazi, Dorothea; Kouvatsis, Vlasis; Epstein, Alberto L; Mavromara, Penelope

    2015-06-01

    Dendritic cells (DCs) turn into the most potent antigen-presenting cells following a complex transforming process, which leads to their maturation. Herpes simplex virus-1 (HSV-1) amplicon vectors represent highly versatile viral vector platforms with the ability to transduce immature DCs at exceedingly high efficiencies, while the efficiency of infection of mature DCs is significantly low. However, the bacterial artificial chromosome (BAC)-dependent (BD) amplicon vectors tested so far do not result in the maturation of mouse bone marrow-derived DCs (BMDCs) in vitro. In this study we investigated the effects of light-helper-dependent (LHD) amplicon vectors produced with the replication-defective HSV-1 LaLΔJ helper virus system. First, we observed that transgene expression in BMDC cultures was equally potent between the LHD and the BD amplicon vectors. We determined that the percentage of transduced cells and the duration of transgene expression were negatively influenced by the presence of increasing levels of helper virus. Second, infection by the LHD amplicon vector as well as the helper HSV-1 LaLΔJ virus alone resulted in the phenotypic maturation of BMDCs and the expression of both interferon-stimulated genes and proinflammatory cytokines. Further comparisons of the gene expression of infected DCs showed that while interferon-stimulated genes such as Ifit1, Ifit3, Mx2, Isg15, and Cxcl10 were induced by both BD and LHD amplicon vectors, early proinflammatory cytokine gene expression (Tnfa, Il1a, Il1b, Il6, Il10, Il12b, Cxcl1, and Cxcl16) and DC maturation were mediated only by the LHD amplicons. PMID:26046494

  6. Mechanisms Regulating Acquisition of Platelet-Derived Factor V/Va by Megakaryocytes.

    PubMed

    Gertz, Jacqueline M; Bouchard, Beth A

    2015-10-01

    Factor Va serves as the nonenzymatic protein cofactor for the prothrombinase complex, which converts prothrombin to thrombin in the events leading to formation of a hemostatic plug. Several observations support the concept that platelet-derived factor V/Va is physically and functionally distinct and plays a more important role in thrombin generation at sites of vascular injury as compared to its plasma counterpart. Platelet-derived factor V/Va is generated following endocytosis of the plasma-derived molecule by the platelet precursor cells, megakaryocytes, via a two receptor system consisting of low density lipoprotein (LDL) receptor-related protein-1 (LRP-1) and an unidentified specific "binding site". More recently, it was suggested that a cell surface-expressed β-galactoside binding protein, galectin-8, was involved in factor V endocytosis. Endocytosed factor V is trafficked through the cell and retailored prior to its storage in α-granules. Given the essential role of platelet-derived factor Va in clot formation, understanding the cellular and molecular mechanisms that regulate how platelets acquire this molecule will be important for the treatment of excessive bleeding or clotting. PMID:25800007

  7. Identification of QTL conditioning partial resistance to white mold in kidney bean line VA19 derived from an interspecific population

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Scarlet-runner bean (Phaseolus coccineus L.), a representative species of the secondary gene pool of common bean, is a potential source of white mold resistance for improving dry bean and snap bean. VA19 is a light-red kidney bean line that possesses resistance to white mold putatively derived from...

  8. Effects of an apovincaminic acid derivative VA-045 on neuronal activity in rat brain stem reticular formation.

    PubMed

    Okuyama, S; Kawashima, N; Araki, H; Otomo, S; Shima, K

    1994-01-01

    Extracellular single unit and spontaneous cortical electroencephalographic (ECoG) recordings were made in the brain stem reticular formation (RF) and the frontal cortex, respectively, of urethane-anesthetized rats. VA-045, an apovincaminic acid derivative had no significant effects on the spontaneous firing rate of the RF neurons and ECoG. Closed head injury (CHI) was induced by dropping a 400 g weight through a tube from 70 cm on a steel helmet placed on the vertex. CHI led to a reduction in the firing rate of RF neurons and ECoG synchronization. VA-045 dose-dependently reversed the CHI-induced decrease in the firing rate of RF and led to ECoG desyncronization. Clonidine, an alpha 2 adrenoceptor agonist, and scopolamine, a muscarinic receptor antagonist, also reduced the firing rate of RF neurons and led to ECoG synchronization. VA-045 dose-dependently antagonized the effects of clonidine, but not the effects of scopolamine on RF neuronal activity and ECoG. Thus, VA-045 has an ameliorating effect on the CHI-induced depression of neuronal activity in the RF. A central alpha 2 adrenoceptor antagonistic action may be involved. PMID:7968229

  9. A Novel Ligustrazine Derivative T-VA Prevents Neurotoxicity in Differentiated PC12 Cells and Protects the Brain against Ischemia Injury in MCAO Rats

    PubMed Central

    Li, Guoling; Tian, Yufei; Zhang, Yuzhong; Hong, Ying; Hao, Yingzhi; Chen, Chunxiao; Wang, Penglong; Lei, Haimin

    2015-01-01

    Broad-spectrum drugs appear to be more promising for the treatment of acute ischemic stroke. In our previous work, a new ligustrazine derivative (3,5,6-trimethylpyrazin-2-yl) methyl 3-methoxy-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzoate (T-VA) showed neuroprotective effect on injured PC12 cells (EC50 = 4.249 µM). In the current study, we show that this beneficial effect was due to the modulation of nuclear transcription factor-κB/p65 (NF-κB/p65) and cyclooxygenase-2 (COX-2) expressions. We also show that T-VA exhibited neuroprotective effect in a rat model of ischemic stroke with concomitant improvement of motor functions. We propose that the protective effect observed in vivo is owing to increased vascular endothelial growth factor (VEGF) expression, decreased oxidative stress, and up-regulation of Ca2+–Mg2+ ATP enzyme activity. Altogether, our results warrant further studies on the utility of T-VA for the potential treatment of ischemic brain injuries, such as stroke. PMID:26370988

  10. The herpes simplex virus amplicon: analyses of cis-acting replication functions.

    PubMed Central

    Spaete, R R; Frenkel, N

    1985-01-01

    Previous studies have shown that defective virus vectors (amplicons) derived from herpes simplex viruses could be efficiently propagated in virus stocks in the presence of trans-acting helper virus functions. The present study established that two separate cis-acting functions--a DNA replication origin and a cleavage/packaging signal--are required for amplicon propagation. Using deleted derivatives of cloned amplicons, we mapped one of the viral DNA replication origins (ori-2 or oriL) at coordinate 0.422 of the standard HSV-1 genome and at an equivalent position within the HSV-2 genome. Images PMID:2983310

  11. Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors

    PubMed Central

    Falcicchia, Chiara; Trempat, Pascal; Binaschi, Anna; Perrier-Biollay, Coline; Roncon, Paolo; Soukupova, Marie; Berthommé, Hervé; Simonato, Michele

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies. PMID:26954758

  12. Co-existence of circular and multiple linear amplicons in methotrexate-resistant Leishmania.

    PubMed Central

    Olmo, A; Arrebola, R; Bernier, V; González-Pacanowska, D; Ruiz-Pérez, L M

    1995-01-01

    Circular and linear amplicons were analyzed in detail in Leishmania tropica cells resistant to methotrexate (MTX). Both types of elements presented sequences related to the H locus and coexisted in resistant cells. The linear amplicons appeared first during the selection process (at 10 microM MTX) and varied with regard to size and structure in cells exposed to increasing concentrations of drug. The circular element was evident at higher concentrations (50 microMs) but was the major amplified DNA in cells resistant to 1000 microM MTX while the level of amplification of the linear elements remained low. The extrachromosomal DNAs were unstable in the absence of drug and their disappearance coincided with an increase in sensitivity to MTX. Mapping of the minichromosomes and the circular element showed that they were all constituted by inverted duplications. The circular amplicon contained an inverted repeat derived from the H locus that encompassed the pteridine reductase gene (PTR1) responsible for MTX resistance. The amplified segment in the linear amplicons was longer and included the pgpB and pgpC genes that encode P-glycoproteins of unknown function previously characterized in different Leishmania species. Images PMID:7659507

  13. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    SciTech Connect

    Gardner, S. N.

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether there are also TaqMan/Luminex probe matches within predicted amplicons.

  14. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    Energy Science and Technology Software Center (ESTSC)

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether theremore » are also TaqMan/Luminex probe matches within predicted amplicons.« less

  15. Virion-associated cofactor high-mobility group DNA-binding protein-1 facilitates transposition from the herpes simplex virus/Sleeping Beauty amplicon vector platform.

    PubMed

    de Silva, Suresh; Lotta, Louis T; Burris, Clark A; Bowers, William J

    2010-11-01

    The development of the integration-competent, herpes simplex virus/Sleeping Beauty (HSV/SB) amplicon vector platform has created a means to efficiently and stably deliver therapeutic transcription units (termed "transgenons") to neurons within the mammalian brain. Furthermore, an investigation into the transposition capacity of the HSV/SB vector system revealed that the amplicon genome provides an optimal substrate for the transposition of transgenons at least 12 kb in length [de Silva, S., Mastrangelo, M.A., Lotta, L.T., Jr., Burris, C.A., Federoff, H.J., and Bowers, W.J. ( 2010 ). Gene Ther. 17, 424-431]. These results prompted an investigation into the factors that may contribute toward efficient transposition from the HSV/SB amplicon. One of the cellular cofactors known to play a key role during SB-mediated transposition is the high-mobility group DNA-binding protein-1 (HMGB1). Our present investigation into the role of HMGB1 during amplicon-based transposition revealed that transposition is not strictly dependent on the presence of cellular HMGB1, contrary to what had been previously demonstrated with plasmid-based SB transposition. We have shown for the first time that during amplicon preparation, biologically active HMGB1 derived from the packaging cell line is copackaged into amplicon vector particles. As a result, HSV/SB amplicon virions arrive prearmed with HMGB1 protein at levels sufficient for facilitating SB-mediated transposition in the transduced mammalian cell. PMID:20568967

  16. Herpes simplex virus amplicon: effect of size on replication of constructed defective genomes containing eucaryotic DNA sequences.

    PubMed Central

    Kwong, A D; Frenkel, N

    1984-01-01

    Previous studies (R. R. Spaete and N. Frenkel, Cell 30:295-304, 1982) have documented the potential use of defective virus vectors (amplicons) derived from herpes simplex virus for the efficient introduction of foreign DNA sequences into eucaryotic cells. Specifically, cotransfection of cells with helper virus DNA and cloned amplicons (8 to 10 kilobases [kb]) containing bacterial plasmid DNA sequences linked to a set of herpes simplex virus cis-acting propagation signals (a replication origin and a cleavage-packaging signal) resulted in the generation of virus stocks containing packaged defective genomes that consisted of uniform head-to-tail reiterations of the chimeric seed amplicon sequences. The chimeric defective genomes could be stably propagated in virus stocks and could thus be used to efficiently infect cells. We now report on additional studies designed to propagate relatively large sets of eucaryotic DNA sequences within chimeric packaged defective genomes. These studies have utilized a 12-kb chicken DNA sequence encoding the chicken ovalbumin gene and cloned by Lai et al. (Proc. Natl. Acad. Sci. U.S.A. 77:244-248, 1980) in the plasmid pOV12. Virus stocks derived from cells cotransfected with helper virus DNA and chimeric amplicons (overall size of 19.8 kb, of which 12 kb corresponded to the chicken DNA) contained defective genomes composed of reiterations of the 19.8-kb seed amplicon sequences. However, in addition to the authentically sized repeat units, defective genomes in the derivative virus stocks contained smaller repeat units representing deleted versions of the seed 19.8-kb amplicons. The recombinational events leading to the formation of deleted repeats did not appear to occur at unique sites, as shown by comparative analyses of multiple, independently generated virus series propagated from separate transfections. In contast, seed amplicons ranging in size from 11 to 15 kb and containing subsets of the 12-kb chicken DNA sequences replicated

  17. Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors

    PubMed Central

    Blanc, Andrea Maria; Berois, Mabel Beatriz; Tomé, Lorena Magalí; Epstein, Alberto L.

    2012-01-01

    Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease. PMID:22437537

  18. Fast, accurate and easy-to-pipeline methods for amplicon sequence processing

    NASA Astrophysics Data System (ADS)

    Antonielli, Livio; Sessitsch, Angela

    2016-04-01

    Next generation sequencing (NGS) technologies established since years as an essential resource in microbiology. While on the one hand metagenomic studies can benefit from the continuously increasing throughput of the Illumina (Solexa) technology, on the other hand the spreading of third generation sequencing technologies (PacBio, Oxford Nanopore) are getting whole genome sequencing beyond the assembly of fragmented draft genomes, making it now possible to finish bacterial genomes even without short read correction. Besides (meta)genomic analysis next-gen amplicon sequencing is still fundamental for microbial studies. Amplicon sequencing of the 16S rRNA gene and ITS (Internal Transcribed Spacer) remains a well-established widespread method for a multitude of different purposes concerning the identification and comparison of archaeal/bacterial (16S rRNA gene) and fungal (ITS) communities occurring in diverse environments. Numerous different pipelines have been developed in order to process NGS-derived amplicon sequences, among which Mothur, QIIME and USEARCH are the most well-known and cited ones. The entire process from initial raw sequence data through read error correction, paired-end read assembly, primer stripping, quality filtering, clustering, OTU taxonomic classification and BIOM table rarefaction as well as alternative "normalization" methods will be addressed. An effective and accurate strategy will be presented using the state-of-the-art bioinformatic tools and the example of a straightforward one-script pipeline for 16S rRNA gene or ITS MiSeq amplicon sequencing will be provided. Finally, instructions on how to automatically retrieve nucleotide sequences from NCBI and therefore apply the pipeline to targets other than 16S rRNA gene (Greengenes, SILVA) and ITS (UNITE) will be discussed.

  19. Systems consequences of amplicon formation in human breast cancer

    PubMed Central

    Inaki, Koichiro; Menghi, Francesca; Woo, Xing Yi; Wagner, Joel P.; Jacques, Pierre-Étienne; Lee, Yi Fang; Shreckengast, Phung Trang; Soon, Wendy WeiJia; Malhotra, Ankit; Teo, Audrey S.M.; Hillmer, Axel M.; Khng, Alexis Jiaying; Ruan, Xiaoan; Ong, Swee Hoe; Bertrand, Denis; Nagarajan, Niranjan; Karuturi, R. Krishna Murthy; Hidalgo Miranda, Alfredo

    2014-01-01

    Chromosomal structural variations play an important role in determining the transcriptional landscape of human breast cancers. To assess the nature of these structural variations, we analyzed eight breast tumor samples with a focus on regions of gene amplification using mate-pair sequencing of long-insert genomic DNA with matched transcriptome profiling. We found that tandem duplications appear to be early events in tumor evolution, especially in the genesis of amplicons. In a detailed reconstruction of events on chromosome 17, we found large unpaired inversions and deletions connect a tandemly duplicated ERBB2 with neighboring 17q21.3 amplicons while simultaneously deleting the intervening BRCA1 tumor suppressor locus. This series of events appeared to be unusually common when examined in larger genomic data sets of breast cancers albeit using approaches with lesser resolution. Using siRNAs in breast cancer cell lines, we showed that the 17q21.3 amplicon harbored a significant number of weak oncogenes that appeared consistently coamplified in primary tumors. Down-regulation of BRCA1 expression augmented the cell proliferation in ERBB2-transfected human normal mammary epithelial cells. Coamplification of other functionally tested oncogenic elements in other breast tumors examined, such as RIPK2 and MYC on chromosome 8, also parallel these findings. Our analyses suggest that structural variations efficiently orchestrate the gain and loss of cancer gene cassettes that engage many oncogenic pathways simultaneously and that such oncogenic cassettes are favored during the evolution of a cancer. PMID:25186909

  20. Systems consequences of amplicon formation in human breast cancer.

    PubMed

    Inaki, Koichiro; Menghi, Francesca; Woo, Xing Yi; Wagner, Joel P; Jacques, Pierre-Étienne; Lee, Yi Fang; Shreckengast, Phung Trang; Soon, Wendy WeiJia; Malhotra, Ankit; Teo, Audrey S M; Hillmer, Axel M; Khng, Alexis Jiaying; Ruan, Xiaoan; Ong, Swee Hoe; Bertrand, Denis; Nagarajan, Niranjan; Karuturi, R Krishna Murthy; Miranda, Alfredo Hidalgo; Liu, Edison T

    2014-10-01

    Chromosomal structural variations play an important role in determining the transcriptional landscape of human breast cancers. To assess the nature of these structural variations, we analyzed eight breast tumor samples with a focus on regions of gene amplification using mate-pair sequencing of long-insert genomic DNA with matched transcriptome profiling. We found that tandem duplications appear to be early events in tumor evolution, especially in the genesis of amplicons. In a detailed reconstruction of events on chromosome 17, we found large unpaired inversions and deletions connect a tandemly duplicated ERBB2 with neighboring 17q21.3 amplicons while simultaneously deleting the intervening BRCA1 tumor suppressor locus. This series of events appeared to be unusually common when examined in larger genomic data sets of breast cancers albeit using approaches with lesser resolution. Using siRNAs in breast cancer cell lines, we showed that the 17q21.3 amplicon harbored a significant number of weak oncogenes that appeared consistently coamplified in primary tumors. Down-regulation of BRCA1 expression augmented the cell proliferation in ERBB2-transfected human normal mammary epithelial cells. Coamplification of other functionally tested oncogenic elements in other breast tumors examined, such as RIPK2 and MYC on chromosome 8, also parallel these findings. Our analyses suggest that structural variations efficiently orchestrate the gain and loss of cancer gene cassettes that engage many oncogenic pathways simultaneously and that such oncogenic cassettes are favored during the evolution of a cancer. PMID:25186909

  1. AmpliconDuo: A Split-Sample Filtering Protocol for High-Throughput Amplicon Sequencing of Microbial Communities

    PubMed Central

    Lange, Anja; Jost, Steffen; Heider, Dominik; Bock, Christina; Budeus, Bettina; Schilling, Elmar; Strittmatter, Axel; Boenigk, Jens; Hoffmann, Daniel

    2015-01-01

    High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches (“AmpliconDuo filter”). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model. PMID:26523925

  2. DNA-based methods to prepare helper virus-free herpes amplicon vectors and versatile design of amplicon vector plasmids.

    PubMed

    Kasai, Kazue; Saeki, Yoshinaga

    2006-06-01

    The herpes simplex virus (HSV) amplicon vector is a versatile plasmid-based gene delivery vehicle with a large transgene capacity (up to 150 kb) and the ability to infect a broad range of cell types. The vector system was originally developed by Frenkel and her colleagues in 1980. Ever since, a great deal of effort by various investigators has been directed at minimizing the toxicity associated with the inevitable contamination by helper virus. In 1996, Fraefel and his colleagues successfully devised a cosmid-based packaging system that was free of contamination by helper virus (so-called helper virus-free packaging), which utilized as helper a set of 5 overlapping cosmid clones that covered the entire HSV genome, which lacked the DNA packaging/cleavage signals. With the helper virus-free system, broader applications of the vector became possible. Cloning of the entire HSV genome in bacteria artificial chromosome (BAC) plasmids enabled stable maintenance and propagation of the helper HSV genome in bacteria. It also allowed for the development of BAC-based helper virus-free packaging systems. In this article, we review various versions of DNA-based methods to prepare HSV amplicon vectors free of helper virus contamination. We also examine recent advances in vector design, including methods of vector construction, hybrid amplicon vectors, and the infectious BAC system. Future directions in improving packaging systems and vector designs are discussed. PMID:16787182

  3. Multiplex amplicon sequencing for microbe identification in community-based culture collections.

    PubMed

    Armanhi, Jaderson Silveira Leite; de Souza, Rafael Soares Correa; de Araújo, Laura Migliorini; Okura, Vagner Katsumi; Mieczkowski, Piotr; Imperial, Juan; Arruda, Paulo

    2016-01-01

    Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC. PMID:27404280

  4. Multiplex amplicon sequencing for microbe identification in community-based culture collections

    PubMed Central

    Armanhi, Jaderson Silveira Leite; de Souza, Rafael Soares Correa; de Araújo, Laura Migliorini; Okura, Vagner Katsumi; Mieczkowski, Piotr; Imperial, Juan; Arruda, Paulo

    2016-01-01

    Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC. PMID:27404280

  5. Mutascope: sensitive detection of somatic mutations from deep amplicon sequencing

    PubMed Central

    Yost, Shawn E.; Alakus, Hakan; Matsui, Hiroko; Schwab, Richard B.; Jepsen, Kristen; Frazer, Kelly A.; Harismendy, Olivier

    2013-01-01

    Summary: We present Mutascope, a sequencing analysis pipeline specifically developed for the identification of somatic variants present at low-allelic fraction from high-throughput sequencing of amplicons from matched tumor-normal specimen. Using datasets reproducing tumor genetic heterogeneity, we demonstrate that Mutascope has a higher sensitivity and generates fewer false-positive calls than tools designed for shotgun sequencing or diploid genomes. Availability: Freely available on the web at http://sourceforge.net/projects/mutascope/. Contact: oharismendy@ucsd.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23712659

  6. INTERACTIONS BETWEEN FLUORESCENT PSEUDOMONADS AND VA MYCORRHIZAL FUNGI

    EPA Science Inventory

    Cucumber seeds were treated with rifampin-resistant derivatives of Pseudomonas puntida (A12, N1R or R-20) or P. fluorescens (2-79 or 3871) and planted in soils with and without added inoculum of the VA mycorrhizal fungi Glomus intraradices Schenck & Smith or G. etunicatum Becker ...

  7. KaVA ESTEMA project

    NASA Astrophysics Data System (ADS)

    Oyadomari, Miyako; Imai, Hiroshi; Cho, Se-Hyung; Asaki, Yoshiharu; Choi, Yoon-Kyong; Kim, Jaeheon; Yun, Youngjoo; Matsumoto, Naoko; Min, Cheul-Hong; Oyama, Tomoaki; Yoon, Sung-Chul; Yoon, Dong-Hwan; Kim, Dong-Jin; Dodson, Richard; Rioja, Maria; Burns, Ross; Orosz, Gabor; Nakagawa, Akiharu; Chibueze O, James; Nakashima, Jun-ichi; Sobolev, Andrey

    2016-07-01

    The ESTEMA (Expanded Study on Stellar Masers) project is one of three Large Programs of the KaVA (the combined array of the Korean VLBI Network and Japanese VLBI Exploration of Radio Astrometry), and conducted in 2015-2016. It aims to publish a database of the largest sample of VLBI images of circumstellar water (H2O) and silicon-monoxide (SiO) maser sources towards circumstellar envelopes (CSEs) of 80 evolved stars in late AGB to early post-AGB phase. Here we present the specifications of the ESTEMA observations and the planned scientific goals in order to share the basic information of the ESTEMA with astronomical community and encourage future collaborations with the ESTEMA and future follow-up observations for the targeted stars.

  8. The VA-Medical School Partnership: The Medical School Perspective.

    ERIC Educational Resources Information Center

    Petersdorf, Robert G.

    1987-01-01

    Issues in the relationship between the Veterans' Administration (VA) and medical schools are discussed, including VA faculty recruitment and retention, ambulatory care in VA teaching hospitals, governance and growth of research within VA medical centers, and effects of cost containment and competition on teaching and training in VA hospitals. (MSE)

  9. 76 FR 75509 - Autopsies at VA Expense

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-02

    ... that amendment, VA promulgated 38 CFR 17.38, on October 6, 1999, 64 FR 54212. Section 17.38, inter alia... procedure; Alcohol abuse; Alcoholism; Claims; Day care; Dental health; Drug abuse; Government contracts...-reference to VA regulations that authorize certain outpatient and ambulatory care. The proposed rule...

  10. 78 FR 32126 - VA Dental Insurance Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-29

    ... in the Federal Register (77 FR 12517) a proposed rule to amend VA regulations to establish VADIP, a... coverage capabilities as determined during the Federal contracting process. See 77 FR 12518. Although VA... 510(b). See 77 FR 12520. We will conduct the Federal contracting process anticipating this...

  11. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    PubMed Central

    Mauk, Michael G.; Liu, Changchun; Song, Jinzhao; Bau, Haim H.

    2015-01-01

    Microfluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction)-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1) nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane”) to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.

  12. Taxonomic Assessment of Rumen Microbiota Using Total RNA and Targeted Amplicon Sequencing Approaches.

    PubMed

    Li, Fuyong; Henderson, Gemma; Sun, Xu; Cox, Faith; Janssen, Peter H; Guan, Le Luo

    2016-01-01

    Taxonomic characterization of active gastrointestinal microbiota is essential to detect shifts in microbial communities and functions under various conditions. This study aimed to identify and quantify potentially active rumen microbiota using total RNA sequencing and to compare the outcomes of this approach with the widely used targeted RNA/DNA amplicon sequencing technique. Total RNA isolated from rumen digesta samples from five beef steers was subjected to Illumina paired-end sequencing (RNA-seq), and bacterial and archaeal amplicons of partial 16S rRNA/rDNA were subjected to 454 pyrosequencing (RNA/DNA Amplicon-seq). Taxonomic assessments of the RNA-seq, RNA Amplicon-seq, and DNA Amplicon-seq datasets were performed using a pipeline developed in house. The detected major microbial phylotypes were common among the three datasets, with seven bacterial phyla, fifteen bacterial families, and five archaeal taxa commonly identified across all datasets. There were also unique microbial taxa detected in each dataset. Elusimicrobia and Verrucomicrobia phyla; Desulfovibrionaceae, Elusimicrobiaceae, and Sphaerochaetaceae families; and Methanobrevibacter woesei were only detected in the RNA-Seq and RNA Amplicon-seq datasets, whereas Streptococcaceae was only detected in the DNA Amplicon-seq dataset. In addition, the relative abundances of four bacterial phyla, eight bacterial families and one archaeal taxon were different among the three datasets. This is the first study to compare the outcomes of rumen microbiota profiling between RNA-seq and RNA/DNA Amplicon-seq datasets. Our results illustrate the differences between these methods in characterizing microbiota both qualitatively and quantitatively for the same sample, and so caution must be exercised when comparing data. PMID:27446027

  13. Taxonomic Assessment of Rumen Microbiota Using Total RNA and Targeted Amplicon Sequencing Approaches

    PubMed Central

    Li, Fuyong; Henderson, Gemma; Sun, Xu; Cox, Faith; Janssen, Peter H.; Guan, Le Luo

    2016-01-01

    Taxonomic characterization of active gastrointestinal microbiota is essential to detect shifts in microbial communities and functions under various conditions. This study aimed to identify and quantify potentially active rumen microbiota using total RNA sequencing and to compare the outcomes of this approach with the widely used targeted RNA/DNA amplicon sequencing technique. Total RNA isolated from rumen digesta samples from five beef steers was subjected to Illumina paired-end sequencing (RNA-seq), and bacterial and archaeal amplicons of partial 16S rRNA/rDNA were subjected to 454 pyrosequencing (RNA/DNA Amplicon-seq). Taxonomic assessments of the RNA-seq, RNA Amplicon-seq, and DNA Amplicon-seq datasets were performed using a pipeline developed in house. The detected major microbial phylotypes were common among the three datasets, with seven bacterial phyla, fifteen bacterial families, and five archaeal taxa commonly identified across all datasets. There were also unique microbial taxa detected in each dataset. Elusimicrobia and Verrucomicrobia phyla; Desulfovibrionaceae, Elusimicrobiaceae, and Sphaerochaetaceae families; and Methanobrevibacter woesei were only detected in the RNA-Seq and RNA Amplicon-seq datasets, whereas Streptococcaceae was only detected in the DNA Amplicon-seq dataset. In addition, the relative abundances of four bacterial phyla, eight bacterial families and one archaeal taxon were different among the three datasets. This is the first study to compare the outcomes of rumen microbiota profiling between RNA-seq and RNA/DNA Amplicon-seq datasets. Our results illustrate the differences between these methods in characterizing microbiota both qualitatively and quantitatively for the same sample, and so caution must be exercised when comparing data. PMID:27446027

  14. A chromosomal region 7p11.2 transcript map: its development and application to the study of EGFR amplicons in glioblastoma.

    PubMed Central

    Eley, Greg D.; Reiter, Jill L.; Pandita, Ajay; Park, Soyeon; Jenkins, Robert B.; Maihle, Nita J.; James, C. David

    2002-01-01

    Cumulative information available about the organization of amplified chromosomal regions in human tumors suggests that the amplification repeat units, or amplicons, can be of a simple or complex nature. For the former, amplified regions generally retain their native chromosomal configuration and involve a single amplification target sequence. For complex amplicons, amplified DNAs usually undergo substantial reorganization relative to the normal chromosomal regions from which they evolve, and the regions subject to amplification may contain multiple target sequences. Previous efforts to characterize the 7p11.2 epidermal growth factor receptor ) amplicon in glioblastoma have relied primarily on the use of markers positioned by linkage analysis and/or radiation hybrid mapping, both of which are known to have the potential for being inaccurate when attempting to order loci over relatively short (<1 Mb) chromosomal regions. Due to the limited resolution of genetic maps that have been established through the use of these approaches, we have constructed a 2-Mb bacterial and P1-derived artificial chromosome (BAC-PAC) contig for the EGFR region and have applied markers positioned on its associated physical map to the analysis of 7p11.2 amplifications in a series of glioblastomas. Our data indicate that EGFR is the sole amplification target within the mapped region, although there are several additional 7p11.2 genes that can be coamplified and overexpressed with EGFR. Furthermore, these results are consistent with EGFR amplicons retaining the same organization as the native chromosome 7p11.2 region from which they are derived. PMID:11916499

  15. Agreement Between HEDIS Performance Assessments in the VA and Medicare Advantage: Is Quality in the Eye of the Beholder?

    PubMed

    Trivedi, Amal N; Wilson, Ira B; Charlton, Mary E; Kizer, Kenneth W

    2016-01-01

    Medicare Advantage (MA) plans and the Veterans Affairs (VA) health care system assess quality of care using standardized Healthcare Effectiveness Data and Information Set (HEDIS) performance measures. Little is known, however, about the relative accuracy of quality indicators for persons receiving care in more than one health care system. Among Veterans dually enrolled in an MA plan, we examined the agreement between MA and VA HEDIS assessments. Our study tested the hypothesis that private health plans underreport quality of care relative to a fully integrated delivery system utilizing a comprehensive electronic health record. Despite assessing the same individuals using identical measure specifications, reported VA performance was significantly better than reported MA performance for all 12 HEDIS measures. The VA's performance advantage ranged from 9.8% (glycosylated hemoglobin [HbA1c] < 7.0% in diabetes) to 54.7% (blood pressure < 140/90 mm Hg in diabetes). The overall agreement between VA and MA HEDIS assessments ranged from 38.5% to 62.6%. Performance rates derived from VA and MA aggregate data were 1.6% to 14.3% higher than those reported by VA alone. This analysis suggests that neither MA plans nor the VA fully capture quality of care information for dually enrolled persons. However, the VA's system-wide electronic health record may allow for more complete capture of quality information across multiple providers and settings. PMID:27033565

  16. Efficient error correction for next-generation sequencing of viral amplicons

    PubMed Central

    2012-01-01

    Background Next-generation sequencing allows the analysis of an unprecedented number of viral sequence variants from infected patients, presenting a novel opportunity for understanding virus evolution, drug resistance and immune escape. However, sequencing in bulk is error prone. Thus, the generated data require error identification and correction. Most error-correction methods to date are not optimized for amplicon analysis and assume that the error rate is randomly distributed. Recent quality assessment of amplicon sequences obtained using 454-sequencing showed that the error rate is strongly linked to the presence and size of homopolymers, position in the sequence and length of the amplicon. All these parameters are strongly sequence specific and should be incorporated into the calibration of error-correction algorithms designed for amplicon sequencing. Results In this paper, we present two new efficient error correction algorithms optimized for viral amplicons: (i) k-mer-based error correction (KEC) and (ii) empirical frequency threshold (ET). Both were compared to a previously published clustering algorithm (SHORAH), in order to evaluate their relative performance on 24 experimental datasets obtained by 454-sequencing of amplicons with known sequences. All three algorithms show similar accuracy in finding true haplotypes. However, KEC and ET were significantly more efficient than SHORAH in removing false haplotypes and estimating the frequency of true ones. Conclusions Both algorithms, KEC and ET, are highly suitable for rapid recovery of error-free haplotypes obtained by 454-sequencing of amplicons from heterogeneous viruses. The implementations of the algorithms and data sets used for their testing are available at: http://alan.cs.gsu.edu/NGS/?q=content/pyrosequencing-error-correction-algorithm PMID:22759430

  17. JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms.

    PubMed

    Petrillo, Mauro; Angers-Loustau, Alexandre; Henriksson, Peter; Bonfini, Laura; Patak, Alex; Kreysa, Joachim

    2015-01-01

    The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/. PMID:26424080

  18. JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms

    PubMed Central

    Petrillo, Mauro; Angers-Loustau, Alexandre; Henriksson, Peter; Bonfini, Laura; Patak, Alex; Kreysa, Joachim

    2015-01-01

    The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/ PMID:26424080

  19. Women's veteran identity and utilization of VA health services.

    PubMed

    Di Leone, Brooke A L; Wang, Joyce M; Kressin, Nancy; Vogt, Dawne

    2016-02-01

    Women have participated in the United States military since its founding. However, until the mid-20th century, there had been limited recognition of women as official members of the military, and women remain a statistical minority within military and veteran populations. It is therefore important to better understand women's veteran identity (which we define here as one's self-concept as derived from their veteran status) and associated implications for service use and experiences in the Department of Veterans Affairs (VA) health care setting. The present research examined the centrality of, and positive regard for, women's veteran identity among 407 female veterans deployed in support of the recent wars in Iraq and Afghanistan. Data were collected via a mailed national survey. Positive regard for veteran identity, but not veteran identity centrality,was positively associated with participants' age and length of time spent in the military. Results also showed that the centrality of women's veteran identity was positively related to their choice to use VA for health care and their feelings of belonging within VA, and that veteran identity centrality and positive regard for veteran identity are differentially associated with participants' military experiences (e.g., combat exposure, deployment sexual harassment) and mental health symptomatology (e.g., depression). PMID:25729892

  20. Integration of complete chloroplast genome sequences with small amplicon datasets improves phylogenetic resolution in Acacia.

    PubMed

    Williams, Anna V; Miller, Joseph T; Small, Ian; Nevill, Paul G; Boykin, Laura M

    2016-03-01

    Combining whole genome data with previously obtained amplicon sequences has the potential to increase the resolution of phylogenetic analyses, particularly at low taxonomic levels or where recent divergence, rapid speciation or slow genome evolution has resulted in limited sequence variation. However, the integration of these types of data for large scale phylogenetic studies has rarely been investigated. Here we conduct a phylogenetic analysis of the whole chloroplast genome and two nuclear ribosomal loci for 65 Acacia species from across the most recent Acacia phylogeny. We then combine this data with previously generated amplicon sequences (four chloroplast loci and two nuclear ribosomal loci) for 508 Acacia species. We use several phylogenetic methods, including maximum likelihood bootstrapping (with and without constraint) and ExaBayes, in order to determine the success of combining a dataset of 4000bp with one of 189,000bp. The results of our study indicate that the inclusion of whole genome data gave a far better resolved and well supported representation of the phylogenetic relationships within Acacia than using only amplicon sequences, with the greatest support observed when using a whole genome phylogeny as a constraint on the amplicon sequences. Our study therefore provides methods for optimal integration of genomic and amplicon sequences. PMID:26702955

  1. Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data.

    PubMed

    Grasso, Catherine; Butler, Timothy; Rhodes, Katherine; Quist, Michael; Neff, Tanaya L; Moore, Stephen; Tomlins, Scott A; Reinig, Erica; Beadling, Carol; Andersen, Mark; Corless, Christopher L

    2015-01-01

    Changes in gene copy number are important in the setting of precision medicine. Recent studies have established that copy number alterations (CNAs) can be detected in sequencing libraries prepared by hybridization-capture, but there has been comparatively little attention given to CNA assessment in amplicon-based libraries prepared by PCR. In this study, we developed an algorithm for detecting CNAs in amplicon-based sequencing data. CNAs determined from the algorithm mirrored those from a hybridization-capture library. In addition, analysis of 14 pairs of matched normal and breast carcinoma tissues revealed that sequence data pooled from normal samples could be substituted for a matched normal tissue without affecting the detection of clinically relevant CNAs (>|2| copies). Comparison of CNAs identified by array comparative genomic hybridization and amplicon-based libraries across 10 breast carcinoma samples showed an excellent correlation. The CNA algorithm also compared favorably with fluorescence in situ hybridization, with agreement in 33 of 38 assessments across four different genes. Factors that influenced the detection of CNAs included the number of amplicons per gene, the average read depth, and, most important, the proportion of tumor within the sample. Our results show that CNAs can be identified in amplicon-based targeted sequencing data, and that their detection can be optimized by ensuring adequate tumor content and read coverage. PMID:25468433

  2. Do Older Rural and Urban Veterans Experience Different Rates of Unplanned Readmission to VA and Non-VA Hospitals?

    ERIC Educational Resources Information Center

    Weeks, William B.; Lee, Richard E.; Wallace, Amy E.; West, Alan N.; Bagian, James P.

    2009-01-01

    Context: Unplanned readmission within 30 days of discharge is an indicator of hospital quality. Purpose: We wanted to determine whether older rural veterans who were enrolled in the VA had different rates of unplanned readmission to VA or non-VA hospitals than their urban counterparts. Methods: We used the combined VA/Medicare dataset to examine…

  3. 75 FR 61859 - Proposed Information Collection (Create Payment Request for the VA Funding Fee Payment System (VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-06

    ....kessinger@va.gov . Please refer to ``OMB Control No. 2900-0474'' in any correspondence. During the comment... AFFAIRS Proposed Information Collection (Create Payment Request for the VA Funding Fee Payment System (VA... (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public comment on...

  4. 75 FR 61252 - Proposed Information Collection (Create Payment Request for the VA Funding Fee Payment System (VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-04

    ....kessinger@va.gov . Please refer to ``OMB Control No. 2900-0474'' in any correspondence. During the comment... AFFAIRS Proposed Information Collection (Create Payment Request for the VA Funding Fee Payment System (VA... (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public comment on...

  5. 78 FR 59771 - Proposed Information Collection (Create Payment Request for the VA Funding Fee Payment System (VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-27

    ...), Department of Veterans Affairs, 810 Vermont Avenue NW., Washington, DC 20420 or email nancy.kessinger@va.gov... AFFAIRS Proposed Information Collection (Create Payment Request for the VA Funding Fee Payment System (VA... (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public comment on...

  6. KENO V.a certification package

    SciTech Connect

    Niemer, K.A.

    1994-04-01

    KENO V.a has been certified. KENO V.a is a multigroup Monte Carlo criticality program used to calculate the k-effective of a 3-D system. It is part of the SCALE modular code system for performing Standardized Computer Analyses for Licensing Evaluation. SCALE was developed for the Nuclear Regulatory Commission to satisfy a need for a standardized method of analysis for the evaluation of nuclear fuel facility and package designs. In its present form, the system has the capability to perform criticality, shielding, and heat transfer analyses using well established functional modules tailored to the SCALE system. KENO V.a will be used at SRS to perform critical calculations related to nuclear criticality safety.

  7. Bacterial metabarcoding by 16S rRNA gene ion torrent amplicon sequencing.

    PubMed

    Fantini, Elio; Gianese, Giulio; Giuliano, Giovanni; Fiore, Alessia

    2015-01-01

    Ion Torrent is a next generation sequencing technology based on the detection of hydrogen ions produced during DNA chain elongation; this technology allows analyzing and characterizing genomes, genes, and species. Here, we describe an Ion Torrent procedure applied to the metagenomic analysis of 16S rRNA gene amplicons to study the bacterial diversity in food and environmental samples. PMID:25343859

  8. Evaluation of Hybridization Capture Versus Amplicon-Based Methods for Whole-Exome Sequencing.

    PubMed

    Samorodnitsky, Eric; Jewell, Benjamin M; Hagopian, Raffi; Miya, Jharna; Wing, Michele R; Lyon, Ezra; Damodaran, Senthilkumar; Bhatt, Darshna; Reeser, Julie W; Datta, Jharna; Roychowdhury, Sameek

    2015-09-01

    Next-generation sequencing has aided characterization of genomic variation. While whole-genome sequencing may capture all possible mutations, whole-exome sequencing remains cost-effective and captures most phenotype-altering mutations. Initial strategies for exome enrichment utilized a hybridization-based capture approach. Recently, amplicon-based methods were designed to simplify preparation and utilize smaller DNA inputs. We evaluated two hybridization capture-based and two amplicon-based whole-exome sequencing approaches, utilizing both Illumina and Ion Torrent sequencers, comparing on-target alignment, uniformity, and variant calling. While the amplicon methods had higher on-target rates, the hybridization capture-based approaches demonstrated better uniformity. All methods identified many of the same single-nucleotide variants, but each amplicon-based method missed variants detected by the other three methods and reported additional variants discordant with all three other technologies. Many of these potential false positives or negatives appear to result from limited coverage, low variant frequency, vicinity to read starts/ends, or the need for platform-specific variant calling algorithms. All methods demonstrated effective copy-number variant calling when evaluated against a single-nucleotide polymorphism array. This study illustrates some differences between whole-exome sequencing approaches, highlights the need for selecting appropriate variant calling based on capture method, and will aid laboratories in selecting their preferred approach. PMID:26110913

  9. Characterizing partial AZFc deletions of the Y chromosome with amplicon-specific sequence markers

    PubMed Central

    Navarro-Costa, Paulo; Pereira, Luísa; Alves, Cíntia; Gusmão, Leonor; Proença, Carmen; Marques-Vidal, Pedro; Rocha, Tiago; Correia, Sónia C; Jorge, Sónia; Neves, António; Soares, Ana P; Nunes, Joaquim; Calhaz-Jorge, Carlos; Amorim, António; Plancha, Carlos E; Gonçalves, João

    2007-01-01

    Background The AZFc region of the human Y chromosome is a highly recombinogenic locus containing multi-copy male fertility genes located in repeated DNA blocks (amplicons). These AZFc gene families exhibit slight sequence variations between copies which are considered to have functional relevance. Yet, partial AZFc deletions yield phenotypes ranging from normospermia to azoospermia, thwarting definite conclusions on their real impact on fertility. Results The amplicon content of partial AZFc deletion products was characterized with novel amplicon-specific sequence markers. Data indicate that partial AZFc deletions are a male infertility risk [odds ratio: 5.6 (95% CI: 1.6–30.1)] and although high diversity of partial deletion products and sequence conversion profiles were recorded, the AZFc marker profiles detected in fertile men were also observed in infertile men. Additionally, the assessment of rearrangement recurrence by Y-lineage analysis indicated that while partial AZFc deletions occurred in highly diverse samples, haplotype diversity was minimal in fertile men sharing identical marker profiles. Conclusion Although partial AZFc deletion products are highly heterogeneous in terms of amplicon content, this plasticity is not sufficient to account for the observed phenotypical variance. The lack of causative association between the deletion of specific gene copies and infertility suggests that AZFc gene content might be part of a multifactorial network, with Y-lineage evolution emerging as a possible phenotype modulator. PMID:17903263

  10. De novo-generated small palindromes are characteristic of amplicon boundary junction of double minutes.

    PubMed

    Zhu, Jing; Yu, Yang; Meng, Xiangning; Fan, Yihui; Zhang, Yu; Zhou, Chunshui; Yue, Zhichao; Jin, Yan; Zhang, Chunyu; Yu, Lisa; Ji, Wei; Jia, Xueyuan; Guan, Rongwei; Wu, Jie; Yu, Jingcui; Bai, Jing; Guan, Xin-Yuan; Wang, Mingrong; Lee, Ki-Young; Sun, Wenjing; Fu, Songbin

    2013-08-15

    Double minutes (DMs) are hallmarks of gene amplification. However, their molecular structure and the mechanisms of formation are largely unknown. To elucidate the structure and underlying molecular mechanism of DMs, we obtained and cloned DMs using microdissection; and degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR) from the ovarian cancer cell line UACC-1598. Two large amplicons, the 284 kb AmpMYCN, originating from locus 2p24.3 and the 391 kb AmpEIF5A2, from locus 3q26.2, were found co-amplified on the same DMs. The two amplicons are joined through a complex 7 kb junction DNA sequence. Analysis of the junction has revealed three de novo created small palindromes surrounding the six breakpoints. Consistent with these observations, we further found that 70% of the 57 reported DM junction sequences have de novo creation of small palindromic sequences surrounding the breakpoints. Together, our findings indicate that de novo-generated small palindromic sequences are characteristic of amplicon boundary junctions on DMs. It is possible that the de novo-generated small palindromic sequences, which may be generated through non-homologous end joining in concert with a novel DNA repair machinery, play a common role in amplicon rejoining and gene amplification. PMID:23382041

  11. De novo origin of VCY2 from autosome to Y-transposed amplicon.

    PubMed

    Cao, Peng-Rong; Wang, Lei; Jiang, Yu-Chao; Yi, Yin-Sha; Qu, Fang; Liu, Tao-Cheng; Lv, Yuan

    2015-01-01

    The formation of new genes is a primary driving force of evolution in all organisms. The de novo evolution of new genes from non-protein-coding genomic regions is emerging as an important additional mechanism for novel gene creation. Y chromosomes underlie sex determination in mammals and contain genes that are required for male-specific functions. In this study, a search was undertaken for Y chromosome de novo genes derived from non-protein-coding sequences. The Y chromosome orphan gene variable charge, Y-linked (VCY)2, is an autosome-derived gene that has sequence similarity to large autosomal fragments but lacks an autosomal protein-coding homolog. VCY2 locates in the amplicon containing long DNA fragments that were transposed from autosomes to the Y chromosome before the ape-monkey split. We confirmed that VCY2 cannot be encoded by autosomes due to the presence of multiple disablers that disrupt the open reading frame, such as the absence of start or stop codons and the presence of premature stop codons. Similar observations have been made for homologs in the autosomes of the chimpanzee, gorilla, rhesus macaque, baboon and out-group marmoset, which suggests that there was a non-protein-coding ancestral VCY2 that was common to apes and monkeys that predated the transposition event. Furthermore, while protein-coding orthologs are absent, a putative non-protein-coding VCY2 with conserved disablers was identified in the rhesus macaque Y chromosome male-specific region. This finding implies that VCY2 might have not acquired its protein-coding ability before the ape-monkey split. VCY2 encodes a testis-specific expressed protein and is involved in the pathologic process of male infertility, and the acquisition of this gene might improve male fertility. This is the first evidence that de novo genes can be generated from transposed autosomal non-protein-coding segments, and this evidence provides novel insights into the evolutionary history of the Y chromosome. PMID

  12. Estimation of copy number using SYBR Green: confounding by AT-rich DNA and by variation in amplicon length.

    PubMed

    Colborn, James M; Byrd, Brian D; Koita, Ousmane A; Krogstad, Donald J

    2008-12-01

    Although SYBR Green is used to estimate copy number, its fluorescence varies with amplicon length and adenine/thymine (AT) content. As a result, threshold cycle (Ct) values obtained using real-time polymerase chain reaction (PCR) are lower for longer amplicons (P<0.001) and amplicons with greater AT content (P<0.001). In contrast, neither amplicon length nor AT content affects the Ct with TaqMan probes or LUX-labeled primers. Because SYBR Green yields lower Cts with AT-rich templates and longer templates, it overestimates copy number for those templates. Therefore, sequence-specific methods such as TaqMan probes or LUX-labeled primers should be considered when using real-time PCR to estimate copy number if the amplicons generated are AT-rich or vary in length. PMID:19052298

  13. 76 FR 56861 - Virginia Disaster #VA-00038

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-14

    ... ADMINISTRATION Virginia Disaster VA-00038 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY... Commonwealth of Virginia (FEMA-4024-DR), dated 09/03/2011. Incident: Hurricane Irene. Incident Period: 08/26..., Southampton, Suffolk City, Sussex, Virginia Beach City, Westmoreland, Williamsburg City, York. The...

  14. 77 FR 73510 - Virginia Disaster #VA-00052

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-10

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster VA-00052 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY... State of Virginia (FEMA- 4092-DR), dated 11/26/2012. Incident: Hurricane Sandy Incident Period:...

  15. 76 FR 70804 - Virginia Disaster #VA-00037

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-15

    ... ADMINISTRATION Virginia Disaster VA-00037 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY: This is a Notice of the Presidential declaration of a major disaster for the Commonwealth of Virginia... Economic Injury Loans): Louisa. Contiguous Counties (Economic Injury Loans Only): Virginia:...

  16. 75 FR 24757 - Virginia Disaster #VA-00029

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-05

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster VA-00029 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY... Commonwealth of Virginia (FEMA-1905-DR), dated 04/27/2010. Incident: Severe Winter Storms and...

  17. 77 FR 47489 - Virginia Disaster #VA-00048

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-08

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster VA-00048 AGENCY: Small Business Administration. ACTION: Notice. SUMMARY: This is... State of Virginia (FEMA- 4072-DR), dated 07/27/2012. Incident: Severe Storms and Straight-line...

  18. 75 FR 9006 - Virginia Disaster #VA-00028

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-26

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster VA-00028 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY... Commonwealth of Virginia (FEMA-1874-DR), dated 02/16/2010. Incident: Severe Winter Storm and...

  19. 76 FR 40766 - Virginia Disaster #VA-00032

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-11

    ... ADMINISTRATION Virginia Disaster VA-00032 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY: This is a notice of an Administrative declaration of a disaster for the Commonwealth of Virginia dated... determined to be adversely affected by the disaster: Primary Counties: Pulaski. Contiguous Counties:...

  20. 76 FR 72020 - Virginia Disaster #VA-00039

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-21

    ... ADMINISTRATION Virginia Disaster VA-00039 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY: This is a notice of an Administrative declaration of a disaster for the Commonwealth of Virginia dated... adversely affected by the disaster: Primary Counties: Fairfax, Prince William. Contiguous Counties:...

  1. 76 FR 59765 - Virginia Disaster # VA-00036

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-27

    ... ADMINISTRATION Virginia Disaster VA-00036 AGENCY: U.S. Small Business Administration. ACTION: Notice SUMMARY: This is a notice of an Administrative declaration of a disaster for the Commonwealth of Virginia dated...: Virginia: Charles City, Chesterfield, Colonial Heights City, Dinwiddie, Hanover, Henrico, James City,...

  2. 76 FR 72022 - Virginia Disaster #VA-00040

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-21

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster VA-00040 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY... State of Virginia (FEMA- 4042-DR), dated 11/10/2011. Incident: Earthquake. Incident Period:...

  3. 76 FR 40765 - Virginia Disaster #VA-00034

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-11

    ... ADMINISTRATION Virginia Disaster VA-00034 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY: This is a notice of an Administrative declaration of a disaster for the Commonwealth of Virginia dated...: Virginia: Bristol City, Grayson, Russell, Scott, Smyth. Tennessee: Johnson, Sullivan. The Interest...

  4. 77 FR 74908 - Virginia Disaster #VA-00051

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-18

    ... ADMINISTRATION Virginia Disaster VA-00051 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY: This is a notice of an Administrative declaration of a disaster for the Commonwealth of Virginia dated... adversely affected by the disaster: Primary Counties: Accomack. Contiguous Counties: Virginia:...

  5. 76 FR 72994 - Virginia Disaster #VA-00041

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-28

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster VA-00041 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY... State of Virginia (FEMA- 4045-DR), dated 11/17/2011. Incident: Remnants of Tropical Storm Lee....

  6. 77 FR 38179 - Autopsies at VA Expense

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-27

    ... document published in the Federal Register on December 2, 2011 (76 FR 75509), VA proposed the above... abuse; Alcoholism; Claims; Day care; Dental health; Drug abuse; Government contracts; Grant programs... statute that previously authorized certain outpatient and ambulatory care, which included...

  7. Telephone Enrollment in the VA Healthcare System. Interim final rule.

    PubMed

    2016-03-16

    This rulemaking amends VA's medical regulations to allow veterans to complete applications for health care enrollment by telephone by providing application information to a VA employee, agreeing to VA's provisions regarding copayment liability and assignment of third-party insurance benefits, and attesting to the accuracy and authenticity of the information provided over the phone. This action will make it easier for veterans to apply to enroll and will speed VA processing of applications. PMID:26987128

  8. Assessing a new approach to verbal autopsy interpretation in a rural Ethiopian community: the InterVA model.

    PubMed Central

    Fantahun, Mesganaw; Fottrell, Edward; Berhane, Yemane; Wall, Stig; Högberg, Ulf; Byass, Peter

    2006-01-01

    OBJECTIVE: Verbal autopsy (VA) -- the interviewing of family members or caregivers about the circumstances of a death after the event -- is an established tool in areas where routine death registration is non-existent or inadequate. We assessed the performance of a probabilistic model (InterVA) for interpreting community-based VA interviews, in order to investigate patterns of cause-specific mortality in a rural Ethiopian community. We compared results with those obtained after review of the VA by local physicians, with a view to validating the model as a community-based tool. METHODS: Two-hundred and eighty-nine VA interviews were successfully completed; these included most deaths occurring in a defined community over a 1-year period. The VA interviews were interpreted by physicians and by the model, and cause-specific mortality fractions were derived for the whole community and for particular age groups using both approaches. FINDINGS: The results of the two approaches to interpretation correlated well in this example from Ethiopia. Four major cause groups accounted for over 60% of all mortality, and patterns within specific age groups were consistent with expectations for an underdeveloped high-mortality community in sub-Saharan Africa. CONCLUSION: Compared with interpretation by physicians, the InterVA model is much less labour intensive and offers 100% consistency. It is a valuable new tool for characterizing patterns of cause-specific mortality in communities without death registration and for comparing patterns of mortality in different populations. PMID:16583079

  9. 48 CFR 819.7109 - VA review of application.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... SOCIOECONOMIC PROGRAMS SMALL BUSINESS PROGRAMS VA Mentor-Protégé Program 819.7109 VA review of application. (a) VA OSDBU will review the information to establish the mentor and protégé eligibility and to ensure... charge to apply for the Mentor-Protégé Program. (b) After OSDBU completes its review and provides...

  10. 48 CFR 819.7109 - VA review of application.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... SOCIOECONOMIC PROGRAMS SMALL BUSINESS PROGRAMS VA Mentor-Protégé Program 819.7109 VA review of application. (a) VA OSDBU will review the information to establish the mentor and protégé eligibility and to ensure... charge to apply for the Mentor-Protégé Program. (b) After OSDBU completes its review and provides...

  11. 48 CFR 819.7109 - VA review of application.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... SOCIOECONOMIC PROGRAMS SMALL BUSINESS PROGRAMS VA Mentor-Protégé Program 819.7109 VA review of application. (a) VA OSDBU will review the information to establish the mentor and protégé eligibility and to ensure... charge to apply for the Mentor-Protégé Program. (b) After OSDBU completes its review and provides...

  12. 48 CFR 819.7109 - VA review of application.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... SOCIOECONOMIC PROGRAMS SMALL BUSINESS PROGRAMS VA Mentor-Protégé Program 819.7109 VA review of application. (a) VA OSDBU will review the information to establish the mentor and protégé eligibility and to ensure... charge to apply for the Mentor-Protégé Program. (b) After OSDBU completes its review and provides...

  13. 48 CFR 819.7109 - VA review of application.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... SOCIOECONOMIC PROGRAMS SMALL BUSINESS PROGRAMS VA Mentor-Protégé Program 819.7109 VA review of application. (a) VA OSDBU will review the information to establish the mentor and protégé eligibility and to ensure... charge to apply for the Mentor-Protégé Program. (b) After OSDBU completes its review and provides...

  14. Simultaneous Amplicon Sequencing to Explore Co-Occurrence Patterns of Bacterial, Archaeal and Eukaryotic Microorganisms in Rumen Microbial Communities

    PubMed Central

    Kittelmann, Sandra; Seedorf, Henning; Walters, William A.; Clemente, Jose C.; Knight, Rob; Gordon, Jeffrey I.; Janssen, Peter H.

    2013-01-01

    Ruminants rely on a complex rumen microbial community to convert dietary plant material to energy-yielding products. Here we developed a method to simultaneously analyze the community's bacterial and archaeal 16S rRNA genes, ciliate 18S rRNA genes and anaerobic fungal internal transcribed spacer 1 genes using 12 DNA samples derived from 11 different rumen samples from three host species (Ovis aries, Bos taurus, Cervus elephas) and multiplex 454 Titanium pyrosequencing. We show that the mixing ratio of the group-specific DNA templates before emulsion PCR is crucial to compensate for differences in amplicon length. This method, in contrast to using a non-specific universal primer pair, avoids sequencing non-targeted DNA, such as plant- or endophyte-derived rRNA genes, and allows increased or decreased levels of community structure resolution for each microbial group as needed. Communities analyzed with different primers always grouped by sample origin rather than by the primers used. However, primer choice had a greater impact on apparent archaeal community structure than on bacterial community structure, and biases for certain methanogen groups were detected. Co-occurrence analysis of microbial taxa from all three domains of life suggested strong within- and between-domain correlations between different groups of microorganisms within the rumen. The approach used to simultaneously characterize bacterial, archaeal and eukaryotic components of a microbiota should be applicable to other communities occupying diverse habitats. PMID:23408926

  15. DADA2: High-resolution sample inference from Illumina amplicon data.

    PubMed

    Callahan, Benjamin J; McMurdie, Paul J; Rosen, Michael J; Han, Andrew W; Johnson, Amy Jo A; Holmes, Susan P

    2016-07-01

    We present the open-source software package DADA2 for modeling and correcting Illumina-sequenced amplicon errors (https://github.com/benjjneb/dada2). DADA2 infers sample sequences exactly and resolves differences of as little as 1 nucleotide. In several mock communities, DADA2 identified more real variants and output fewer spurious sequences than other methods. We applied DADA2 to vaginal samples from a cohort of pregnant women, revealing a diversity of previously undetected Lactobacillus crispatus variants. PMID:27214047

  16. Molecular cloning and characterization of a 20q13.2 amplicon in breast carcinoma

    SciTech Connect

    Collins, C.; Froula, J.; Kowbel, D.

    1994-09-01

    Comparative genomic hybridization (CGH) has identified an amplification event involving chromosome band 20q13.2 in 15-20% of primary breast carcinomas. The application of FISH to the study of tumor interphase nuclei using 33 locus specific cosmid and P1 probes revealed amplification of band 20q13.2 in 35% of breast cancer cell lines and 8% of primary tumors. Moreover, this study localized the amplification event to the 1.5Mb interval defined by (Flpter 0.80-0.84.) and excluded all known genes in the region as candidates for the putative oncogene(s). To both identify the putative oncogene(s) and characterize the amplicon, a 12 member 4Mb YAC contig has been assembled by STS mapping that spans the core of the amplicon. The YAC contig is now being converted to a P1 contig to facilitate sequencing, exon trapping and direct selection of cDNAs. This is being accomplished by performing interAlu PCR reactions on individual YACs and sequencing the reaction products to create 5-10 new STSs per megaYAC. The DuPont P1 library is then screened for these STSs by the PCR. To date 21 P1 clones, forming 6 contigs, have been isolated by screening the DuPont P1 library for existing and/or newly created STSs. The ends of the 21 P1 clones are being sequenced to facilitate contig alignment and to enable chromosome walking. In collaboration with the Human Genome Center at the Lawrence Berkeley Laboratory we have initiated the directed sequencing of two P1 contigs, localized within the amplicon core, and ultimately will sequence the entire 1-2Mb amplicon.

  17. Massively parallel sequencing of 17 commonly used forensic autosomal STRs and amelogenin with small amplicons.

    PubMed

    Kim, Eun Hye; Lee, Hwan Young; Yang, In Seok; Jung, Sang-Eun; Yang, Woo Ick; Shin, Kyoung-Jin

    2016-05-01

    The next-generation sequencing (NGS) method has been utilized to analyze short tandem repeat (STR) markers, which are routinely used for human identification purposes in the forensic field. Some researchers have demonstrated the successful application of the NGS system to STR typing, suggesting that NGS technology may be an alternative or additional method to overcome limitations of capillary electrophoresis (CE)-based STR profiling. However, there has been no available multiplex PCR system that is optimized for NGS analysis of forensic STR markers. Thus, we constructed a multiplex PCR system for the NGS analysis of 18 markers (13CODIS STRs, D2S1338, D19S433, Penta D, Penta E and amelogenin) by designing amplicons in the size range of 77-210 base pairs. Then, PCR products were generated from two single-sources, mixed samples and artificially degraded DNA samples using a multiplex PCR system, and were prepared for sequencing on the MiSeq system through construction of a subsequent barcoded library. By performing NGS and analyzing the data, we confirmed that the resultant STR genotypes were consistent with those of CE-based typing. Moreover, sequence variations were detected in targeted STR regions. Through the use of small-sized amplicons, the developed multiplex PCR system enables researchers to obtain successful STR profiles even from artificially degraded DNA as well as STR loci which are analyzed with large-sized amplicons in the CE-based commercial kits. In addition, successful profiles can be obtained from mixtures up to a 1:19 ratio. Consequently, the developed multiplex PCR system, which produces small size amplicons, can be successfully applied to STR NGS analysis of forensic casework samples such as mixtures and degraded DNA samples. PMID:26799314

  18. Characterization of FGFR1 Locus in sqNSCLC Reveals a Broad and Heterogeneous Amplicon

    PubMed Central

    Rooney, Claire; Geh, Catherine; Williams, Victoria; Heuckmann, Johannes M.; Menon, Roopika; Schneider, Petra; Al-Kadhimi, Katherine; Dymond, Michael; Smith, Neil R.; Baker, Dawn; French, Tim; Smith, Paul D.; Harrington, Elizabeth A.; Barrett, J. Carl; Kilgour, Elaine

    2016-01-01

    FGFR1 amplification occurs in ~20% of sqNSCLC and trials with FGFR inhibitors have selected FGFR1 amplified patients by FISH. Lung cancer cell lines were profiled for sensitivity to AZD4547, a potent, selective inhibitor of FGFRs 1–3. Sensitivity to FGFR inhibition was associated with but not wholly predicted by increased FGFR1 gene copy number. Additional biomarker assays evaluating expression of FGFRs and correlation between amplification and expression in clinical tissues are therefore warranted. We validated nanoString for mRNA expression analysis of 194 genes, including FGFRs, from clinical tumour tissue. In a panel of sqNSCLC tumours 14.4% (13/90) were FGFR1 amplified by FISH. Although mean FGFR1 expression was significantly higher in amplified samples, there was significant overlap in the range of expression levels between the amplified and non-amplified cohorts with several non-amplified samples expressing FGFR1 to levels equivalent to amplified samples. Statistical analysis revealed increased expression of FGFR1 neighboring genes on the 8p12 amplicon (BAG4, LSM1 and WHSC1L1) in FGFR1 amplified tumours, suggesting a broad rather than focal amplicon and raises the potential for codependencies. High resolution aCGH analysis of pre-clinical and clinical samples supported the presence of a broad and heterogeneous amplicon around the FGFR1 locus. In conclusion, the range of FGFR1 expression levels in both FGFR1 amplified and non-amplified NSCLC tissues, together with the breadth and intra-patient heterogeneity of the 8p amplicon highlights the need for gene expression analysis of clinical samples to inform the understanding of determinants of response to FGFR inhibitors. In this respect the nanoString platform provides an attractive option for RNA analysis of FFPE clinical samples. PMID:26905262

  19. Ioncopy: a novel method for calling copy number alterations in amplicon sequencing data including significance assessment

    PubMed Central

    Budczies, Jan; Pfarr, Nicole; Stenzinger, Albrecht; Treue, Denise; Endris, Volker; Ismaeel, Fakher; Bangemann, Nikola; Blohmer, Jens-Uwe; Dietel, Manfred; Loibl, Sibylle; Klauschen, Frederick; Weichert, Wilko; Denkert, Carsten

    2016-01-01

    Recently, it has been demonstrated that calling of copy number alterations (CNAs) from amplicon sequencing (AS) data is feasible. Most approaches, however, require non-tumor (germline) DNA for data normalization. Here, we present the method Ioncopy for CNA detection which requires no normal controls and includes a significance assessment for each detected alteration. Ioncopy was evaluated in a cohort of 184 clinically annotated breast carcinomas. A total number of 252 amplifications were detected, of which 183 (72.6%) could be validated by a call of an additional amplicon interrogating the same gene. Moreover, a total number of 33 deletions were found, whereof 27 (81.8%) could be validated. Analyzing the 16 most frequently amplified genes, validation rates of over 89% could be achieved for 11 of these genes. 11 of the top 16 genes showed significant overexpression in the amplified tumors. 89.5% of the HER2-amplified tumors were GRB7 and STARD3 co-amplified, whereas 68.4% of the HER2-amplified tumors had additional MED1 amplifications. Correlations between CNAs measured by amplicons in HER2 exons 19, 20 and 21 were strong (all R > 0.93). AS based detection of HER2 amplifications had a sensitivity of 90.0% and a specificity of 98.8% compared to the gold standard of HER2 immunohistochemistry combined with in situ hybridization. In summary, we developed and validated a novel method for detection and significance assessment of CNAs in amplicon sequencing data. Using Ioncopy, AS offers a straightforward and efficient approach to simultaneously analyze gene amplifications and gene deletions together with simple somatic mutations in a single assay. PMID:26910888

  20. Electrochemical DNA sensor for anthrax toxin activator gene atxA-detection of PCR amplicons.

    PubMed

    Das, Ritu; Goel, Ajay K; Sharma, Mukesh K; Upadhyay, Sanjay

    2015-12-15

    We report the DNA probe functionalized electrochemical genosensor for the detection of Bacillus anthracis, specific towards the regulatory gene atxA. The DNA sensor is fabricated on electrochemically deposited gold nanoparticle on self assembled layer of (3-Mercaptopropyl) trimethoxysilane (MPTS) on GC electrode. DNA hybridization is monitored by differential pulse voltammogram (DPV). The modified GC electrode is characterized by atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) method. We also quantified the DNA probe density on electrode surface by the chronocoulometric method. The detection is specific and selective for atxA gene by DNA probe on the electrode surface. No report is available for the detection of B. anthracis by using atxA an anthrax toxin activator gene. In the light of real and complex sample, we have studied the PCR amplicons of 303, 361 and 568 base pairs by using symmetric and asymmetric PCR approaches. The DNA probe of atxA gene efficiently hybridizes with different base pairs of PCR amplicons. The detection limit is found to be 1.0 pM (S/N ratio=3). The results indicate that the DNA sensor is able to detect synthetic target as well as PCR amplicons of different base pairs. PMID:26257186

  1. From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows

    PubMed Central

    Murray, Dáithí C.; Coghlan, Megan L.; Bunce, Michael

    2015-01-01

    Amplicon sequencing has been the method of choice in many high-throughput DNA sequencing (HTS) applications. To date there has been a heavy focus on the means by which to analyse the burgeoning amount of data afforded by HTS. In contrast, there has been a distinct lack of attention paid to considerations surrounding the importance of sample preparation and the fidelity of library generation. No amount of high-end bioinformatics can compensate for poorly prepared samples and it is therefore imperative that careful attention is given to sample preparation and library generation within workflows, especially those involving multiple PCR steps. This paper redresses this imbalance by focusing on aspects pertaining to the benchtop within typical amplicon workflows: sample screening, the target region, and library generation. Empirical data is provided to illustrate the scope of the problem. Lastly, the impact of various data analysis parameters is also investigated in the context of how the data was initially generated. It is hoped this paper may serve to highlight the importance of pre-analysis workflows in achieving meaningful, future-proof data that can be analysed appropriately. As amplicon sequencing gains traction in a variety of diagnostic applications from forensics to environmental DNA (eDNA) it is paramount workflows and analytics are both fit for purpose. PMID:25902146

  2. Analysing 454 amplicon resequencing experiments using the modular and database oriented Variant Identification Pipeline

    PubMed Central

    2010-01-01

    Background Next-generation amplicon sequencing enables high-throughput genetic diagnostics, sequencing multiple genes in several patients together in one sequencing run. Currently, no open-source out-of-the-box software solution exists that reliably reports detected genetic variations and that can be used to improve future sequencing effectiveness by analyzing the PCR reactions. Results We developed an integrated database oriented software pipeline for analysis of 454/Roche GS-FLX amplicon resequencing experiments using Perl and a relational database. The pipeline enables variation detection, variation detection validation, and advanced data analysis, which provides information that can be used to optimize PCR efficiency using traditional means. The modular approach enables customization of the pipeline where needed and allows researchers to adopt their analysis pipeline to their experiments. Clear documentation and training data is available to test and validate the pipeline prior to using it on real sequencing data. Conclusions We designed an open-source database oriented pipeline that enables advanced analysis of 454/Roche GS-FLX amplicon resequencing experiments using SQL-statements. This modular database approach allows easy coupling with other pipeline modules such as variant interpretation or a LIMS system. There is also a set of standard reporting scripts available. PMID:20487544

  3. Sample Preparation for Fungal Community Analysis by High-Throughput Sequencing of Barcode Amplicons.

    PubMed

    Clemmensen, Karina Engelbrecht; Ihrmark, Katarina; Durling, Mikael Brandström; Lindahl, Björn D

    2016-01-01

    Fungal species participate in vast numbers of processes in the landscape around us. However, their often cryptic growth, inside various substrates and in highly diverse species assemblages, has been a major obstacle to thorough analysis of fungal communities, hampering exhaustive description of the fungal kingdom. Recent technological developments allowing rapid, high-throughput sequencing of mixed communities from many samples at once are currently having a tremendous impact in fungal community ecology. Universal DNA extraction followed by amplification and sequencing of fungal species-level barcodes such as the nuclear internal transcribed spacer (ITS) region now enable identification and relative quantification of fungal community members across well-replicated experimental settings. Here, we present the sample preparation procedure presently used in our laboratory for fungal community analysis by high-throughput sequencing of amplified ITS2 markers. We focus on the procedure optimized for studies of total fungal communities in humus-rich soils, wood, and litter. However, this procedure can be applied to other sample types and markers. We focus on the laboratory-based part of sample preparation, that is, the procedure from the point where samples enter the laboratory until amplicons are submitted for sequencing. Our procedure comprises four main parts: (1) universal DNA extraction, (2) optimization of PCR conditions, (3) production of tagged ITS amplicons, and (4) preparation of the multiplexed amplicon mix to be sequenced. The presented procedure is independent of the specific high-throughput sequencing technology used, which makes it highly versatile. PMID:26791497

  4. How Involved are Non-VA Chaplains in Supporting Veterans?

    PubMed

    Kopacz, Marek S; Feldstein, Bruce D; Asekoff, Cecille Allman; Kaprow, Maurice S; Smith-Coggins, Rebecca; Rasmussen, Kathy A

    2016-08-01

    In terms of supporting veteran populations, little is known of the experiences of chaplains professionally active outside of Department of Veterans Affairs (VA) healthcare settings. The present study looks to examine how involved non-VA chaplains are in supporting veterans as well as their familiarity with the VA. An online survey was distributed in a convenience sample of chaplains, of which n = 39 met the inclusion criterion for this study (i.e., no past or present VA affiliation). The results find that most of the non-VA chaplains encounter veteran service users either on a weekly or monthly basis. Though familiar with VA services, non-VA chaplains were not sure of their veteran service users' VA enrollment status nor did they feel able to adequately advise their veteran service users on VA enrollment. The results suggest that non-VA chaplains actively support veteran populations. Opportunities for enhancing chaplaincy services and VA outreach programs are discussed. PMID:27023459

  5. 48 CFR 853.215-70 - VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ..., Application for Furnishing Nursing Home Care to Beneficiaries of VA. 853.215-70 Section 853.215-70 Federal... 853.215-70 VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA. VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA, will be used...

  6. 48 CFR 853.215-70 - VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ..., Application for Furnishing Nursing Home Care to Beneficiaries of VA. 853.215-70 Section 853.215-70 Federal... 853.215-70 VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA. VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA, will be used...

  7. 48 CFR 853.215-70 - VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ..., Application for Furnishing Nursing Home Care to Beneficiaries of VA. 853.215-70 Section 853.215-70 Federal... 853.215-70 VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA. VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA, will be used...

  8. 48 CFR 853.215-70 - VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ..., Application for Furnishing Nursing Home Care to Beneficiaries of VA. 853.215-70 Section 853.215-70 Federal... 853.215-70 VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA. VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA, will be used...

  9. 48 CFR 853.215-70 - VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ..., Application for Furnishing Nursing Home Care to Beneficiaries of VA. 853.215-70 Section 853.215-70 Federal... 853.215-70 VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA. VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA, will be used...

  10. Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing

    PubMed Central

    2012-01-01

    Background High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. Results We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. Conclusions The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons. PMID:22866951

  11. Quantitative and qualitative differences in celiac disease epitopes among durum wheat varieties identified through deep RNA-amplicon sequencing

    PubMed Central

    2013-01-01

    Background Wheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are also the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses in the small intestine. Various peptides with three major T cell epitopes involved in CD are derived from alpha-gliadin fraction of gluten. Alpha-gliadins are encoded by a large multigene family and amino acid variation in the CD epitopes is known to influence the immunogenicity of individual gene family members. Current commercial methods of gluten detection are unable to distinguish between immunogenic and non-immunogenic CD epitope variants and thus to accurately quantify the overall CD epitope load of a given wheat variety. Such quantification is indispensable for correct selection of wheat varieties with low potential to cause CD. Results A 454 RNA-amplicon sequencing method was developed for alpha-gliadin transcripts encompassing the three major CD epitopes and their variants. The method was used to screen developing grains on plants of 61 different durum wheat cultivars and accessions. A dedicated sequence analysis pipeline returned a total of 304 unique alpha-gliadin transcripts, corresponding to a total of 171 ‘unique deduced protein fragments’ of alpha-gliadins. The numbers of these fragments obtained in each plant were used to calculate quantitative and quantitative differences between the CD epitopes expressed in the endosperm of these wheat plants. A few plants showed a lower fraction of CD epitope-encoding alpha-gliadin transcripts, but none were free of CD epitopes. Conclusions The dedicated 454 RNA-amplicon sequencing method enables 1) the grouping of wheat plants according to the genetic variation in alpha-gliadin transcripts, and 2) the screening for plants which are potentially less CD-immunogenic. The resulting alpha-gliadin sequence database will

  12. Application of PCR amplicon sequencing using a single primer pair in PCR amplification to assess variations in Helicobacter pylori CagA EPIYA tyrosine phosphorylation motifs

    PubMed Central

    2010-01-01

    Background The presence of various EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this study, a unique PCR assay and sequencing strategy was developed to establish the number and variation of cagA EPIYA motifs. Findings MDA-DNA derived from gastric biopsy specimens from eleven subjects with gastritis was used with M13- and T7-sequence-tagged primers for amplification of the cagA EPIYA motif region. Automated capillary electrophoresis using a high resolution kit and amplicon sequencing confirmed variations in the cagA EPIYA motif region. In nine cases, sequencing revealed the presence of AB, ABC, or ABCC (Western type) cagA EPIYA motif, respectively. In two cases, double cagA EPIYA motifs were detected (ABC/ABCC or ABC/AB), indicating the presence of two H. pylori strains in the same biopsy. Conclusion Automated capillary electrophoresis and Amplicon sequencing using a single, M13- and T7-sequence-tagged primer pair in PCR amplification enabled a rapid molecular typing of cagA EPIYA motifs. Moreover, the techniques described allowed for a rapid detection of mixed H. pylori strains present in the same biopsy specimen. PMID:20181142

  13. Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay

    PubMed Central

    Cousins, Matthew M.; Donnell, Deborah; Eshleman, Susan H.

    2013-01-01

    We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change < 3-bp insertion < 9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology. PMID:23178437

  14. Medical Student Psychiatry Examination Performance at VA and Non-VA Clerkship Sites

    ERIC Educational Resources Information Center

    Tucker, Phebe; von Schlageter, Margo Shultes; Park, EunMi; Rosenberg, Emily; Benjamin, Ashley B.; Nawar, Ola

    2009-01-01

    Objective: The authors examined the effects of medical student assignment to U.S. Department of Veterans Affairs (VA) Medical Center inpatient and outpatient psychiatry clerkship sites versus other university and community sites on the performance outcome measure of National Board of Medical Examiners (NBME) subject examination scores. Methods:…

  15. The bias associated with amplicon sequencing does not affect the quantitative assessment of bacterial community dynamics.

    PubMed

    Ibarbalz, Federico M; Pérez, María Victoria; Figuerola, Eva L M; Erijman, Leonardo

    2014-01-01

    The performance of two sets of primers targeting variable regions of the 16S rRNA gene V1-V3 and V4 was compared in their ability to describe changes of bacterial diversity and temporal turnover in full-scale activated sludge. Duplicate sets of high-throughput amplicon sequencing data of the two 16S rRNA regions shared a collection of core taxa that were observed across a series of twelve monthly samples, although the relative abundance of each taxon was substantially different between regions. A case in point was the changes in the relative abundance of filamentous bacteria Thiothrix, which caused a large effect on diversity indices, but only in the V1-V3 data set. Yet the relative abundance of Thiothrix in the amplicon sequencing data from both regions correlated with the estimation of its abundance determined using fluorescence in situ hybridization. In nonmetric multidimensional analysis samples were distributed along the first ordination axis according to the sequenced region rather than according to sample identities. The dynamics of microbial communities indicated that V1-V3 and the V4 regions of the 16S rRNA gene yielded comparable patterns of: 1) the changes occurring within the communities along fixed time intervals, 2) the slow turnover of activated sludge communities and 3) the rate of species replacement calculated from the taxa-time relationships. The temperature was the only operational variable that showed significant correlation with the composition of bacterial communities over time for the sets of data obtained with both pairs of primers. In conclusion, we show that despite the bias introduced by amplicon sequencing, the variable regions V1-V3 and V4 can be confidently used for the quantitative assessment of bacterial community dynamics, and provide a proper qualitative account of general taxa in the community, especially when the data are obtained over a convenient time window rather than at a single time point. PMID:24923665

  16. Using Amplicon Sequencing To Characterize and Monitor Bacterial Diversity in Drinking Water Distribution Systems

    PubMed Central

    Shaw, Jennifer L. A.; Weyrich, Laura S.; Sawade, Emma; Drikas, Mary; Cooper, Alan J.

    2015-01-01

    Drinking water assessments use a variety of microbial, physical, and chemical indicators to evaluate water treatment efficiency and product water quality. However, these indicators do not allow the complex biological communities, which can adversely impact the performance of drinking water distribution systems (DWDSs), to be characterized. Entire bacterial communities can be studied quickly and inexpensively using targeted metagenomic amplicon sequencing. Here, amplicon sequencing of the 16S rRNA gene region was performed alongside traditional water quality measures to assess the health, quality, and efficiency of two distinct, full-scale DWDSs: (i) a linear DWDS supplied with unfiltered water subjected to basic disinfection before distribution and (ii) a complex, branching DWDS treated by a four-stage water treatment plant (WTP) prior to disinfection and distribution. In both DWDSs bacterial communities differed significantly after disinfection, demonstrating the effectiveness of both treatment regimes. However, bacterial repopulation occurred further along in the DWDSs, and some end-user samples were more similar to the source water than to the postdisinfection water. Three sample locations appeared to be nitrified, displaying elevated nitrate levels and decreased ammonia levels, and nitrifying bacterial species, such as Nitrospira, were detected. Burkholderiales were abundant in samples containing large amounts of monochloramine, indicating resistance to disinfection. Genera known to contain pathogenic and fecal-associated species were also identified in several locations. From this study, we conclude that metagenomic amplicon sequencing is an informative method to support current compliance-based methods and can be used to reveal bacterial community interactions with the chemical and physical properties of DWDSs. PMID:26162884

  17. Clinical impact of targeted amplicon sequencing for meningioma as a practical clinical-sequencing system.

    PubMed

    Yuzawa, Sayaka; Nishihara, Hiroshi; Yamaguchi, Shigeru; Mohri, Hiromi; Wang, Lei; Kimura, Taichi; Tsuda, Masumi; Tanino, Mishie; Kobayashi, Hiroyuki; Terasaka, Shunsuke; Houkin, Kiyohiro; Sato, Norihiro; Tanaka, Shinya

    2016-07-01

    Recent genetic analyses using next-generation sequencers have revealed numerous genetic alterations in various tumors including meningioma, which is the most common primary brain tumor. However, their use as routine laboratory examinations in clinical applications for tumor genotyping is not cost effective. To establish a clinical sequencing system for meningioma and investigate the clinical significance of genotype, we retrospectively performed targeted amplicon sequencing on 103 meningiomas and evaluated the association with clinicopathological features. We designed amplicon-sequencing panels targeting eight genes including NF2 (neurofibromin 2), TRAF7, KLF4, AKT1, and SMO. Libraries prepared with genomic DNA extracted from PAXgene-fixed paraffin-embedded tissues of 103 meningioma specimens were sequenced using the Illumina MiSeq. NF2 loss in some cases was also confirmed by interphase-fluorescent in situ hybridization. We identified NF2 loss and/or at least one mutation in NF2, TRAF7, KLF4, AKT1, and SMO in 81 out of 103 cases (79%) by targeted amplicon sequencing. On the basis of genetic status, we categorized meningiomas into three genotype groups: NF2 type, TRAKLS type harboring mutation in TRAF7, AKT1, KLF4, and/or SMO, and 'not otherwise classified' type. Genotype significantly correlated with tumor volume, tumor location, and magnetic resonance imaging findings such as adjacent bone change and heterogeneous gadolinium enhancement, as well as histopathological subtypes. In addition, multivariate analysis revealed that genotype was independently associated with risk of recurrence. In conclusion, we established a rapid clinical sequencing system that enables final confirmation of meningioma genotype within 7 days turnaround time. Our method will bring multiple benefits to neuropathologists and neurosurgeons for accurate diagnosis and appropriate postoperative management. PMID:27102344

  18. Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers

    PubMed Central

    Pabinger, Stephan; Ernst, Karina; Pulverer, Walter; Kallmeyer, Rainer; Valdes, Ana M.; Metrustry, Sarah; Katic, Denis; Nuzzo, Angelo; Kriegner, Albert; Vierlinger, Klemens; Weinhaeusel, Andreas

    2016-01-01

    Targeted sequencing of PCR amplicons generated from bisulfite deaminated DNA is a flexible, cost-effective way to study methylation of a sample at single CpG resolution and perform subsequent multi-target, multi-sample comparisons. Currently, no platform specific protocol, support, or analysis solution is provided to perform targeted bisulfite sequencing on a Personal Genome Machine (PGM). Here, we present a novel tool, called TABSAT, for analyzing targeted bisulfite sequencing data generated on Ion Torrent sequencers. The workflow starts with raw sequencing data, performs quality assessment, and uses a tailored version of Bismark to map the reads to a reference genome. The pipeline visualizes results as lollipop plots and is able to deduce specific methylation-patterns present in a sample. The obtained profiles are then summarized and compared between samples. In order to assess the performance of the targeted bisulfite sequencing workflow, 48 samples were used to generate 53 different Bisulfite-Sequencing PCR amplicons from each sample, resulting in 2,544 amplicon targets. We obtained a mean coverage of 282X using 1,196,822 aligned reads. Next, we compared the sequencing results of these targets to the methylation level of the corresponding sites on an Illumina 450k methylation chip. The calculated average Pearson correlation coefficient of 0.91 confirms the sequencing results with one of the industry-leading CpG methylation platforms and shows that targeted amplicon bisulfite sequencing provides an accurate and cost-efficient method for DNA methylation studies, e.g., to provide platform-independent confirmation of Illumina Infinium 450k methylation data. TABSAT offers a novel way to analyze data generated by Ion Torrent instruments and can also be used with data from the Illumina MiSeq platform. It can be easily accessed via the Platomics platform, which offers a web-based graphical user interface along with sample and parameter storage. TABSAT is freely

  19. Quick diagnosis of human brain meningitis using omp85 gene amplicon as a genetic marker.

    PubMed

    Dash, Sandip K; Sharma, Minakshi; Khare, Shashi; Kumar, Ashok

    2013-06-01

    The usual diagnosis of life-threatening human brain bacterial meningitis are expensive, time consuming or non-confirmatory. A quick PCR based diagnosis of meningitis in cerebrospinal fluids (CSF) using specific primers of virulent Omp85 gene of Neisseria meningitidis can detect as low as 1.0 ng of genomic DNA (G-DNA) in 80 min for confirmation of bacterial meningitis caused by N. meningitidis infection. The 257 bp amplicon of Omp85 gene does not show homology with other suspected pathogens in CSF and can be used as a specific genetic marker for diagnosis of the disease. PMID:24426115

  20. Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers.

    PubMed

    Pabinger, Stephan; Ernst, Karina; Pulverer, Walter; Kallmeyer, Rainer; Valdes, Ana M; Metrustry, Sarah; Katic, Denis; Nuzzo, Angelo; Kriegner, Albert; Vierlinger, Klemens; Weinhaeusel, Andreas

    2016-01-01

    Targeted sequencing of PCR amplicons generated from bisulfite deaminated DNA is a flexible, cost-effective way to study methylation of a sample at single CpG resolution and perform subsequent multi-target, multi-sample comparisons. Currently, no platform specific protocol, support, or analysis solution is provided to perform targeted bisulfite sequencing on a Personal Genome Machine (PGM). Here, we present a novel tool, called TABSAT, for analyzing targeted bisulfite sequencing data generated on Ion Torrent sequencers. The workflow starts with raw sequencing data, performs quality assessment, and uses a tailored version of Bismark to map the reads to a reference genome. The pipeline visualizes results as lollipop plots and is able to deduce specific methylation-patterns present in a sample. The obtained profiles are then summarized and compared between samples. In order to assess the performance of the targeted bisulfite sequencing workflow, 48 samples were used to generate 53 different Bisulfite-Sequencing PCR amplicons from each sample, resulting in 2,544 amplicon targets. We obtained a mean coverage of 282X using 1,196,822 aligned reads. Next, we compared the sequencing results of these targets to the methylation level of the corresponding sites on an Illumina 450k methylation chip. The calculated average Pearson correlation coefficient of 0.91 confirms the sequencing results with one of the industry-leading CpG methylation platforms and shows that targeted amplicon bisulfite sequencing provides an accurate and cost-efficient method for DNA methylation studies, e.g., to provide platform-independent confirmation of Illumina Infinium 450k methylation data. TABSAT offers a novel way to analyze data generated by Ion Torrent instruments and can also be used with data from the Illumina MiSeq platform. It can be easily accessed via the Platomics platform, which offers a web-based graphical user interface along with sample and parameter storage. TABSAT is freely

  1. Parallel tagged amplicon sequencing of relatively long PCR products using the Illumina HiSeq platform and transcriptome assembly.

    PubMed

    Feng, Yan-Jie; Liu, Qing-Feng; Chen, Meng-Yun; Liang, Dan; Zhang, Peng

    2016-01-01

    In phylogenetics and population genetics, a large number of loci are often needed to accurately resolve species relationships. Normally, loci are enriched by PCR and sequenced by Sanger sequencing, which is expensive when the number of amplicons is large. Next-generation sequencing (NGS) techniques are increasingly used for parallel amplicon sequencing, which reduces sequencing costs tremendously, but has not reduced preparation costs very much. Moreover, for most current NGS methods, amplicons need to be purified and quantified before sequencing and their lengths are also restricted (normally <700 bp). Here, we describe an approach to sequence pooled amplicons of any length using the Illumina platform. Using this method, amplicons are pooled at equal volume rather than at equal concentration, thus eliminating the laborious purification and quantification steps. We then shear the pooled amplicons, repair the ends, add sample identifying linkers and pool multiple samples prior to Illumina library preparation. Data are then assembled using the transcriptome assembly program trinity, which is optimized to deal with templates of highly varying quantities. We demonstrated the utility of our approach by recovering 93.5% of the target amplicons (size up to 1650 bp) in full length for a 16 taxa × 101 loci project, using ~2.0 GB of Illumina HiSeq paired-end 90-bp data. Overall, we validate a rapid, cost-effective and scalable approach to sequence a large number of targeted loci from a large number of samples that is particularly suitable for both phylogenetics and population genetics studies that require a modest scale of data. PMID:25959587

  2. Herpes simplex virus amplicon: cleavage of concatemeric DNA is linked to packaging and involves amplification of the terminally reiterated a sequence.

    PubMed Central

    Deiss, L P; Frenkel, N

    1986-01-01

    Herpes simplex virus-infected cells contain large concatemeric DNA molecules arising from replication of the viral genome. The large concatemers are cleaved to generate unit-length molecules terminating at both ends with the a sequence. We have used constructed defective virus vectors (amplicons) derived from herpes simplex virus to study the mechanism of cleavage of viral DNA concatemers and the packaging of viral DNA into nucleocapsids. These studies revealed that (i) a 248-base-pair a sequence contained the signal(s) required for cleavage-packaging, (ii) the cleavage of viral DNA concatemers was coupled to packaging, (iii) the a sequence contained the information required for its own amplification, and (iv) cleavage-packaging occurred by a novel process involving the amplification of the a sequence. Images PMID:3005637

  3. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing.

    PubMed

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S; Perkins, David L

    2016-01-22

    The human microbiome has emerged as a major player in regulating human health and disease. Translational studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using whole genome shotgun sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1 × 10(6) reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that whole genome shotgun sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection. PMID:26718401

  4. Bioinformatic Amplicon Read Processing Strategies Strongly Affect Eukaryotic Diversity and the Taxonomic Composition of Communities.

    PubMed

    Majaneva, Markus; Hyytiäinen, Kirsi; Varvio, Sirkka Liisa; Nagai, Satoshi; Blomster, Jaanika

    2015-01-01

    Amplicon read sequencing has revolutionized the field of microbial diversity studies. The technique has been developed for bacterial assemblages and has undergone rigorous testing with mock communities. However, due to the great complexity of eukaryotes and the numbers of different rDNA copies, analyzing eukaryotic diversity is more demanding than analyzing bacterial or mock communities, so studies are needed that test the methods of analyses on taxonomically diverse natural communities. In this study, we used 20 samples collected from the Baltic Sea ice, slush and under-ice water to investigate three program packages (UPARSE, mothur and QIIME) and 18 different bioinformatic strategies implemented in them. Our aim was to assess the impact of the initial steps of bioinformatic strategies on the results when analyzing natural eukaryotic communities. We found significant differences among the strategies in resulting read length, number of OTUs and estimates of diversity as well as clear differences in the taxonomic composition of communities. The differences arose mainly because of the variable number of chimeric reads that passed the pre-processing steps. Singleton removal and denoising substantially lowered the number of errors. Our study showed that the initial steps of the bioinformatic amplicon read processing strategies require careful consideration before applying them to eukaryotic communities. PMID:26047335

  5. Application of novel "mini-amplicon" STR multiplexes to high volume casework on degraded skeletal remains.

    PubMed

    Parsons, Thomas J; Huel, Rene; Davoren, Jon; Katzmarzyk, Cheryl; Milos, Ana; Selmanović, Arijana; Smajlović, Lejla; Coble, Michael D; Rizvić, Adnan

    2007-06-01

    The International Commission on Missing Persons (ICMP) conducts high throughput STR profiling on degraded skeletal remains, primarily recovered from mass graves relating to conflicts from 1992 to 1999 in the former Yugoslavia. To date, over 11,000 individuals have been identified through comparison of bone profiles to a large database of profiles from family members of the missing. To increase success rates in STR recovery, three short amplicon STR multiplexes (a 7-plex, a 6-plex, and a 5-plex) have been devised and implemented. These target loci from large commercial multiplexes, with an average decrease in amplicon size of 144 bp. The ICMP "miniplexes" have proven to provide substantially greater recovery of DNA data from a certain subset of difficult samples. However, the circumstances under which miniplexes provide additional data are restricted, and their advantages do not outweigh those of large commercial multiplexes for a majority of cases. The miniplexes, however, also have a very powerful use in DNA testing to support large scale reassociation of commingled, partial skeletons recovered from secondary mass graves. PMID:19083751

  6. Amplicon-based metagenomics identified candidate organisms in soils that caused yield decline in strawberry

    PubMed Central

    Xu, Xiangming; Passey, Thomas; Wei, Feng; Saville, Robert; Harrison, Richard J.

    2015-01-01

    A phenomenon of yield decline due to weak plant growth in strawberry was recently observed in non-chemo-fumigated soils, which was not associated with the soil fungal pathogen Verticillium dahliae, the main target of fumigation. Amplicon-based metagenomics was used to profile soil microbiota in order to identify microbial organisms that may have caused the yield decline. A total of 36 soil samples were obtained in 2013 and 2014 from four sites for metagenomic studies; two of the four sites had a yield-decline problem, the other two did not. More than 2000 fungal or bacterial operational taxonomy units (OTUs) were found in these samples. Relative abundance of individual OTUs was statistically compared for differences between samples from sites with or without yield decline. A total of 721 individual comparisons were statistically significant – involving 366 unique bacterial and 44 unique fungal OTUs. Based on further selection criteria, we focused on 34 bacterial and 17 fungal OTUs and found that yield decline resulted probably from one or more of the following four factors: (1) low abundance of Bacillus and Pseudomonas populations, which are well known for their ability of supressing pathogen development and/or promoting plant growth; (2) lack of the nematophagous fungus (Paecilomyces species); (3) a high level of two non-specific fungal root rot pathogens; and (4) wet soil conditions. This study demonstrated the usefulness of an amplicon-based metagenomics approach to profile soil microbiota and to detect differential abundance in microbes. PMID:26504572

  7. Photochemical immobilization of anthraquinone conjugated oligonucleotides and PCR amplicons on solid surfaces.

    PubMed

    Koch, T; Jacobsen, N; Fensholdt, J; Boas, U; Fenger, M; Jakobsen, M H

    2000-01-01

    Ligand immobilization on solid surfaces is an essential step in fields such as diagnostics, bio sensor manufacturing, and new material sciences in general. In this paper a photochemical approach based on anthraquinone as the chromophore is presented. Photochemical procedures offer special advantages as they are able to generate highly reactive species in an orientation specific manner. As presented here, anthraquinone (AQ) mediated covalent DNA immobilization appears to be superior to currently known procedures. A synthetic procedure providing AQ-phosphoramidites is presented. These reagents facilitate AQ conjugation during routine DNA synthesis, thus enabling the AQ-oligonucleotides to be immobilized in a very convenient and efficient manner. AQ-conjugated PCR primers can be used directly in PCR. When the PCR is performed in solution, the amplicons can be immobilized after the PCR. Moreover, when the primers are immobilized prior to the PCR, a solid-phase PCR can be performed and the amplicons are thus produced directly on the solid support. PMID:10898568

  8. AMPLISAS: a web server for multilocus genotyping using next-generation amplicon sequencing data.

    PubMed

    Sebastian, Alvaro; Herdegen, Magdalena; Migalska, Magdalena; Radwan, Jacek

    2016-03-01

    Next-generation sequencing (NGS) technologies are revolutionizing the fields of biology and medicine as powerful tools for amplicon sequencing (AS). Using combinations of primers and barcodes, it is possible to sequence targeted genomic regions with deep coverage for hundreds, even thousands, of individuals in a single experiment. This is extremely valuable for the genotyping of gene families in which locus-specific primers are often difficult to design, such as the major histocompatibility complex (MHC). The utility of AS is, however, limited by the high intrinsic sequencing error rates of NGS technologies and other sources of error such as polymerase amplification or chimera formation. Correcting these errors requires extensive bioinformatic post-processing of NGS data. Amplicon Sequence Assignment (AMPLISAS) is a tool that performs analysis of AS results in a simple and efficient way, while offering customization options for advanced users. AMPLISAS is designed as a three-step pipeline consisting of (i) read demultiplexing, (ii) unique sequence clustering and (iii) erroneous sequence filtering. Allele sequences and frequencies are retrieved in excel spreadsheet format, making them easy to interpret. AMPLISAS performance has been successfully benchmarked against previously published genotyped MHC data sets obtained with various NGS technologies. PMID:26257385

  9. Amplicon-based metagenomics identified candidate organisms in soils that caused yield decline in strawberry.

    PubMed

    Xu, Xiangming; Passey, Thomas; Wei, Feng; Saville, Robert; Harrison, Richard J

    2015-01-01

    A phenomenon of yield decline due to weak plant growth in strawberry was recently observed in non-chemo-fumigated soils, which was not associated with the soil fungal pathogen Verticillium dahliae, the main target of fumigation. Amplicon-based metagenomics was used to profile soil microbiota in order to identify microbial organisms that may have caused the yield decline. A total of 36 soil samples were obtained in 2013 and 2014 from four sites for metagenomic studies; two of the four sites had a yield-decline problem, the other two did not. More than 2000 fungal or bacterial operational taxonomy units (OTUs) were found in these samples. Relative abundance of individual OTUs was statistically compared for differences between samples from sites with or without yield decline. A total of 721 individual comparisons were statistically significant - involving 366 unique bacterial and 44 unique fungal OTUs. Based on further selection criteria, we focused on 34 bacterial and 17 fungal OTUs and found that yield decline resulted probably from one or more of the following four factors: (1) low abundance of Bacillus and Pseudomonas populations, which are well known for their ability of supressing pathogen development and/or promoting plant growth; (2) lack of the nematophagous fungus (Paecilomyces species); (3) a high level of two non-specific fungal root rot pathogens; and (4) wet soil conditions. This study demonstrated the usefulness of an amplicon-based metagenomics approach to profile soil microbiota and to detect differential abundance in microbes. PMID:26504572

  10. Bioinformatic Amplicon Read Processing Strategies Strongly Affect Eukaryotic Diversity and the Taxonomic Composition of Communities

    PubMed Central

    Majaneva, Markus; Hyytiäinen, Kirsi; Varvio, Sirkka Liisa; Nagai, Satoshi; Blomster, Jaanika

    2015-01-01

    Amplicon read sequencing has revolutionized the field of microbial diversity studies. The technique has been developed for bacterial assemblages and has undergone rigorous testing with mock communities. However, due to the great complexity of eukaryotes and the numbers of different rDNA copies, analyzing eukaryotic diversity is more demanding than analyzing bacterial or mock communities, so studies are needed that test the methods of analyses on taxonomically diverse natural communities. In this study, we used 20 samples collected from the Baltic Sea ice, slush and under-ice water to investigate three program packages (UPARSE, mothur and QIIME) and 18 different bioinformatic strategies implemented in them. Our aim was to assess the impact of the initial steps of bioinformatic strategies on the results when analyzing natural eukaryotic communities. We found significant differences among the strategies in resulting read length, number of OTUs and estimates of diversity as well as clear differences in the taxonomic composition of communities. The differences arose mainly because of the variable number of chimeric reads that passed the pre-processing steps. Singleton removal and denoising substantially lowered the number of errors. Our study showed that the initial steps of the bioinformatic amplicon read processing strategies require careful consideration before applying them to eukaryotic communities. PMID:26047335

  11. ICO amplicon NGS data analysis: a Web tool for variant detection in common high-risk hereditary cancer genes analyzed by amplicon GS Junior next-generation sequencing.

    PubMed

    Lopez-Doriga, Adriana; Feliubadaló, Lídia; Menéndez, Mireia; Lopez-Doriga, Sergio; Morón-Duran, Francisco D; del Valle, Jesús; Tornero, Eva; Montes, Eva; Cuesta, Raquel; Campos, Olga; Gómez, Carolina; Pineda, Marta; González, Sara; Moreno, Victor; Capellá, Gabriel; Lázaro, Conxi

    2014-03-01

    Next-generation sequencing (NGS) has revolutionized genomic research and is set to have a major impact on genetic diagnostics thanks to the advent of benchtop sequencers and flexible kits for targeted libraries. Among the main hurdles in NGS are the difficulty of performing bioinformatic analysis of the huge volume of data generated and the high number of false positive calls that could be obtained, depending on the NGS technology and the analysis pipeline. Here, we present the development of a free and user-friendly Web data analysis tool that detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high-risk hereditary cancer genes using amplicon libraries run in a GS Junior System. The Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. We believe that this tool will greatly facilitate the use of the NGS approach in routine laboratories. PMID:24227591

  12. Differences in the methylation patterns of the VaSTS1 and VaSTS10 genes of Vitis amurensis Rupr.

    PubMed

    Tyunin, A P; Kiselev, K V; Karetin, Y A

    2013-09-01

    Resveratrol is a plant-derived phenol but the mechanism that regulates its biosynthesis remains unidentified. Stilbene synthase (STS) catalyzes resveratrol formation in vivo and we have proposed that inducers of resveratrol production affect STS expression through an unidentified epigenetic mechanism. To investigate the role of DNA methylation in resveratrol biosynthesis, we treated both rolB transgenic and empty vector control Vitis amurensis cell cultures with the DNA demethylation agent, 5-azacytidine. Treated cells had increased resveratrol production through activation of VaSTS10 expression. The lowest levels of cytosine methylation were at the 5'- and 3'-ends of the VaSTS1 protein-coding sequence. Cytosine methylation decreased mostly at the 5'- and 3'-ends of VaSTS10 after azaC treatment with an intriguing regularity in the number of cytosine nucleotides within the 5'- and 3'- ends of the protein-coding sequences. Thus, cytosine methylation is crucial for the regulation of the resveratrol biosynthetic pathway. PMID:23690043

  13. Identification and Evaluation of Single-Nucleotide Polymorphisms in Allotetraploid Peanut (Arachis hypogaea L.) Based on Amplicon Sequencing Combined with High Resolution Melting (HRM) Analysis

    PubMed Central

    Hong, Yanbin; Pandey, Manish K.; Liu, Ying; Chen, Xiaoping; Liu, Hong; Varshney, Rajeev K.; Liang, Xuanqiang; Huang, Shangzhi

    2015-01-01

    The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut. PMID:26697032

  14. Identification and Evaluation of Single-Nucleotide Polymorphisms in Allotetraploid Peanut (Arachis hypogaea L.) Based on Amplicon Sequencing Combined with High Resolution Melting (HRM) Analysis.

    PubMed

    Hong, Yanbin; Pandey, Manish K; Liu, Ying; Chen, Xiaoping; Liu, Hong; Varshney, Rajeev K; Liang, Xuanqiang; Huang, Shangzhi

    2015-01-01

    The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut. PMID:26697032

  15. 38 CFR 1.203 - Information to be reported to VA Police.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... reported to VA Police. 1.203 Section 1.203 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS... be reported to VA Police. Information about actual or possible violations of criminal laws related to... occurs on VA premises, will be reported by VA management officials to the VA police component...

  16. 38 CFR 1.203 - Information to be reported to VA Police.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... reported to VA Police. 1.203 Section 1.203 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS... be reported to VA Police. Information about actual or possible violations of criminal laws related to... occurs on VA premises, will be reported by VA management officials to the VA police component...

  17. 38 CFR 1.203 - Information to be reported to VA Police.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... reported to VA Police. 1.203 Section 1.203 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS... be reported to VA Police. Information about actual or possible violations of criminal laws related to... occurs on VA premises, will be reported by VA management officials to the VA police component...

  18. 38 CFR 1.203 - Information to be reported to VA Police.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... reported to VA Police. 1.203 Section 1.203 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS... be reported to VA Police. Information about actual or possible violations of criminal laws related to... occurs on VA premises, will be reported by VA management officials to the VA police component...

  19. 38 CFR 1.203 - Information to be reported to VA Police.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... reported to VA Police. 1.203 Section 1.203 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS... be reported to VA Police. Information about actual or possible violations of criminal laws related to... occurs on VA premises, will be reported by VA management officials to the VA police component...

  20. Home Health Care and Patterns of Subsequent VA and Medicare Health Care Utilization for Veterans

    ERIC Educational Resources Information Center

    Van Houtven, Courtney Harold; Jeffreys, Amy S.; Coffman, Cynthia J.

    2008-01-01

    Purpose: The Veterans Affairs or VA health care system is in the process of significantly expanding home health care (HOC) nationwide. We describe VA HHC use in 2003 for all VA HHC users from 2002; we examine whether VA utilization across a broad spectrum of services differed for a sample of VA HHC users and their propensity-score-matched…

  1. Membrane-dependent Interaction of Factor Xa and Prothrombin with Factor Va in the Prothrombinase Complex

    PubMed Central

    Qureshi, Shabir H.; Yang, Likui; Manithody, Chandrashekhara; Rezaie, Alireza R.

    2009-01-01

    Because all three protein components of prothrombinase, factors (f) Xa and Va and prothrombin, bind to negatively charged membrane phospholipids, the exact role of the membrane in the prothrombinase reaction has not been fully understood. In this study, we prepared deletion derivatives of fXa and prothrombin in which both the Gla and first EGF-like domains of the protease (E2-fXa) as well as the Gla and both kringle domains of the substrate (prethrombin-2) were deleted. The fVa-mediated catalytic activity of E2-fXa toward prethrombin-2 was analyzed in both the absence and presence of phospholipids composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS). PCPS markedly accelerated the initial rate of prethrombin-2 activation by E2-fXa, with the cofactor exhibiting saturation only in the presence of phospholipids (apparent Kd ∼60 nM). Competitive kinetic studies in the presence of the two exosite-1-specific ligands Tyr63-sulfated hirudin54-65 and TM456 suggested that while both peptides are highly effective inhibitors of the fVa-mediated activation of prethrombin-2 by E2-fXa in the absence of PCPS, they are ineffective competitors in the presence of phospholipids. Since neither E2-fXa nor prethrombin-2 can interact with membranes, these results suggest that fVa interaction with PCPS improves the affinity of the activation complex for the proexosite-1 of the substrate. Direct binding studies employing OG488-EGR-labeled fXa and E2-fXa revealed that the interaction of the Gla-domain of fXa with PCPS also induces conformational changes in the protease to facilitate its high-affinity interaction with fVa. PMID:19378973

  2. DOE Spent Nuclear Fuel Information In Support of TSPA-VA

    SciTech Connect

    A. Brewer; D. Cresap; D. Fillmore; H. Loo; M. Ebner; R. McCormack

    1998-09-01

    RW has started the viability assessment (VA) effort to determine the feasibility of Yucca Mountain as the first geologic repository for spent nuclear fuel (SNF) and high-level waste. One component of the viability assessment will be a total system performance assessment (TSPA), based on the design concept and the scientific data and analysis available, describing the repository's probable behavior relative to the overall system performance standards. Thus, all the data collected from the Exploratory Studies Facility to-date have been incorporated into the latest TSPA model. In addition, the Repository Integration Program, an integrated probabilistic simulator, used in the TSPA has also been updated by Golder Associates Incorporated at December 1997. To ensure that the Department of Energy-owned (DOE-owned) SNF continues to be acceptable for disposal in the repository, it will be included in the TSPA-VA evaluation. A number of parameters are needed in the TSPA-VA models to predict the performance of the DOE-owned SNF materials placed into the potential repository. This report documents all of the basis and/or derivation for each of these parameters. A number of properties were not readily available at the time the TSPA-VA data was requested. Thus, expert judgement and opinion was utilized to determine a best property value. The performance of the DOE-owned SNF will be published as part of the TSPA-VA report. Each DOE site will be collecting better data as the DOE SNF program moves closer to repository license application. As required by the RW-0333P, the National Spent Nuclear Fuel Program will be assisting each site in qualifying the information used to support the performance assessment evaluations.

  3. 12 CFR 3.205 - VaR-based measure.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... portfolio of correlation trading positions that is modeled under § 3.209. A national bank or Federal savings... proxies are used. (b) Quantitative requirements for VaR-based measure. (1) The VaR-based measure must be... association's trading portfolio over a full business cycle. A national bank or Federal savings...

  4. 12 CFR 324.205 - VaR-based measure.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... of correlation trading positions that is modeled under § 324.209. An FDIC-supervised institution may.... (b) Quantitative requirements for VaR-based measure. (1) The VaR-based measure must be calculated on... of at least six months representing the volatility of the FDIC-supervised institution's...

  5. 38 CFR 17.71 - Revocation of VA approval.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... revocation of VA approval, VA health care personnel shall: (1) Cease referring veterans to the community residential care facility; and, (2) Notify any veteran residing in the community residential care facility of... health care personnel shall notify that person or entity of the community residential care...

  6. 38 CFR 17.71 - Revocation of VA approval.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... revocation of VA approval, VA health care personnel shall: (1) Cease referring veterans to the community residential care facility; and, (2) Notify any veteran residing in the community residential care facility of... health care personnel shall notify that person or entity of the community residential care...

  7. 38 CFR 17.71 - Revocation of VA approval.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... revocation of VA approval, VA health care personnel shall: (1) Cease referring veterans to the community residential care facility; and, (2) Notify any veteran residing in the community residential care facility of... health care personnel shall notify that person or entity of the community residential care...

  8. 38 CFR 17.71 - Revocation of VA approval.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... revocation of VA approval, VA health care personnel shall: (1) Cease referring veterans to the community residential care facility; and, (2) Notify any veteran residing in the community residential care facility of... health care personnel shall notify that person or entity of the community residential care...

  9. 76 FR 60713 - Establishment of Class E Airspace; Bumpass, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-30

    ... Airspace at Bumpass, VA, to accommodate the new Standard Instrument Approach Procedures serving Lake Anna... establish Class E airspace at Bumpass, VA (76 FR 45479) Docket No. FAA-2011-0377. Interested parties were... for Lake Anna Airport. This action is necessary for the safety and management of IFR operations at...

  10. 78 FR 76061 - Authorization for Non-VA Medical Services

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-16

    ..., Health professions, Health records, Homeless, Mental health programs, Nursing homes, Reporting and.... SUPPLEMENTARY INFORMATION: On November 28, 2012, VA proposed a rule in the Federal Register, at 77 FR 70967, to... for that disability. On the same date, VA published a companion direct final rule at 77 FR 70893...

  11. 78 FR 63143 - VA Dental Insurance Program-Federalism

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-23

    ... AFFAIRS 38 CFR Part 17 RIN 2900-AO86 VA Dental Insurance Program--Federalism AGENCY: Department of... its regulations related to the VA Dental Insurance Program (VADIP), a pilot program to offer premium-based dental insurance to enrolled veterans and certain survivors and dependents of...

  12. 76 FR 34576 - Amendment of Class E Airspace; Waynesboro, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-14

    ...) to amend Class E airspace at Eagle's Nest Airport, Waynesboro, VA (75 FR 14820) Docket No. FAA-2010..., 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71.1 0 2. The incorporation... Federal Aviation Administration 14 CFR Part 71 Amendment of Class E Airspace; Waynesboro, VA...

  13. 77 FR 25592 - Drawbridge Operation Regulations; James River, Hopewell, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-01

    ... SECURITY Coast Guard 33 CFR Part 117 Drawbridge Operation Regulations; James River, Hopewell, VA AGENCY... Memorial Bridge, at mile 65.0, across the James River, at Hopewell, VA. This deviation is necessary to... schedule, the SR 156/Benjamin Harrison Memorial Bridge, at mile 65.0, across the James River, at...

  14. 76 FR 52230 - Establishment of Class E Airspace; Forest, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-22

    ... Airspace at Forest, VA, to accommodate the new Area Navigation (RNAV) Global Positioning System (GPS..., VA (76 FR 34196) Docket No. FAA-2011-0378. Interested parties were invited to participate in this... Procedures (44 FR 11034; February 26, 1979); and (3) does not warrant preparation of a Regulatory...

  15. 33 CFR 80.510 - Chesapeake Bay Entrance, VA.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Chesapeake Bay Entrance, VA. 80.510 Section 80.510 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Fifth District § 80.510 Chesapeake Bay Entrance, VA....

  16. FACILITIES FOR EDUCATION IN VA HOSPITALS. FINAL REPORT.

    ERIC Educational Resources Information Center

    GREEN, ALAN C.; AND OTHERS

    THIS STUDY WAS AUTHORIZED BY THE VA DEPARTMENT OF MEDICINE AND SURGERY FOR THE PURPOSE OF IDENTIFYING AND DETERMINING THE FACILITIES NEEDED TO PROPERLY HOUSE AND SUPPORT EDUCATION ACTIVITIES IN EXISTING AND FUTURE VA HOSPITALS AND TO PRODUCE ARCHITECTURAL GUIDANCE IN THE DESIGN OF THE FACILITIES. CURRENT PRACTICES AND SIGNIFICANT TRENDS IN MEDICAL…

  17. Single-step conversion of cells to retrovirus vector producers with herpes simplex virus-Epstein-Barr virus hybrid amplicons.

    PubMed

    Sena-Esteves, M; Saeki, Y; Camp, S M; Chiocca, E A; Breakefield, X O

    1999-12-01

    We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV-Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 10(6) and 10(7) transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery. PMID:10559361

  18. 76 FR 79067 - Payment or Reimbursement for Emergency Treatment Furnished by Non-VA Providers in Non-VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-21

    ...- connected conditions. As explained in a notice of proposed rulemaking published on June 11, 2010 (75 FR... with Non- VA Outpatient Care''. 75 FR 7218 (Feb. 18, 2010). Second, section 402(b)(3) made the... proposed rule published on June 11, 2010 (75 FR 33216), we proposed to amend the following VA...

  19. The Bias Associated with Amplicon Sequencing Does Not Affect the Quantitative Assessment of Bacterial Community Dynamics

    PubMed Central

    Figuerola, Eva L. M.; Erijman, Leonardo

    2014-01-01

    The performance of two sets of primers targeting variable regions of the 16S rRNA gene V1–V3 and V4 was compared in their ability to describe changes of bacterial diversity and temporal turnover in full-scale activated sludge. Duplicate sets of high-throughput amplicon sequencing data of the two 16S rRNA regions shared a collection of core taxa that were observed across a series of twelve monthly samples, although the relative abundance of each taxon was substantially different between regions. A case in point was the changes in the relative abundance of filamentous bacteria Thiothrix, which caused a large effect on diversity indices, but only in the V1–V3 data set. Yet the relative abundance of Thiothrix in the amplicon sequencing data from both regions correlated with the estimation of its abundance determined using fluorescence in situ hybridization. In nonmetric multidimensional analysis samples were distributed along the first ordination axis according to the sequenced region rather than according to sample identities. The dynamics of microbial communities indicated that V1–V3 and the V4 regions of the 16S rRNA gene yielded comparable patterns of: 1) the changes occurring within the communities along fixed time intervals, 2) the slow turnover of activated sludge communities and 3) the rate of species replacement calculated from the taxa–time relationships. The temperature was the only operational variable that showed significant correlation with the composition of bacterial communities over time for the sets of data obtained with both pairs of primers. In conclusion, we show that despite the bias introduced by amplicon sequencing, the variable regions V1–V3 and V4 can be confidently used for the quantitative assessment of bacterial community dynamics, and provide a proper qualitative account of general taxa in the community, especially when the data are obtained over a convenient time window rather than at a single time point. PMID:24923665

  20. The depiction of Alboran Sea Gyre during Donde Va? using remote sensing and conventional data

    NASA Technical Reports Server (NTRS)

    Laviolette, P. E.

    1984-01-01

    Experienced oceanographic investigators have come to realize that remote sensing techniques are most successful when applied as part of programs of integrated measurements aimed at solving specific oceanographic problems. A good example of such integration occurred during the multi-platform international experiment, Donde Va? in the Alboran Sea during the period June through October, 1982. The objective of Donde Va? was to derive the interrelationship of the Atlantic waters entering the Mediterranean Sea and the Alboran Sea Gyre. The experimental plan conceived solely with this objective in mind consisted of a variety of remote sensing and conventional platforms: three ships, three aircraft, five current moorings, two satellites and a specialized beach radar (CODAR). Integrated analyses of these multiple-data sets are still being conducted. However, the initial results show detailed structure of the incoming Atlantic jet and Alboran Sea Gyre that would not have been possible by conventional means.

  1. Pitfalls of haplotype phasing from amplicon-based long-read sequencing

    PubMed Central

    Laver, Thomas W.; Caswell, Richard C.; Moore, Karen A.; Poschmann, Jeremie; Johnson, Matthew B.; Owens, Martina M.; Ellard, Sian; Paszkiewicz, Konrad H.; Weedon, Michael N.

    2016-01-01

    The long-read sequencers from Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT) offer the opportunity to phase mutations multiple kilobases apart directly from sequencing reads. In this study, we used long-range PCR with ONT and PacBio sequencing to phase two variants 9 kb apart in the RET gene. We also re-analysed data from a recent paper which had apparently successfully used ONT to phase clinically important haplotypes at the CYP2D6 and HLA loci. From these analyses, we demonstrate PCR-chimera formation during PCR amplification and reference alignment bias are pitfalls that need to be considered when attempting to phase variants using amplicon-based long-read sequencing technologies. These methodological pitfalls need to be avoided if the opportunities provided by long-read sequencers are to be fully exploited. PMID:26883533

  2. Amplicon restriction patterns associated with nitrogenase activity of root nodules for selection of superior Myrica seedlings.

    PubMed

    Yanthan, Mhathung; Misra, Arvind K

    2013-11-01

    Trees of Myrica sp. grow abundantly in the forests of Meghalaya, India. These trees are actinorhizal and harbour nitrogen-fixing Frankia in their root nodules and contribute positively towards the enhancement of nitrogen status of forest areas. They can be used in rejuvenation of mine spoils and nitrogen-depleted fallow lands generated due to slash and burn agriculture practiced in the area. We have studied the association of amplicon restriction patterns (ARPs) of Myrica ribosomal RNA gene and internal transcribed spacer (ITS) region and nitrogenase activity of its root nodules. We found that ARPs thus obtained could be used as markers for early screening of seedlings that could support strains of Frankia that fix atmospheric nitrogen more efficiently. PMID:24287658

  3. 16S rRNA amplicon sequencing dataset for conventionalized and conventionally raised zebrafish larvae.

    PubMed

    Davis, Daniel J; Bryda, Elizabeth C; Gillespie, Catherine H; Ericsson, Aaron C

    2016-09-01

    Data presented here contains metagenomic analysis regarding the sequential conventionalization of germ-free zebrafish embryos. Zebrafish embryos that underwent a germ-free sterilization process immediately after fertilization were promptly exposed to and raised to larval stage in conventional fish water. At 6 days postfertilization (dpf), these "conventionalized" larvae were compared to zebrafish larvae that were raised in conventional fish water never undergoing the initial sterilization process. Bacterial 16S rRNA amplicon sequencing was performed on DNA isolated from homogenates of the larvae revealing distinct microbiota variations between the two groups. The dataset described here is also related to the research article entitled "Microbial modulation of behavior and stress responses in zebrafish larvae" (Davis et al., 2016) [1]. PMID:27508247

  4. Integrating metagenomic and amplicon databases to resolve the phylogenetic and ecological diversity of the Chlamydiae.

    PubMed

    Lagkouvardos, Ilias; Weinmaier, Thomas; Lauro, Federico M; Cavicchioli, Ricardo; Rattei, Thomas; Horn, Matthias

    2014-01-01

    In the era of metagenomics and amplicon sequencing, comprehensive analyses of available sequence data remain a challenge. Here we describe an approach exploiting metagenomic and amplicon data sets from public databases to elucidate phylogenetic diversity of defined microbial taxa. We investigated the phylum Chlamydiae whose known members are obligate intracellular bacteria that represent important pathogens of humans and animals, as well as symbionts of protists. Despite their medical relevance, our knowledge about chlamydial diversity is still scarce. Most of the nine known families are represented by only a few isolates, while previous clone library-based surveys suggested the existence of yet uncharacterized members of this phylum. Here we identified more than 22,000 high quality, non-redundant chlamydial 16S rRNA gene sequences in diverse databases, as well as 1900 putative chlamydial protein-encoding genes. Even when applying the most conservative approach, clustering of chlamydial 16S rRNA gene sequences into operational taxonomic units revealed an unexpectedly high species, genus and family-level diversity within the Chlamydiae, including 181 putative families. These in silico findings were verified experimentally in one Antarctic sample, which contained a high diversity of novel Chlamydiae. In our analysis, the Rhabdochlamydiaceae, whose known members infect arthropods, represents the most diverse and species-rich chlamydial family, followed by the protist-associated Parachlamydiaceae, and a putative new family (PCF8) with unknown host specificity. Available information on the origin of metagenomic samples indicated that marine environments contain the majority of the newly discovered chlamydial lineages, highlighting this environment as an important chlamydial reservoir. PMID:23949660

  5. Long amplicon (LA)-qPCR for the discrimination of infectious and noninfectious phix174 bacteriophages after UV inactivation.

    PubMed

    Ho, Johannes; Seidel, Michael; Niessner, Reinhard; Eggers, Jutta; Tiehm, Andreas

    2016-10-15

    Waterborne viruses are increasingly being considered in risk assessment schemes. In general, virus detection by culture methods is time consuming. In contrast, detection by quantitative polymerase chain reaction (qPCR) is more rapid and therefore, more suitable for monitoring. At present, qPCR lacks the essential ability for discriminating between infectious and non-infectious viruses, thus limiting its applicability for monitoring disinfection processes. In this study, a method was developed to quantify UV inactivation by long amplicon (LA)-qPCR. Bacteriophage phiX174 was used as a surrogate for human pathogenic viruses. A qPCR protocol was developed with new sets of primers, resulting in amplicon lengths of 108, 250, 456, 568, 955, 1063, 1544, and 1764 nucleotides. The log reduction of gene copies increased with increasing amplicon length. Additional treatment with the intercalating dye, PMA, had no effect, indicating that the bacteriophage capsids were not damaged by low pressure UV irradiation. A qPCR of nearly the complete genome (approx. 5000 nucleotides) showed similar results to the plaque assay. The log reduction in qPCR correlates with [specific amplicon length x UV dose]. The normalized DNA effect constant can be applied to calculate phiX174 inactivation based on qPCR detection. PMID:27450352

  6. Policy Recommendations to VA Leave NAS at Odds with Congress

    ERIC Educational Resources Information Center

    Walsh, John

    1978-01-01

    Describes adverse congressional reaction to a recent National Academy of Sciences (NAS) recommendation that the Veteran's Administration's (VA) health care system be phased into a general health care system. (SL)

  7. Exploration Day at Busch Gardens, Williamsburg, Va. - Aug. 5, 2011

    NASA Video Gallery

    Friday, August 8, was NASA Days at Busch Gardens Williamsburg, Va. NASA exhibits and educational specialists worked to inspire young and old, and NASA astronaut Susan Kilrain -- a veteran of two Sp...

  8. 76 FR 72838 - Amendment of Class E Airspace; Luray, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-28

    ... Luray, VA, to accommodate the new Area Navigation (RNAV) Global Positioning System (GPS) Standard... accommodate the new Area Navigation (RNAV) Global Positioning System (GPS) Standard Instrument Approach... FR 52292). Interested parties were invited to participate in this rulemaking effort by...

  9. 78 FR 42455 - Medications Prescribed by Non-VA Providers

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-16

    ... Administrative practice and procedure, Alcohol abuse, Alcoholism, Claims, Day care, Dental health, Drug abuse... section to read as follows: Sec. 17.96 Medication prescribed by non-VA physicians. * * * * * (a) * * *...

  10. 76 FR 48204 - Fund Availability Under VA's Homeless Providers Grant and Per Diem Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-08

    ... availability of funds for currently operational fiscal year (FY) 2009 VA Grant and Per Diem Special Need Grant Recipients in conjunction with their collaborative VA Special Need partners and currently operational VA... under VA's Homeless Providers Grant and Per Diem Program for FY 2009 operational Grant and Per...

  11. 78 FR 38452 - Agency Information Collection (VA Police Officer Pre-Employment Screening Checklist) Activities...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-26

    ... AFFAIRS Agency Information Collection (VA Police Officer Pre-Employment Screening Checklist) Activities... ``OMB Control No. 2900-0524.'' SUPPLEMENTARY INFORMATION: Title: VA Police Officer Pre-Employment... checks on applicants seeking employment as VA police officers. VA will use the data collected...

  12. 78 FR 18425 - Proposed Information Collection VA Police Officer Pre-Employment Screening Checklist); Comment...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-26

    ... AFFAIRS Proposed Information Collection VA Police Officer Pre-Employment Screening Checklist); Comment... applicant's qualification and suitability as a VA police officer. DATES: Written comments and... information technology. Title: VA Police Officer Pre-Employment Screening Checklist, VA Form 0120. OMB...

  13. Polymicrobial Nature of Chronic Diabetic Foot Ulcer Biofilm Infections Determined Using Bacterial Tag Encoded FLX Amplicon Pyrosequencing (bTEFAP)

    PubMed Central

    Dowd, Scot E.; Wolcott, Randall D.; Sun, Yan; McKeehan, Trevor; Smith, Ethan; Rhoads, Daniel

    2008-01-01

    Background Diabetic extremity ulcers are associated with chronic infections. Such ulcer infections are too often followed by amputation because there is little or no understanding of the ecology of such infections or how to control or eliminate this type of chronic infection. A primary impediment to the healing of chronic wounds is biofilm phenotype infections. Diabetic foot ulcers are the most common, disabling, and costly complications of diabetes. Here we seek to derive a better understanding of the polymicrobial nature of chronic diabetic extremity ulcer infections. Methods and Findings Using a new bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) approach we have evaluated the bacterial diversity of 40 chronic diabetic foot ulcers from different patients. The most prevalent bacterial genus associated with diabetic chronic wounds was Corynebacterium spp. Findings also show that obligate anaerobes including Bacteroides, Peptoniphilus, Fingoldia, Anaerococcus, and Peptostreptococcus spp. are ubiquitous in diabetic ulcers, comprising a significant portion of the wound biofilm communities. Other major components of the bacterial communities included commonly cultured genera such as Streptococcus, Serratia, Staphylococcus and Enterococcus spp. Conclusions In this article, we highlight the patterns of population diversity observed in the samples and introduce preliminary evidence to support the concept of functional equivalent pathogroups (FEP). Here we introduce FEP as consortia of genotypically distinct bacteria that symbiotically produce a pathogenic community. According to this hypothesis, individual members of these communities when they occur alone may not cause disease but when they coaggregate or consort together into a FEP the synergistic effect provides the functional equivalence of well-known pathogens, such as Staphylococcus aureus, giving the biofilm community the factors necessary to maintain chronic biofilm infections. Further work is

  14. Extracellular DNA amplicon sequencing reveals high levels of benthic eukaryotic diversity in the central Red Sea.

    PubMed

    Pearman, John K; Irigoien, Xabier; Carvalho, Susana

    2016-04-01

    The present study aims to characterize the benthic eukaryotic biodiversity patterns at a coarse taxonomic level in three areas of the central Red Sea (a lagoon, an offshore area in Thuwal and a shallow coastal area near Jeddah) based on extracellular DNA. High-throughput amplicon sequencing targeting the V9 region of the 18S rRNA gene was undertaken for 32 sediment samples. High levels of alpha-diversity were detected with 16,089 operational taxonomic units (OTUs) being identified. The majority of the OTUs were assigned to Metazoa (29.2%), Alveolata (22.4%) and Stramenopiles (17.8%). Stramenopiles (Diatomea) and Alveolata (Ciliophora) were frequent in a lagoon and in shallower coastal stations, whereas metazoans (Arthropoda: Maxillopoda) were dominant in deeper offshore stations. Only 24.6% of total OTUs were shared among all areas. Beta-diversity was generally lower between the lagoon and Jeddah (nearshore) than between either of those and the offshore area, suggesting a nearshore-offshore biodiversity gradient. The current approach allowed for a broad-range of benthic eukaryotic biodiversity to be analysed with significantly less labour than would be required by other traditional taxonomic approaches. Our findings suggest that next generation sequencing techniques have the potential to provide a fast and standardised screening of benthic biodiversity at large spatial and temporal scales. PMID:26525270

  15. Differential amplicons (ΔAmp)—a new molecular method to assess RNA integrity

    PubMed Central

    Björkman, J.; Švec, D.; Lott, E.; Kubista, M.; Sjöback, R.

    2015-01-01

    Integrity of the mRNA in clinical samples has major impact on the quality of measured expression levels. This is independent of the measurement technique being next generation sequencing (NGS), Quantitative real-time PCR (qPCR) or microarray profiling. If mRNA is highly degraded or damaged, measured data will be very unreliable and the whole study is likely a waste of time and money. It is therefore common strategy to test the quality of RNA in samples before conducting large and costly studies. Most methods today to assess the quality of RNA are ignorant to the nature of the RNA and, therefore, reflect the integrity of ribosomal RNA, which is the dominant species, rather than of mRNAs, microRNAs and long non-coding RNAs, which usually are the species of interest. Here, we present a novel molecular approach to assess the quality of the targeted RNA species by measuring the differential amplification (ΔAmp) of an Endogenous RNase Resistant (ERR) marker relative to a reference gene, optionally combined with the measurement of two amplicons of different lengths. The combination reveals any mRNA degradation caused by ribonucleases as well as physical, chemical or UV damage. ΔAmp has superior sensitivity to common microfluidic electrophoretic methods, senses the integrity of the actual targeted RNA species, and allows for a smoother and more cost efficient workflow. PMID:27077042

  16. Targeted amplicon sequencing (TAS): a scalable next-gen approach to multilocus, multitaxa phylogenetics.

    PubMed

    Bybee, Seth M; Bracken-Grissom, Heather; Haynes, Benjamin D; Hermansen, Russell A; Byers, Robert L; Clement, Mark J; Udall, Joshua A; Wilcox, Edward R; Crandall, Keith A

    2011-01-01

    Next-gen sequencing technologies have revolutionized data collection in genetic studies and advanced genome biology to novel frontiers. However, to date, next-gen technologies have been used principally for whole genome sequencing and transcriptome sequencing. Yet many questions in population genetics and systematics rely on sequencing specific genes of known function or diversity levels. Here, we describe a targeted amplicon sequencing (TAS) approach capitalizing on next-gen capacity to sequence large numbers of targeted gene regions from a large number of samples. Our TAS approach is easily scalable, simple in execution, neither time-nor labor-intensive, relatively inexpensive, and can be applied to a broad diversity of organisms and/or genes. Our TAS approach includes a bioinformatic application, BarcodeCrucher, to take raw next-gen sequence reads and perform quality control checks and convert the data into FASTA format organized by gene and sample, ready for phylogenetic analyses. We demonstrate our approach by sequencing targeted genes of known phylogenetic utility to estimate a phylogeny for the Pancrustacea. We generated data from 44 taxa using 68 different 10-bp multiplexing identifiers. The overall quality of data produced was robust and was informative for phylogeny estimation. The potential for this method to produce copious amounts of data from a single 454 plate (e.g., 325 taxa for 24 loci) significantly reduces sequencing expenses incurred from traditional Sanger sequencing. We further discuss the advantages and disadvantages of this method, while offering suggestions to enhance the approach. PMID:22002916

  17. Amplicon pyrosequencing reveals the soil microbial diversity associated with invasive Japanese barberry (Berberis thunbergii DC.).

    PubMed

    Coats, V C; Pelletreau, K N; Rumpho, M E

    2014-03-01

    The soil microbial community acts as a reservoir of microbes that directly influences the structure and composition of the aboveground plant community, promotes plant growth, increases stress tolerance and mediates local patterns of nutrient cycling. Direct interactions between plants and rhizosphere-dwelling microorganisms occur at, or near, the surface of the root. Upon introduction and establishment, invasive plants modify the soil microbial communities and soil biochemistry affecting bioremediation efforts and future plant communities. Here, we used tag-encoded FLX amplicon 454 pyrosequencing (TEFAP) to characterize the bacterial and fungal community diversity in the rhizosphere of Berberis thunbergii DC. (Japanese barberry) from invasive stands in coastal Maine to investigate effects of soil type, soil chemistry and surrounding plant cover on the soil microbial community structure. Acidobacteria, Actinobacteria, Proteobacteria and Verrucomicrobia were the dominant bacterial phyla, whereas fungal communities were comprised mostly of Ascomycota and Basidiomycota phyla members, including Agaricomycetes and Sordariomycetes. Bulk soil chemistry had more effect on the bacterial community structure than the fungal community. An effect of geographic location was apparent in the rhizosphere microbial communities, yet it was less significant than the effect of surrounding plant cover. These data demonstrate a high degree of spatial variation in the rhizosphere microbial communities of Japanese barberry with apparent effects of soil chemistry, location and canopy cover on the microbial community structure. PMID:24118303

  18. Shedding Light on the Microbial Community of the Macropod Foregut Using 454-Amplicon Pyrosequencing

    PubMed Central

    Gulino, Lisa-Maree; Ouwerkerk, Diane; Kang, Alicia Y. H.; Maguire, Anita J.; Kienzle, Marco; Klieve, Athol V.

    2013-01-01

    Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as ‘shared’ OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry. PMID:23626688

  19. Rare amplicons implicate frequent deregulation of cell fate specification pathways in oral squamous cell carcinoma.

    PubMed

    Snijders, Antoine M; Schmidt, Brian L; Fridlyand, Jane; Dekker, Nusi; Pinkel, Daniel; Jordan, Richard C K; Albertson, Donna G

    2005-06-16

    Genomes of solid tumors are characterized by gains and losses of regions, which may contribute to tumorigenesis by altering gene expression. Often the aberrations are extensive, encompassing whole chromosome arms, which makes identification of candidate genes in these regions difficult. Here, we focused on narrow regions of gene amplification to facilitate identification of genetic pathways important in oral squamous cell carcinoma (SCC) development. We used array comparative genomic hybridization (array CGH) to define minimum common amplified regions and then used expression analysis to identify candidate driver genes in amplicons that spanned <3 Mb. We found genes involved in integrin signaling (TLN1), survival (YAP1, BIRC2), and adhesion and migration (TLN1, LAMA3, MMP7), as well as members of the hedgehog (GLI2) and notch (JAG1, RBPSUH, FJX1) pathways to be amplified and overexpressed. Deregulation of these and other members of the hedgehog and notch pathways (HHIP, SMO, DLL1, NOTCH4) implicates deregulation of developmental and differentiation pathways, cell fate misspecification, in oral SCC development. PMID:15824737

  20. 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

  1. Flow cytometry community fingerprinting and amplicon sequencing for the assessment of landfill leachate cellulolytic bioaugmentation.

    PubMed

    Kinet, R; Dzaomuho, P; Baert, J; Taminiau, B; Daube, G; Nezer, C; Brostaux, Y; Nguyen, F; Dumont, G; Thonart, P; Delvigne, F

    2016-08-01

    Flow cytometry (FCM) is a high throughput single cell technology that is actually becoming widely used for studying phenotypic and genotypic diversity among microbial communities. This technology is considered in this work for the assessment of a bioaugmentation treatment in order to enhance cellulolytic potential of landfill leachate. The experimental results reveal the relevant increase of leachate cellulolytic potential due to bioaugmentation. Cytometric monitoring of microbial dynamics along these assays is then realized. The flow FP package is used to establish microbial samples fingerprint from initial 2D cytometry histograms. This procedure allows highlighting microbial communities' variation along the assays. Cytometric and 16S rRNA gene sequencing fingerprinting methods are then compared. The two approaches give same evidence about microbial dynamics throughout digestion assay. There are however a lack of significant correlation between cytometric and amplicon sequencing fingerprint at genus or species level. Same phenotypical profiles of microbiota during assays matched to several 16S rRNA gene sequencing ones. Flow cytometry fingerprinting can thus be considered as a promising routine on-site method suitable for the detection of stability/variation/disturbance of complex microbial communities involved in bioprocesses. PMID:27160955

  2. Comparative analyses of amplicon migration behavior in differing denaturing gradient gel electrophoresis (DGGE) systems

    NASA Astrophysics Data System (ADS)

    Thornhill, D. J.; Kemp, D. W.; Sampayo, E. M.; Schmidt, G. W.

    2010-03-01

    Denaturing gradient gel electrophoresis (DGGE) is commonly utilized to identify and quantify microbial diversity, but the conditions required for different electrophoretic systems to yield equivalent results and optimal resolution have not been assessed. Herein, the influence of different DGGE system configuration parameters on microbial diversity estimates was tested using Symbiodinium, a group of marine eukaryotic microbes that are important constituents of coral reef ecosystems. To accomplish this, bacterial clone libraries were constructed and sequenced from cultured isolates of Symbiodinium for the ribosomal DNA internal transcribed spacer 2 (ITS2) region. From these, 15 clones were subjected to PCR with a GC clamped primer set for DGGE analyses. Migration behaviors of the resulting amplicons were analyzed using a range of conditions, including variation in the composition of the denaturing gradient, electrophoresis time, and applied voltage. All tests were conducted in parallel on two commercial DGGE systems, a C.B.S. Scientific DGGE-2001, and the Bio-Rad DCode system. In this context, identical nucleotide fragments exhibited differing migration behaviors depending on the model of apparatus utilized, with fragments denaturing at a lower gradient concentration and applied voltage on the Bio-Rad DCode system than on the C.B.S. Scientific DGGE-2001 system. Although equivalent PCR-DGGE profiles could be achieved with both brands of DGGE system, the composition of the denaturing gradient and application of electrophoresis time × voltage must be appropriately optimized to achieve congruent results across platforms.

  3. Digital fragment analysis of short tandem repeats by high-throughput amplicon sequencing.

    PubMed

    Darby, Brian J; Erickson, Shay F; Hervey, Samuel D; Ellis-Felege, Susan N

    2016-07-01

    High-throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high-throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high-throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost-effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence-aware microsatellite data. PMID:27386092

  4. Swarm v2: highly-scalable and high-resolution amplicon clustering

    PubMed Central

    Quince, Christopher; de Vargas, Colomban; Dunthorn, Micah

    2015-01-01

    Previously we presented Swarm v1, a novel and open source amplicon clustering program that produced fine-scale molecular operational taxonomic units (OTUs), free of arbitrary global clustering thresholds and input-order dependency. Swarm v1 worked with an initial phase that used iterative single-linkage with a local clustering threshold (d), followed by a phase that used the internal abundance structures of clusters to break chained OTUs. Here we present Swarm v2, which has two important novel features: (1) a new algorithm for d = 1 that allows the computation time of the program to scale linearly with increasing amounts of data; and (2) the new fastidious option that reduces under-grouping by grafting low abundant OTUs (e.g., singletons and doubletons) onto larger ones. Swarm v2 also directly integrates the clustering and breaking phases, dereplicates sequencing reads with d = 0, outputs OTU representatives in fasta format, and plots individual OTUs as two-dimensional networks. PMID:26713226

  5. A Multiplexed Amplicon Approach for Detecting Gene Fusions by Next-Generation Sequencing.

    PubMed

    Beadling, Carol; Wald, Abigail I; Warrick, Andrea; Neff, Tanaya L; Zhong, Shan; Nikiforov, Yuri E; Corless, Christopher L; Nikiforova, Marina N

    2016-03-01

    Chromosomal rearrangements that result in oncogenic gene fusions are clinically important drivers of many cancer types. Rapid and sensitive methods are therefore needed to detect a broad range of gene fusions in clinical specimens that are often of limited quantity and quality. We describe a next-generation sequencing approach that uses a multiplex PCR-based amplicon panel to interrogate fusion transcripts that involve 19 driver genes and 94 partners implicated in solid tumors. The panel also includes control assays that evaluate the 3'/5' expression ratios of 12 oncogenic kinases, which might be used to infer gene fusion events when the partner is unknown or not included on the panel. There was good concordance between the solid tumor fusion gene panel and other methods, including fluorescence in situ hybridization, real-time PCR, Sanger sequencing, and other next-generation sequencing panels, because 40 specimens known to harbor gene fusions were correctly identified. No specific fusion reads were observed in 59 fusion-negative specimens. The 3'/5' expression ratio was informative for fusions that involved ALK, RET, and NTRK1 but not for BRAF or ROS1 fusions. However, among 37 ALK or RET fusion-negative specimens, four exhibited elevated 3'/5' expression ratios, indicating that fusions predicted solely by 3'/5' read ratios require confirmatory testing. PMID:26747586

  6. Drug and drug-related supply promotion by pharmaceutical company representatives at VA facilities. Final rule.

    PubMed

    2012-03-01

    This final rule amends the Department of Veterans Affairs (VA) regulations regarding access to VA facilities by pharmaceutical company representatives. The purposes of the rule are to reduce or eliminate any potential for disruption in the patient care environment, manage activities and promotions at VA facilities, and provide pharmaceutical company representatives with a consistent standard of permissible business practice at VA facilities. The amendments will facilitate mutually beneficial relationships between VA and pharmaceutical company representatives. PMID:22420057

  7. VA opsin-based photoreceptors in the hypothalamus of birds.

    PubMed

    Halford, Stephanie; Pires, Susana S; Turton, Michael; Zheng, Lei; González-Menéndez, Irene; Davies, Wayne L; Peirson, Stuart N; García-Fernández, José M; Hankins, Mark W; Foster, Russell G

    2009-08-25

    Studies in the 1930s demonstrated that birds possess photoreceptors that are located within the hypothalamus and regulate photoperiodic responses to day length. Most recently, photoperiod has been shown to alter the activity of the pars tuberalis to release thyrotrophin, which ultimately drives a reproductive response. Despite these significant findings, the cellular and molecular identity of the hypothalamic photoreceptors has remained a mystery. Action spectra implicated an opsin-based photopigment system, but further identification based on rod- or cone-opsin probes failed, suggesting the utilization of a novel opsin. The vertebrate ancient (VA) opsin photopigments were isolated in 1997 but were thought to have a restricted taxonomic distribution, confined to the agnatha and teleost fish. Here, we report the isolation of VA opsin from chicken and show that the two isoforms spliced from this gene (cVAL and cVA) are capable of forming functional photopigments. Further, we show that VA opsin is expressed within a population of hypothalamic neurons with extensive projections to the median eminence. These results provide the most complete cellular and molecular description of a deep brain photoreceptor in any vertebrate and strongly implicate VA opsin in mediating the avian photoperiodic response. PMID:19664923

  8. Ambient temperature detection of PCR amplicons with a novel sequence-specific nucleic acid lateral flow biosensor.

    PubMed

    Ang, Geik Yong; Yu, Choo Yee; Yean, Chan Yean

    2012-01-01

    In the field of diagnostics, molecular amplification targeting unique genetic signature sequences has been widely used for rapid identification of infectious agents, which significantly aids physicians in determining the choice of treatment as well as providing important epidemiological data for surveillance and disease control assessment. We report the development of a rapid nucleic acid lateral flow biosensor (NALFB) in a dry-reagent strip format for the sequence-specific detection of single-stranded polymerase chain reaction (PCR) amplicons at ambient temperature (22-25°C). The NALFB was developed in combination with a linear-after-the-exponential PCR assay and the applicability of this biosensor was demonstrated through detection of the cholera toxin gene from diarrheal-causing toxigenic Vibrio cholerae. Amplification using the advanced asymmetric PCR boosts the production of fluorescein-labeled single-stranded amplicons, allowing capture probes immobilized on the NALFB to hybridize specifically with complementary targets in situ on the strip. Subsequent visual formation of red lines is achieved through the binding of conjugated gold nanoparticles to the fluorescein label of the captured amplicons. The visual detection limit observed with synthetic target DNA was 0.3 ng and 1 pg with pure genomic DNA. Evaluation of the NALFB with 164 strains of V. cholerae and non-V. cholerae bacteria recorded 100% for both sensitivity and specificity. The whole procedure of the low-cost NALFB, which is performed at ambient temperature, eliminates the need for preheated buffers or additional equipment, greatly simplifying the protocol for sequence-specific PCR amplicon analysis. PMID:22705404

  9. Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing

    PubMed Central

    Polson, Shawn W.; Wommack, K. Eric; Williamson, Shannon J.; McDonald, Ian R.; Cary, S. Craig

    2012-01-01

    Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa–a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3–V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure. PMID:22970184

  10. Strengths and Limitations of 16S rRNA Gene Amplicon Sequencing in Revealing Temporal Microbial Community Dynamics

    PubMed Central

    Poretsky, Rachel; Rodriguez-R, Luis M.; Luo, Chengwei; Tsementzi, Despina; Konstantinidis, Konstantinos T.

    2014-01-01

    This study explored the short-term planktonic microbial community structure and resilience in Lake Lanier (GA, USA) while simultaneously evaluating the technical aspects of identifying taxa via 16S rRNA gene amplicon and metagenomic sequence data. 16S rRNA gene amplicons generated from four temporally discrete samples were sequenced with 454 GS-FLX-Ti yielding ∼40,000 rRNA gene sequences from each sample and representing ∼300 observed OTUs. Replicates obtained from the same biological sample clustered together but several biases were observed, linked to either the PCR or sequencing-preparation steps. In comparisons with companion whole-community shotgun metagenome datasets, the estimated number of OTUs at each timepoint was concordant, but 1.5 times and ∼10 times as many phyla and genera, respectively, were identified in the metagenomes. Our analyses showed that the 16S rRNA gene captures broad shifts in community diversity over time, but with limited resolution and lower sensitivity compared to metagenomic data. We also identified OTUs that showed marked shifts in abundance over four close timepoints separated by perturbations and tracked these taxa in the metagenome vs. 16S rRNA amplicon data. A strong summer storm had less of an effect on community composition than did seasonal mixing, which revealed a distinct succession of organisms. This study provides insights into freshwater microbial communities and advances the approaches for assessing community diversity and dynamics in situ. PMID:24714158

  11. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing.

    PubMed

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  12. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing

    PubMed Central

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  13. The Impact of Private Insurance Coverage on Veterans' Use of VA Care: Insurance and Selection Effects

    PubMed Central

    Shen, Yujing; Hendricks, Ann; Wang, Fenghua; Gardner, John; Kazis, Lewis E

    2008-01-01

    Objective To examine private insurance coverage and its impact on use of Veterans Health Administration (VA) care among VA enrollees without Medicare coverage. Data Sources The 1999 National Health Survey of Veteran Enrollees merged with VA administrative data, with other information drawn from American Hospital Association data and the Area Resource File. Study Design We modeled VA enrollees' decision of having private insurance coverage and its impact on use of VA care controlling for sociodemographic information, patients' health status, VA priority status and access to VA and non-VA alternatives. We estimated the true impact of insurance on the use of VA care by teasing out potential selection bias. Bias came from two sources: a security selection effect (sicker enrollees purchase private insurance for extra security and use more VA and non-VA care) and a preference selection effect (VA enrollees who prefer non-VA care may purchase private insurance and use less VA care). Principal Findings VA enrollees with private insurance coverage were less likely to use VA care. Security selection dominated preference selection and naïve models that did not control for selection effects consistently underestimated the insurance effect. Conclusions Our results indicate that prior research, which has not controlled for insurance selection effects, may have underestimated the potential impact of any private insurance policy change, which may in turn affect VA enrollees' private insurance coverage and consequently their use of VA care. From the decline in private insurance coverage from 1999 to 2002, we projected an increase of 29,400 patients and 158 million dollars for VA health care services. PMID:18211529

  14. The cost of hospital readmissions: evidence from the VA.

    PubMed

    Carey, Kathleen; Stefos, Theodore

    2016-09-01

    This paper is an examination of hospital 30-day readmission costs using data from 119 acute care hospitals operated by the U.S. Veterans Administration (VA) in fiscal year 2011. We applied a two-part model that linked readmission probability to readmission cost to obtain patient level estimates of expected readmission cost for VA patients overall, and for patients discharged for three prevalent conditions with relatively high readmission rates. Our focus was on the variable component of direct patient cost. Overall, managers could expect to save $2140 for the average 30-day readmission avoided. For heart attack, heart failure, and pneumonia patients, expected readmission cost estimates were $3432, $2488 and $2278. Patient risk of illness was the dominant driver of readmission cost in all cases. The VA experience has implications for private sector hospitals that treat a high proportion of chronically ill and/or low income patients, or that are contemplating adopting bundled payment mechanisms. PMID:25576391

  15. Amplicon-based semiconductor sequencing of human exomes: performance evaluation and optimization strategies.

    PubMed

    Damiati, E; Borsani, G; Giacopuzzi, Edoardo

    2016-05-01

    The Ion Proton platform allows to perform whole exome sequencing (WES) at low cost, providing rapid turnaround time and great flexibility. Products for WES on Ion Proton system include the AmpliSeq Exome kit and the recently introduced HiQ sequencing chemistry. Here, we used gold standard variants from GIAB consortium to assess the performances in variants identification, characterize the erroneous calls and develop a filtering strategy to reduce false positives. The AmpliSeq Exome kit captures a large fraction of bases (>94 %) in human CDS, ClinVar genes and ACMG genes, but with 2,041 (7 %), 449 (13 %) and 11 (19 %) genes not fully represented, respectively. Overall, 515 protein coding genes contain hard-to-sequence regions, including 90 genes from ClinVar. Performance in variants detection was maximum at mean coverage >120×, while at 90× and 70× we measured a loss of variants of 3.2 and 4.5 %, respectively. WES using HiQ chemistry showed ~71/97.5 % sensitivity, ~37/2 % FDR and ~0.66/0.98 F1 score for indels and SNPs, respectively. The proposed low, medium or high-stringency filters reduced the amount of false positives by 10.2, 21.2 and 40.4 % for indels and 21.2, 41.9 and 68.2 % for SNP, respectively. Amplicon-based WES on Ion Proton platform using HiQ chemistry emerged as a competitive approach, with improved accuracy in variants identification. False-positive variants remain an issue for the Ion Torrent technology, but our filtering strategy can be applied to reduce erroneous variants. PMID:27003585

  16. Prerequisites for amplicon pyrosequencing of microbial methanol utilizers in the environment

    PubMed Central

    Kolb, Steffen; Stacheter, Astrid

    2013-01-01

    The commercial availability of next generation sequencing (NGS) technologies facilitated the assessment of functional groups of microorganisms in the environment with high coverage, resolution, and reproducibility. Soil methylotrophs were among the first microorganisms in the environment that were assessed with molecular tools, and nowadays, as well with NGS technologies. Studies in the past years re-attracted notice to the pivotal role of methylotrophs in global conversions of methanol, which mainly originates from plants, and is involved in oxidative reactions and ozone formation in the atmosphere. Aerobic methanol utilizers belong to Bacteria, yeasts, Ascomycota, and molds. Numerous bacterial methylotrophs are facultatively aerobic, and also contribute to anaerobic methanol oxidation in the environment, whereas strict anaerobic methanol utilizers belong to methanogens and acetogens. The diversity of enzymes catalyzing the initial oxidation of methanol is considerable, and comprises at least five different enzyme types in aerobes, and one in strict anaerobes. Only the gene of the large subunit of pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH; mxaF) has been analyzed by environmental pyrosequencing. To enable a comprehensive assessment of methanol utilizers in the environment, new primers targeting genes of the PQQ MDH in Methylibium (mdh2), of the nicotinamide adenine dinucleotide-dependent MDH (mdh), of the methanol oxidoreductase of Actinobacteria (mdo), of the fungal flavin adenine nucleotide-dependent alcohol oxidase (mod1, mod2, and homologs), and of the gene of the large subunit of the methanol:corrinoid methyltransferases (mtaC) in methanogens and acetogens need to be developed. Combined stable isotope probing of nucleic acids or proteins with amplicon-based NGS are straightforward approaches to reveal insights into functions of certain methylotrophic taxa in the global methanol cycle. PMID:24046766

  17. Voltammetric detection of sequence-selective DNA hybridization related to Toxoplasma gondii in PCR amplicons.

    PubMed

    Gokce, Gultekin; Erdem, Arzum; Ceylan, Cagdas; Akgöz, Muslum

    2016-03-01

    This work describes the single-use electrochemical DNA biosensor technology developed for voltammetric detection of sequence selective DNA hybridization related to important human and veterinary pathogen; Toxoplasma gondii. In the principle of electrochemical label-free detection assay, the duplex of DNA hybrid formation was detected by measuring guanine oxidation signal occured in the presence of DNA hybridization. The biosensor design consisted of the immobilization of an inosine-modified (guanine-free) probe onto the surface of pencil graphite electrode (PGE), and the detection of the duplex formation in connection with the differential pulse voltammetry(DPV) by measuring the guanine signal. Toxoplasma gondii capture probe was firstly immobilized onto the surface of the activated PGE by wet adsorption. The extent of hybridization at PGE surface between the probe and the target was then determined by measuring the guanine signal observed at +1.0V. The electrochemical monitoring of optimum DNA hybridization has been performed in the target concentration of 40µg/mL in 50min of hybridization time. The specificity of the electrochemical biosensor was then tested using non-complementary, or mismatch short DNA sequences. Under the optimum conditions, the guanine oxidation signal indicating full hybridization was measured in various target concentration from 0.5 to 25µg/mL and a detection limit was found to be 1.78µg/mL. This single-use biosensor platform was successfully applied for the voltammetric detection of DNA hybridization related to Toxoplasma gondii in PCR amplicons. PMID:26717837

  18. Analysis of a Drosophila amplicon in follicle cells highlights the diversity of metazoan replication origins.

    PubMed

    Kim, Jane C; Orr-Weaver, Terry L

    2011-10-01

    To investigate the properties of metazoan replication origins, recent studies in cell culture have adopted the strategy of identifying origins using genome-wide approaches and assessing correlations with such features as transcription and histone modifications. Drosophila amplicon in follicle cells (DAFCs), genomic regions that undergo repeated rounds of DNA replication to increase DNA copy number, serve as powerful in vivo model replicons. Because there are six DAFCs, compared with thousands of origins activated in the typical S phase, close molecular characterization of all DAFCs is possible. To determine the extent to which the six DAFCs are different or similar, we investigated the developmental and replication properties of the newly identified DAFC-34B. DAFC-34B contains two genes expressed in follicle cells, although the timing and spatial patterns of expression suggest that amplification is not a strategy to promote high expression at this locus. Like the previously characterized DAFC-62D, DAFC-34B displays origin activation at two separate stages of development. However, unlike DAFC-62D, amplification at the later stage is not transcription-dependent. We mapped the DAFC-34B amplification origin to 1 kb by nascent strand analysis and delineated cis requirements for origin activation, finding that a 6-kb region, but not the 1-kb origin alone, is sufficient for amplification. We analyzed the developmental localization of the origin recognition complex (ORC) and the minichromosome maintenance (MCM)2-7 complex, the replicative helicase. Intriguingly, the final round of origin activation at DAFC-34B occurs in the absence of detectable ORC, although MCMs are present, suggesting a new amplification initiation mechanism. PMID:21933960

  19. Herpes Simplex Virus Type 1/Adeno-Associated Virus rep+ Hybrid Amplicon Vector Improves the Stability of Transgene Expression in Human Cells by Site-Specific Integration

    PubMed Central

    Wang, Y.; Camp, S. M.; Niwano, M.; Shen, X.; Bakowska, J. C.; Breakefield, X. O.; Allen, P. D.

    2002-01-01

    Herpes simplex virus type 1 (HSV-1) amplicon vectors are promising gene delivery tools, but their utility in gene therapy has been impeded to some extent by their inability to achieve stable transgene expression. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). A rep− HSV/AAV hybrid amplicon vector was made by inserting a transgene cassette flanked with AAV ITRs into an HSV-1 amplicon backbone, and a rep+ HSV/AAV hybrid amplicon was made by inserting rep68/78 outside the rep− vector 3′ AAV ITR sequence. Both vectors also had a pair of loxP sites flanking the ITRs. The resulting hybrid amplicon vectors were successfully packaged and compared to a standard amplicon vector for stable transduction frequency (STF) in human 293 and Gli36 cell lines and primary myoblasts. The rep+, but not the rep−, hybrid vector improved STF in all three types of cells; 84% of Gli36 and 40% of 293 stable clones transduced by the rep+ hybrid vector integrated the transgene into the AAVS1 site. Due to the difficulty in expanding primary myoblasts, we did not assess site-specific integration in these cells. A strategy to attempt further improvement of STF by “deconcatenating” the hybrid amplicon DNA via Cre-loxP recombination was tested, but it did not increase STF. These data demonstrate that introducing the integrating elements of AAV into HSV-1 amplicon vectors can significantly improve their ability to achieve stable gene transduction by conferring the AAV-like capability of site-specific genomic integration in dividing cells. PMID:12072515

  20. Gene-specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases.

    PubMed

    Itoh, Nobuya; Kazama, Miki; Takeuchi, Nami; Isotani, Kentaro; Kurokawa, Junji

    2016-06-01

    Screening gene-specific amplicons from metagenomes (S-GAM) is a highly promising technique for the isolation of genes encoding enzymes for biochemical and industrial applications. From metagenomes, we isolated phenylacetaldehyde reductase (par) genes, which code for an enzyme that catalyzes the production of various Prelog's chiral alcohols. Nearly full-length par genes were amplified by PCR from metagenomic DNA, the products of which were fused with engineered par sequences at both terminal regions of the expression vector to ensure proper expression and then used to construct Escherichia coli plasmid libraries. Sequence- and activity-based screening of these libraries identified different homologous par genes, Hpar-001 to -036, which shared more than 97% amino acid sequence identity with PAR. Comparative characterization of these active homologs revealed a wide variety of enzymatic properties including activity, substrate specificity, and thermal stability. Moreover, amino acid substitutions in these genes coincided with those of Sar268 and Har1 genes, which were independently engineered by error-prone PCR to exhibit increased activity in the presence of concentrated 2-propanol. The comparative data from both approaches suggest that sequence information from homologs isolated from metagenomes is quite useful for enzyme engineering. Furthermore, by examining the GAM-based sequence dataset derived from soil metagenomes, we easily found amino acid substitutions that increase the thermal stability of PAR/PAR homologs. Thus, GAM-based approaches can provide not only useful homologous enzymes but also an alternative to directed evolution methodologies. PMID:27419059

  1. Analysis of β-Subunit-dependent GABAA Receptor Modulation and Behavioral Effects of Valerenic Acid Derivatives.

    PubMed

    Khom, S; Hintersteiner, J; Luger, D; Haider, M; Pototschnig, G; Mihovilovic, M D; Schwarzer, C; Hering, S

    2016-06-01

    Valerenic acid (VA)-a β2/3-selective GABA type A (GABAA) receptor modulator-displays anxiolytic and anticonvulsive effects in mice devoid of sedation, making VA an interesting drug candidate. Here we analyzed β-subunit-dependent enhancement of GABA-induced chloride currents (IGABA) by a library of VA derivatives and studied their effects on pentylenetetrazole (PTZ)-induced seizure threshold and locomotion. Compound-induced IGABA enhancement was determined in oocytes expressing α1β1γ2S, α1β2γ2S, or α1β3γ2S receptors. Effects on seizure threshold and locomotion were studied using C57BL/6N mice and compared with saline-treated controls. β2/3-selective VA derivatives such as VA-amide (VA-A) modulating α1β3γ2S (VA-A: Emax = 972 ± 69%, n = 6, P < 0.05) and α1β2γ2S receptors (Emax = 1119 ± 72%, n = 6, P < 0.05) more efficaciously than VA (α1β3γ2S: VA: Emax = 632 ± 88%, n = 9 versus α1β2γ2S: VA: Emax = 721 ± 68%, n = 6) displayed significantly more pronounced seizure threshold elevation than VA (saline control: 40.4 ± 1.4 mg/kg PTZ versus VA 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ versus VA-A 3 mg/kg: 57.9 ± 1.9 mg/kg PTZ, P < 0.05). Similarly, VA's methylamide (VA-MA) enhancing IGABA through β3-containing receptors more efficaciously than VA (Emax = 1043 ± 57%, P < 0.01, n = 6) displayed stronger anticonvulsive effects. Increased potency of IGABA enhancement and anticonvulsive effects at lower doses compared with VA were observed for VA-tetrazole (α1β3γ2S: VA-TET: EC50 = 6.0 ± 1.0 μM, P < 0.05; VA-TET: 0.3 mg/kg: 47.3 ± 0.5 mg/kg PTZ versus VA: 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ, P < 0.05). At higher doses (≥10 mg/kg), VA-A, VA-MA, and VA-TET reduced locomotion. In contrast, unselective VA derivatives induced anticonvulsive effects only at high doses (30 mg/kg) or did not display any behavioral effects. Our data indicate that the β2/3-selective compounds VA-A, VA-MA, and VA-TET induce anticonvulsive effects at low doses (≤10 mg/kg), whereas

  2. VA Library Service--Today's look at Tomorrow's Library.

    ERIC Educational Resources Information Center

    Veterans Administration, Washington, DC.

    The Conference Poceedings are divided into three broad topics: systems planning, audiovisuals in biomedical communication, and automation and networking. Speakers from within the Veterans Administration (VA), from the National Medical Audiovisual Center, and the Lister Hill National Center for Biomedical Communications, National Library of…

  3. 77 FR 67063 - VA Directive 0005 on Scientific Integrity

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-08

    ... April 9, 2012 (77 FR 21158). FOR FURTHER INFORMATION CONTACT: Billy E. Jones, M.D., Senior Advisor to... was announced in the Federal Register on April 9, 2012 (77 FR 21158). All of the public comments have... AFFAIRS VA Directive 0005 on Scientific Integrity AGENCY: Office of Policy and Planning, Department...

  4. 76 FR 77270 - Board Meeting; January 9, 2012, Arlington, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-12

    ... REVIEW BOARD Board Meeting; January 9, 2012, Arlington, VA The U.S. Nuclear Waste Technical Review Board... Waste Technical Review Board will hold a public meeting in Arlington, Virginia, on Monday, January 9... on technical issues related to nuclear waste management and to review the technical validity of...

  5. 78 FR 62441 - VA Dental Insurance Program-Federalism

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-22

    ... of veterans. Specifically, this rule will add language to clarify the limited preemptive effect of... CFR 17.169 to add language to clarify the limited preemptive effect of certain criteria in the VA... business of insurance. Although section 510 does not include express preemption language, Congress...

  6. 77 FR 70967 - Authorization for Non-VA Medical Services

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-28

    ... regulatory action under Executive Order 12866. Unfunded Mandates Reform Act The Unfunded Mandates Reform Act... specify veterans' eligibility for non-VA care. Administrative Procedure Act Concurrent with this proposed... documents will speed notice and comment rulemaking under section 553 of the Administrative Procedure...

  7. 75 FR 41247 - Virginia Disaster Number VA-00028

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-15

    ... ADMINISTRATION Virginia Disaster Number VA-00028 AGENCY: U.S. Small Business Administration. ACTION: Amendment 4... Only for the Commonwealth of Virginia (FEMA-1874-DR), dated 02/16/2010. Incident: Severe Winter Storm... disaster declaration for Private Non-Profit organizations in the Commonwealth of Virginia, dated...

  8. 76 FR 58557 - Virginia Disaster Number VA-00038

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-21

    ... ADMINISTRATION Virginia Disaster Number VA-00038 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Only for the Commonwealth of Virginia (FEMA-4024-DR), dated 09/03/2011. Incident: Hurricane Irene... Private Non-Profit organizations in the Commonwealth of Virginia, dated 09/03/2011, is hereby amended...

  9. 77 FR 530 - Virginia Disaster Number VA-00037

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-05

    ... ADMINISTRATION Virginia Disaster Number VA-00037 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Virginia (FEMA-4042-DR), dated 11/04/2011. Incident: Earthquake. Incident Period: 08/23/2011 through 10/25... major disaster declaration for the Commonwealth of Virginia, dated 11/04/2011 is hereby amended...

  10. 77 FR 7229 - Virginia Disaster Number VA-00037

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-10

    ... ADMINISTRATION Virginia Disaster Number VA-00037 AGENCY: U.S. Small Business Administration. ACTION: Amendment 3... Virginia (FEMA-4042-DR), dated 11/04/2011. Incident: Earthquake. Incident Period: 08/23/2011 through 10/25... disaster declaration for the Commonwealth of Virginia, dated 11/04/2011 is hereby amended to include...

  11. 76 FR 62132 - Virginia Disaster Number VA-00038

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-06

    ... ADMINISTRATION Virginia Disaster Number VA-00038 AGENCY: U.S. Small Business Administration. ACTION: Amendment 2... only for the State of Virginia (FEMA-4024-DR), dated 09/03/2011. Incident: Hurricane Irene. Incident... Non-Profit organizations in the State of Virginia, dated 09/03/2011, is hereby amended to include...

  12. 75 FR 41559 - Virginia Disaster Number VA-00029

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-16

    ... ADMINISTRATION Virginia Disaster Number VA-00029 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Only for the Commonwealth of Virginia (FEMA-1905-DR), dated 04/27/2010. Incident: Severe winter storms... disaster declaration for Private Non-Profit organizations in the Commonwealth of Virginia, dated...

  13. 75 FR 26813 - VIRGINIA Disaster Number VA-00028

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-12

    ... ADMINISTRATION VIRGINIA Disaster Number VA-00028 AGENCY: U.S. Small Business Administration. ACTION: Amendment 2... Only for the Commonwealth of VIRGINIA (FEMA-1874-DR), dated 02/16/2010. Incident: Severe Winter Storm... disaster declaration for Private Non-Profit organizations in the Commonwealth of VIRGINIA, dated...

  14. 77 FR 51100 - Virginia Disaster No. VA-00048

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-23

    ... ADMINISTRATION Virginia Disaster No. VA-00048 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Only for the State of VIRGINIA (FEMA-4072-DR), dated 07/27/2012. Incident: Severe Storms and Straight... Private Non-Profit organizations in the State of VIRGINIA, dated 07/27/2012, is hereby amended to...

  15. 76 FR 74837 - Virginia Disaster Number VA-00040

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-01

    ... ADMINISTRATION Virginia Disaster Number VA-00040 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Only for the State of Virginia (FEMA-4042-DR), dated 11/10/2011. Incident: Earthquake. Incident Period... non-profit organizations in the State of Virginia, dated 11/10/2011, is hereby amended to include...

  16. 75 FR 2895 - Virginia Disaster Number VA-00027

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-19

    ... ADMINISTRATION Virginia Disaster Number VA-00027 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Only for the Commonwealth of Virginia (FEMA-1862-DR), dated 12/09/2009. Incident: Severe Storms and... the Commonwealth of ] Virginia, dated 12/09/2009, is hereby amended to establish the incident...

  17. 77 FR 1547 - Virginia Disaster Number VA-00040

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-10

    ... ADMINISTRATION Virginia Disaster Number VA-00040 AGENCY: U.S. Small Business Administration. ACTION: Amendment 3... Only for the Commonwealth of Virginia (FEMA--4042-DR), dated 11/10/2011. Incident: Earthquake. Incident... Non-Profit organizations in the Commonwealth of Virginia, dated 11/10/2011, is hereby amended...

  18. 78 FR 2708 - Virginia Disaster Number VA-00052

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-14

    ... ADMINISTRATION Virginia Disaster Number VA-00052 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Only for the State of Virginia (FEMA--4092--DR), dated 11/26/2012 . Incident: Hurricane Sandy Incident... Non-Profit organizations in the State of Virginia, dated 11/26/2012, is hereby amended to include...

  19. 75 FR 35511 - Virginia Disaster Number VA-00028

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-22

    ... ADMINISTRATION Virginia Disaster Number VA-00028 AGENCY: Small Business Administration. ACTION: Amendment 3... Only for the Commonwealth of Virginia (FEMA-1874-DR), dated 02/16/2010. Incident: Severe Winter Storm... disaster declaration for Private Non-Profit organizations in the Commonwealth of Virginia, dated...

  20. 77 FR 1547 - Virginia Disaster Number VA-00037

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-10

    ... ADMINISTRATION Virginia Disaster Number VA-00037 AGENCY: U.S. Small Business Administration. ACTION: Amendment 2... Virginia (FEMA-4042-DR), dated 11/04/ 2011. Incident: Earthquake. Incident Period: 08/23/2011 Through 10/25... disaster declaration for the State of Virginia, dated 11/04/2011 is hereby amended to include the...

  1. 75 FR 21370 - Virginia Disaster Number VA-00028

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-23

    ... ADMINISTRATION Virginia Disaster Number VA-00028 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Only for the Commonwealth of Virginia (FEMA-1874-DR), dated 02/16/2010. Incident: Severe Winter Storm... disaster declaration for Private Non-Profit organizations in the Commonwealth of Virginia, dated...

  2. 76 FR 78957 - Virginia Disaster Number VA-00040

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-20

    ... ADMINISTRATION Virginia Disaster Number VA-00040 AGENCY: U.S. Small Business Administration. ACTION: Amendment 2... Only for the Commonwealth of Virginia (FEMA--4042--DR), dated 11/10/2011. Incident: Earthquake... Private Non-Profit organizations in the Commonwealth of Virginia, dated 11/10/2011, is hereby amended...

  3. Geropsychology Training in a VA Nursing Home Setting

    ERIC Educational Resources Information Center

    Karel, Michele J.; Moye, Jennifer

    2005-01-01

    There is a growing need for professional psychology training in nursing home settings, and nursing homes provide a rich environment for teaching geropsychology competencies. We describe the nursing home training component of our Department of Veterans Affairs (VA) Predoctoral Internship and Geropsychology Postdoctoral Fellowship programs. Our…

  4. 76 FR 30534 - Establishment of Class E Airspace; Kenbridge, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-26

    ... proposed rulemaking to amend Class E airspace 700 feet above the surface, at Kenbridge, VA (75 FR 14823...: Authority: 49 U.S.C. 106(g); 40103, 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389... Policies and Procedures (44 FR 11034; February 26, 1979); and (3) does not warrant preparation of...

  5. 75 FR 79295 - Establishment of Class E Airspace; Crewe, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-20

    ... Federal Register a notice of proposed rulemaking to establish Class E airspace at Crewe, VA (75 FR 57215...: Authority: 49 U.S.C. 106(g); 40103, 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389... ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034; February 26, 1979); and (3)...

  6. 32 CFR 105.10 - SARC and SAPR VA procedures.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... directly to the installation commander in accordance with 32 CFR part 103, to include providing regular... CIVILIAN SEXUAL ASSAULT PREVENTION AND RESPONSE PROGRAM PROCEDURES § 105.10 SARC and SAPR VA procedures. (a) SARC procedures. The SARC shall: (1) Serve as the single point of contact to coordinate sexual...

  7. 76 FR 43575 - Amendment of Class E Airspace; Staunton, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-21

    ... Valley Regional Airport, Staunton, VA (76 FR 14822) Docket No. FAA- 2010-1285. Interested parties were... Executive Order 12866; (2) is not a ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR... FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71.1 0 2. The incorporation by reference in 14 CFR...

  8. Military and VA General Dentistry Training: A National Resource.

    ERIC Educational Resources Information Center

    Atchison, Kathryn A.; Bachand, William; Buchanan, C. Richard; Lefever, Karen H.; Lin, Sylvia; Engelhardt, Rita

    2002-01-01

    Compared the program characteristics of the postgraduate general dentistry (PGD) training programs sponsored by the military and the Veterans Health Administration (VA). Gathered information on program infrastructure and emphasis, resident preparation prior to entering the program, and patients served and types of services provided. Programs…

  9. 77 FR 2348 - Agency Information Collection (VA Enrollment Certification): Activity Under OMB Review

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-17

    ... or email denise.mclamb@va.gov . Please refer to ``OMB Control No. 2900-0073.'' SUPPLEMENTARY... AFFAIRS Agency Information Collection (VA Enrollment Certification): Activity Under OMB Review AGENCY..., 2012. ADDRESSES: Submit written comments on the collection of information through...

  10. 31. FELGATES CREEK BRIDGE (HAER No. VA48I), VIEW FROM WEST ...

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

    31. FELGATES CREEK BRIDGE (HAER No. VA-48-I), VIEW FROM WEST END. NOTE ALUMINUM RAILING ON EAST SIDE FOR CANTILEVERED BIKE PATH. - Colonial Parkway, Yorktown to Jamestown Island, Yorktown, York County, VA

  11. Genotyping-in-Thousands by sequencing (GT-seq): A cost effective SNP genotyping method based on custom amplicon sequencing.

    PubMed

    Campbell, Nathan R; Harmon, Stephanie A; Narum, Shawn R

    2015-07-01

    Genotyping-in-Thousands by sequencing (GT-seq) is a method that uses next-generation sequencing of multiplexed PCR products to generate genotypes from relatively small panels (50-500) of targeted single-nucleotide polymorphisms (SNPs) for thousands of individuals in a single Illumina HiSeq lane. This method uses only unlabelled oligos and PCR master mix in two thermal cycling steps for amplification of targeted SNP loci. During this process, sequencing adapters and dual barcode sequence tags are incorporated into the amplicons enabling thousands of individuals to be pooled into a single sequencing library. Post sequencing, reads from individual samples are split into individual files using their unique combination of barcode sequences. Genotyping is performed with a simple perl script which counts amplicon-specific sequences for each allele, and allele ratios are used to determine the genotypes. We demonstrate this technique by genotyping 2068 individual steelhead trout (Oncorhynchus mykiss) samples with a set of 192 SNP markers in a single library sequenced in a single Illumina HiSeq lane. Genotype data were 99.9% concordant to previously collected TaqMan(™) genotypes at the same 192 loci, but call rates were slightly lower with GT-seq (96.4%) relative to Taqman (99.0%). Of the 192 SNPs, 187 were genotyped in ≥90% of the individual samples and only 3 SNPs were genotyped in <70% of samples. This study demonstrates amplicon sequencing with GT-seq greatly reduces the cost of genotyping hundreds of targeted SNPs relative to existing methods by utilizing a simple library preparation method and massive efficiency of scale. PMID:25476721

  12. Analysis of β-Subunit-dependent GABAA Receptor Modulation and Behavioral Effects of Valerenic Acid Derivatives

    PubMed Central

    Hintersteiner, J.; Luger, D.; Haider, M.; Pototschnig, G.; Mihovilovic, M. D.; Schwarzer, C.; Hering, S.

    2016-01-01

    Valerenic acid (VA)—a β2/3-selective GABA type A (GABAA) receptor modulator—displays anxiolytic and anticonvulsive effects in mice devoid of sedation, making VA an interesting drug candidate. Here we analyzed β-subunit-dependent enhancement of GABA-induced chloride currents (IGABA) by a library of VA derivatives and studied their effects on pentylenetetrazole (PTZ)-induced seizure threshold and locomotion. Compound-induced IGABA enhancement was determined in oocytes expressing α1β1γ2S, α1β2γ2S, or α1β3γ2S receptors. Effects on seizure threshold and locomotion were studied using C57BL/6N mice and compared with saline-treated controls. β2/3-selective VA derivatives such as VA-amide (VA-A) modulating α1β3γ2S (VA-A: Emax = 972 ± 69%, n = 6, P < 0.05) and α1β2γ2S receptors (Emax = 1119 ± 72%, n = 6, P < 0.05) more efficaciously than VA (α1β3γ2S: VA: Emax = 632 ± 88%, n = 9 versus α1β2γ2S: VA: Emax = 721 ± 68%, n = 6) displayed significantly more pronounced seizure threshold elevation than VA (saline control: 40.4 ± 1.4 mg/kg PTZ versus VA 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ versus VA-A 3 mg/kg: 57.9 ± 1.9 mg/kg PTZ, P < 0.05). Similarly, VA’s methylamide (VA-MA) enhancing IGABA through β3-containing receptors more efficaciously than VA (Emax = 1043 ± 57%, P < 0.01, n = 6) displayed stronger anticonvulsive effects. Increased potency of IGABA enhancement and anticonvulsive effects at lower doses compared with VA were observed for VA-tetrazole (α1β3γ2S: VA-TET: EC50 = 6.0 ± 1.0 μM, P < 0.05; VA-TET: 0.3 mg/kg: 47.3 ± 0.5 mg/kg PTZ versus VA: 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ, P < 0.05). At higher doses (≥10 mg/kg), VA-A, VA-MA, and VA-TET reduced locomotion. In contrast, unselective VA derivatives induced anticonvulsive effects only at high doses (30 mg/kg) or did not display any behavioral effects. Our data indicate that the β2/3-selective compounds VA-A, VA-MA, and VA-TET induce anticonvulsive effects at low doses (≤10 mg

  13. 12 CFR 3.206 - Stressed VaR-based measure.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 12 Banks and Banking 1 2014-01-01 2014-01-01 false Stressed VaR-based measure. 3.206 Section 3.206... Risk-Weighted Assets-Market Risk § 3.206 Stressed VaR-based measure. (a) General requirement. At least... calculate its VaR-based measure to calculate a stressed VaR-based measure. (b) Quantitative requirements...

  14. 48 CFR 852.219-71 - VA mentor-protégé program.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false VA mentor-protégÃ....219-71 VA mentor-protégé program. As prescribed in 819.7115(a), insert the following clause: VA Mentor-Protégé Program (DEC 2009) (a) Large businesses are encouraged to participate in the VA...

  15. 48 CFR 852.219-71 - VA mentor-protégé program.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false VA mentor-protégÃ....219-71 VA mentor-protégé program. As prescribed in 819.7115(a), insert the following clause: VA Mentor-Protégé Program (DEC 2009) (a) Large businesses are encouraged to participate in the VA...

  16. 48 CFR 852.219-71 - VA mentor-protégé program.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false VA mentor-protégÃ....219-71 VA mentor-protégé program. As prescribed in 819.7115(a), insert the following clause: VA Mentor-Protégé Program (DEC 2009) (a) Large businesses are encouraged to participate in the VA...

  17. 48 CFR 852.219-71 - VA mentor-protégé program.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false VA mentor-protégÃ....219-71 VA mentor-protégé program. As prescribed in 819.7115(a), insert the following clause: VA Mentor-Protégé Program (DEC 2009) (a) Large businesses are encouraged to participate in the VA...

  18. 48 CFR 852.219-71 - VA mentor-protégé program.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false VA mentor-protégÃ....219-71 VA mentor-protégé program. As prescribed in 819.7115(a), insert the following clause: VA Mentor-Protégé Program (DEC 2009) (a) Large businesses are encouraged to participate in the VA...

  19. The cytological manifestation of gene amplification in multidrug-resistant mouse leukemia P388 sublines is correlated with amplicon content

    SciTech Connect

    Il`inskaya, G.V.; Kopnin, B.P.; Demidova, N.S.

    1995-10-01

    Previously, we showed that development of multidrug resistance (MDR) in mouse P388 leukemia cells is often associated with the appearance of newly-formed chromosomelike structures that contain amplified copies of the mdr1 gene. In the present study, we compared amplicon content in P388 sublines showing different types of these structures. A strong correlation between the formation of specific acentric markers consisting of two identical arms and the absence of the sorcin gene coamplification was found. In all the sublines containing other types of chromosomelike structures, the sorcin gene is coamplified. 9 refs., 2 figs., 1 tab.

  20. The Impact of VA's Geriatric Research, Education and Clinical Centers on Academic Affiliates

    ERIC Educational Resources Information Center

    Bragg, Elizabeth J.; Meganathan, Karthikeyan; Shay, Kenneth; Gilman, Stuart C.; Zeiss, Robert A.; Hettler, Debbie L.

    2011-01-01

    The education mission of the Department of Veterans Affairs (VA) is to train health professionals to benefit VA and the United States. One approach for achieving that mission, along with VA's research and clinical missions, was the establishment of Geriatric Research, Education and Clinical Centers (GRECCs) in 1975. These were developed at VA…

  1. 78 FR 48543 - Veterans Health Administration Fund Availability Under the VA's Homeless Providers Grant and Per...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-08

    ... Veterans Affairs (VA) is announcing the availability of 1-year renewal funding for currently operational... Special Need partners and currently operational VA GPD Special Need Grant Recipients which do not involve... under VA's Homeless Providers GPD Program for FY 2011 operational GPD Special Need grant recipients...

  2. 12 CFR 324.206 - Stressed VaR-based measure.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 12 Banks and Banking 5 2014-01-01 2014-01-01 false Stressed VaR-based measure. 324.206 Section 324... Stressed VaR-based measure. (a) General requirement. At least weekly, an FDIC-supervised institution must use the same internal model(s) used to calculate its VaR-based measure to calculate a stressed...

  3. 78 FR 6849 - Agency Information Collection (Verification of VA Benefits) Activity Under OMB Review

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-31

    ... AFFAIRS Agency Information Collection (Verification of VA Benefits) Activity Under OMB Review AGENCY... abstracted below to the Office of Management and Budget (OMB) for review and comment. The PRA submission... VA Benefits, VA Form 26-8937. OMB Control Number: 2900-0406. ] Type of Review: Extension of...

  4. 75 FR 17832 - Proposed Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-07

    ... nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0021'' in any correspondence. During the... AFFAIRS Proposed Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity.... SUMMARY: The Veterans Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing...

  5. 77 FR 60746 - Proposed Information Collection (VA/DOD Joint Disability Evaluation Board Claim) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-04

    ... 20420 or email to ] nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0704'' in any... AFFAIRS Proposed Information Collection (VA/DOD Joint Disability Evaluation Board Claim) Activity: Comment...: The Veterans Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing...

  6. 77 FR 64383 - Proposed Information Collection (Verification of VA Benefits) Activity: Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-19

    ... AFFAIRS Proposed Information Collection (Verification of VA Benefits) Activity: Comment Request AGENCY... Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public... name prior to the closing of any VA-guaranteed loans on an automatic basis. DATES: Written comments...

  7. 75 FR 60171 - Proposed Information Collection (Credit Underwriting Standards and Procedures for Processing VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-29

    ...., Washington, DC 20420 or e-mail nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0521'' in any... AFFAIRS Proposed Information Collection (Credit Underwriting Standards and Procedures for Processing VA... Affairs (VA), is announcing an opportunity for public comment on the proposed collection of...

  8. 78 FR 79563 - Proposed Information Collection (VA MATIC Authorization); Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-30

    ...), Department of Veterans Affairs, 810 Vermont Avenue NW., Washington, DC 20420 or email nancy.kessinger@va.gov... AFFAIRS Proposed Information Collection (VA MATIC Authorization); Comment Request AGENCY: Veterans... Administration (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public comment on...

  9. 78 FR 53506 - Agency Information Collection (HUD/VA Addendum to Uniform Residential Loan Application) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-29

    ....rennie@va.gov . Please refer to ``OMB Control No. 2900-0144.'' SUPPLEMENTARY INFORMATION: Title: HUD/VA... AFFAIRS Agency Information Collection (HUD/VA Addendum to Uniform Residential Loan Application) Activity... information through www.Regulations.gov , or to Office of Information and Regulatory Affairs, Office...

  10. 76 FR 24570 - Proposed Information Collection (Application for VA Education Benefits) Activity; Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-02

    ...., Washington, DC 20420 or email to nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0154'' in... AFFAIRS Proposed Information Collection (Application for VA Education Benefits) Activity; Comment Request... Veterans Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing an...

  11. 78 FR 60379 - Proposed Information Collection (Credit Underwriting Standards and Procedures for Processing VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-01

    ...., Washington, DC 20420 or email nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0521'' in any... AFFAIRS Proposed Information Collection (Credit Underwriting Standards and Procedures for Processing VA... Affairs (VA), is announcing an opportunity for public comment on the proposed collection of...

  12. 76 FR 67561 - Proposed Information Collection (VA Enrollment Certification) Activity: Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-01

    ..., 810 Vermont Avenue NW., Washington, DC 20420 or email to nancy.kessinger@va.gov . Please refer to... AFFAIRS Proposed Information Collection (VA Enrollment Certification) Activity: Comment Request AGENCY... Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for...

  13. 75 FR 54965 - Proposed Information Collection (Statement of Purchaser or Owner Assuming Seller's Loans, VA Form...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-09

    ...., Washington, DC 20420 or e-mail nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0111'' in any... AFFAIRS Proposed Information Collection (Statement of Purchaser or Owner Assuming Seller's Loans, VA Form... Affairs (VA), is announcing an opportunity for public comment on the proposed collection of...

  14. 76 FR 27381 - Proposed Information Collection (Notice of Waiver of VA Compensation or Pension To Receive...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-11

    ...., Washington, DC 20420 or e-mail to nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0463'' in... AFFAIRS Proposed Information Collection (Notice of Waiver of VA Compensation or Pension To Receive... of Veterans Affairs (VA), is announcing an opportunity for public comment on the proposed...

  15. 78 FR 29436 - Proposed Information Collection (HUD/VA Addendum to Uniform Residential Loan Application...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-20

    ...), Department of Veterans Affairs, 810 Vermont Avenue NW., Washington, DC 20420 or email nancy.kessinger@va.gov... AFFAIRS Proposed Information Collection (HUD/VA Addendum to Uniform Residential Loan Application) Activity.... SUMMARY: The Veterans Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing...

  16. 77 FR 56711 - Proposed Information Collection (VA MATIC Enrollment/Change); Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-13

    ... Affairs, 810 Vermont Avenue NW., Washington, DC 20420 or email nancy.kessinger@va.gov . Please refer to... AFFAIRS Proposed Information Collection (VA MATIC Enrollment/Change); Comment Request AGENCY: Veterans... Administration (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public comment on...

  17. 76 FR 67557 - Proposed Information Collection (Survey of Veteran Enrollees' Health and Reliance Upon VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-01

    ..., 810 Vermont Avenue NW., Washington, DC 20420; or email: cynthia.harvey-pryor@va.gov . Please refer to... AFFAIRS Proposed Information Collection (Survey of Veteran Enrollees' Health and Reliance Upon VA...: Notice. SUMMARY: The Veterans Health Administration (VHA), Department of Veterans Affairs (VA),...

  18. 75 FR 33898 - Agency Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-15

    ... through http://www.Regulations.gov ; or to VA's OMB Desk Officer, OMB Human Resources and Housing Branch..., (202) 461-7485, FAX (202) 273-0443 or e-mail denise.mclamb@va.gov . Please refer to ``OMB Control No... AFFAIRS Agency Information Collection (VA Loan Electronic Reporting Interface (VALERI) System)...

  19. 78 FR 36642 - Proposed Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-18

    ... nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0021'' in any correspondence. During the... AFFAIRS Proposed Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity.... SUMMARY: The Veterans Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing...

  20. 75 FR 48413 - Agency Information Collection (HUD/VA Addendum to Uniform Residential Loan Application) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-10

    ... information through www.Regulations.gov ; or to VA's OMB Desk Officer, OMB Human Resources and Housing Branch..., (202) 461-7485, FAX (202) 273-0443 or e-mail denise.mclamb@va.gov . Please refer to ``OMB Control No... AFFAIRS Agency Information Collection (HUD/VA Addendum to Uniform Residential Loan Application)...

  1. 78 FR 21817 - Amendment of Restricted Area R-6601; Fort A.P. Hill, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-12

    ... limits and increase the time of designation of restricted area R-6601, Fort A.P. Hill, VA, (77 FR 35308... Administration 14 CFR Part 73 RIN 2120-AA66 Amendment of Restricted Area R-6601; Fort A.P. Hill, VA AGENCY... limits and time of designation of restricted area R-6601, Fort A.P. Hill, VA. The U.S. Army...

  2. 75 FR 78808 - Agency Information Collection (VA Request for Determination of Reasonable Value) Activity Under...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-16

    ... AFFAIRS Agency Information Collection (VA Request for Determination of Reasonable Value) Activity Under.... SUMMARY: In compliance with the Paperwork Reduction Act (PRA) of 1995 (44 U.S.C. 3501-3521), this notice... INFORMATION: Title: VA Request for Determination of Reasonable Value VA Form 26- 1805 and 26-1805-1....

  3. 38 CFR 26.7 - VA environmental decision making and documents.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2014-07-01 2014-07-01 false VA environmental decision making and documents. 26.7 Section 26.7 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) ENVIRONMENTAL EFFECTS OF THE DEPARTMENT OF VETERANS AFFAIRS (VA) ACTIONS § 26.7 VA environmental decision making and...

  4. 48 CFR 853.236-70 - VA Form 10-6298, Architect-Engineer Fee Proposal.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false VA Form 10-6298, Architect... VETERANS AFFAIRS CLAUSES AND FORMS FORMS Prescription of Forms 853.236-70 VA Form 10-6298, Architect-Engineer Fee Proposal. VA Form 10-6298, Architect-Engineer Fee Proposal, shall be used as prescribed in...

  5. 48 CFR 853.236-70 - VA Form 10-6298, Architect-Engineer Fee Proposal.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false VA Form 10-6298, Architect... VETERANS AFFAIRS CLAUSES AND FORMS FORMS Prescription of Forms 853.236-70 VA Form 10-6298, Architect-Engineer Fee Proposal. VA Form 10-6298, Architect-Engineer Fee Proposal, shall be used as prescribed in...

  6. 48 CFR 853.236-70 - VA Form 10-6298, Architect-Engineer Fee Proposal.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false VA Form 10-6298, Architect... VETERANS AFFAIRS CLAUSES AND FORMS FORMS Prescription of Forms 853.236-70 VA Form 10-6298, Architect-Engineer Fee Proposal. VA Form 10-6298, Architect-Engineer Fee Proposal, shall be used as prescribed in...

  7. 78 FR 11094 - Drawbridge Operation Regulation; James River, Between Isle of Wight and Newport News, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-15

    ... and Newport News, VA AGENCY: Coast Guard, DHS. ACTION: Notice of deviation from drawbridge regulation... News, VA. This deviation is necessary to facilitate generator replacement on the James River Draw... operating schedule, the James River Bridge, mile 5.0, between Isle of Isle and Newport News, VA opens...

  8. 77 FR 58773 - Drawbridge Operation Regulations; James River, Newport News, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-24

    ... SECURITY Coast Guard 33 CFR Part 117 Drawbridge Operation Regulations; James River, Newport News, VA AGENCY... James River, mile 5.0, at Newport News, VA. The deviation is necessary to facilitate the structural... regular operating schedule, the US 17/258 Bridge over the James River, mile 5.0, at Newport News, VA...

  9. 78 FR 26250 - Payment for Home Health Services and Hospice Care to Non-VA Providers

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-06

    ... November 21, 2011 (76 FR 71920), VA proposed to amend its regulations concerning the billing methodology... AFFAIRS 38 CFR Part 17 RIN 2900-AN98 Payment for Home Health Services and Hospice Care to Non-VA Providers... services and hospice care. Because the newly applicable methodology cannot supersede rates for which VA...

  10. 38 CFR 21.1032 - VA has a duty to assist claimants in obtaining evidence.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false VA has a duty to assist claimants in obtaining evidence. 21.1032 Section 21.1032 Pensions, Bonuses, and Veterans' Relief DEPARTMENT... Educational Assistance Claims § 21.1032 VA has a duty to assist claimants in obtaining evidence. (a) VA's...

  11. 38 CFR 21.33 - VA has a duty to assist claimants in obtaining evidence.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false VA has a duty to assist... Employment Under 38 U.S.C. Chapter 31 Claims § 21.33 VA has a duty to assist claimants in obtaining evidence... claimant either orally or in writing when VA: (i) Has made reasonable efforts to obtain relevant...

  12. 38 CFR 26.9 - Information on and public participation in VA environmental process.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... participation in VA environmental process. 26.9 Section 26.9 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) ENVIRONMENTAL EFFECTS OF THE DEPARTMENT OF VETERANS AFFAIRS (VA) ACTIONS § 26.9 Information on and public participation in VA environmental process. (a) During...

  13. 38 CFR 26.9 - Information on and public participation in VA environmental process.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... participation in VA environmental process. 26.9 Section 26.9 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) ENVIRONMENTAL EFFECTS OF THE DEPARTMENT OF VETERANS AFFAIRS (VA) ACTIONS § 26.9 Information on and public participation in VA environmental process. (a) During...

  14. 38 CFR 26.9 - Information on and public participation in VA environmental process.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... participation in VA environmental process. 26.9 Section 26.9 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) ENVIRONMENTAL EFFECTS OF THE DEPARTMENT OF VETERANS AFFAIRS (VA) ACTIONS § 26.9 Information on and public participation in VA environmental process. (a) During...

  15. 38 CFR 26.9 - Information on and public participation in VA environmental process.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... participation in VA environmental process. 26.9 Section 26.9 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) ENVIRONMENTAL EFFECTS OF THE DEPARTMENT OF VETERANS AFFAIRS (VA) ACTIONS § 26.9 Information on and public participation in VA environmental process. (a) During...

  16. 38 CFR 26.9 - Information on and public participation in VA environmental process.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... participation in VA environmental process. 26.9 Section 26.9 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) ENVIRONMENTAL EFFECTS OF THE DEPARTMENT OF VETERANS AFFAIRS (VA) ACTIONS § 26.9 Information on and public participation in VA environmental process. (a) During...

  17. 77 FR 23204 - VA Acquisition Regulation: Electronic Submission of Payment Requests

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-18

    ... using under VA's current interim electronic invoicing clause. See 74 FR 32223. VA's Electronic Invoice... INFORMATION: On July 7, 2009, VA published a notice, in the Federal Register at 74 FR 32223, of a class... interchange (EDI) formats; or (3) another electronic form as prescribed by the contract administration...

  18. 48 CFR 852.219-9 - VA Small business subcontracting plan minimum requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false VA Small business... Provisions and Clauses 852.219-9 VA Small business subcontracting plan minimum requirements. As prescribed in subpart 819.709, insert the following clause: VA Small Business Subcontracting Plan Minimum...

  19. 38 CFR 17.106 - VA collection rules; third-party payers.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2013-07-01 2013-07-01 false VA collection rules; third-party payers. 17.106 Section 17.106 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS MEDICAL Charges, Waivers, and Collections § 17.106 VA collection rules; third-party payers. (a)(1) General rule. VA has the right to...

  20. 38 CFR 17.106 - VA collection rules; third-party payers.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2014-07-01 2014-07-01 false VA collection rules; third-party payers. 17.106 Section 17.106 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS MEDICAL Charges, Waivers, and Collections § 17.106 VA collection rules; third-party payers. (a)(1) General rule. VA has the right to...

  1. 76 FR 63357 - VA National Academic Affiliations Council; Notice of Establishment

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-12

    ... AFFAIRS VA National Academic Affiliations Council; Notice of Establishment As required by Section 9(a)(2... establishment of the Department of Veterans Affairs (VA) National Academic Affiliations Council. The Secretary... partnerships between VA and its academic affiliates. The Council will provide a forum for discussion and...

  2. VA-086 methacrylate gelatine photopolymerizable hydrogels: A parametric study for highly biocompatible 3D cell embedding.

    PubMed

    Occhetta, Paola; Visone, Roberta; Russo, Laura; Cipolla, Laura; Moretti, Matteo; Rasponi, Marco

    2015-06-01

    The ability to replicate in vitro the native extracellular matrix (ECM) features and to control the three-dimensional (3D) cell organization plays a fundamental role in obtaining functional engineered bioconstructs. In tissue engineering (TE) applications, hydrogels have been successfully implied as biomatrices for 3D cell embedding, exhibiting high similarities to the natural ECM and holding easily tunable mechanical properties. In the present study, we characterized a promising photocrosslinking process to generate cell-laden methacrylate gelatin (GelMA) hydrogels in the presence of VA-086 photoinitiator using a ultraviolet LED source. We investigated the influence of prepolymer concentration and light irradiance on mechanical and biomimetic properties of resulting hydrogels. In details, the increasing of gelatin concentration resulted in enhanced rheological properties and shorter polymerization time. We then defined and validated a reliable photopolymerization protocol for cell embedding (1.5% VA-086, LED 2 mW/cm2) within GelMA hydrogels, which demonstrated to support bone marrow stromal cells viability when cultured up to 7 days. Moreover, we showed how different mechanical properties, derived from different crosslinking parameters, strongly influence cell behavior. In conclusion, this protocol can be considered a versatile tool to obtain biocompatible cell-laden hydrogels with properties easily adaptable for different TE applications. PMID:25294368

  3. HSV-1 amplicon vectors that direct the in situ production of foot-and-mouth disease virus antigens in mammalian cells can be used for genetic immunization.

    PubMed

    D'Antuono, Alejandra; Laimbacher, Andrea S; La Torre, Jose; Tribulatti, Virginia; Romanutti, Carina; Zamorano, Patricia; Quattrocchi, Valeria; Schraner, Elisabeth M; Ackermann, Mathias; Fraefel, Cornel; Mattion, Nora

    2010-10-28

    HSV-1 amplicon vectors encoding heterologous antigens were capable to mediate in situ generation of protein synthesis and to generate a specific immune response to the corresponding antigens. In this study, foot-and-mouth disease (FMD) virus antigens were used to generate a genetic vaccine prototype. The amplicons were designed to provide a high safety profile as they do not express any HSV-1 genes when packaged using a helper virus-free system, and they are able to encapsidate several copies of the transgene or allow the simultaneous expression of different genes. Virus-like particles were produced after cell processing of the delivered DNA. Inoculation of mice with 5 × 10(5) transducing units of amplicon vectors resulted in FMDV-specific humoral responses in the absence of adjuvants, which were dependent on the in situ de novo production of the vector-encoded antigens. Challenge of mice vaccinated with these amplicons with a high dose of live virus, resulted in partial protection, with a significant reduction of viremia. This work highlights the potential use of a HSV-1 amplicon vector platform for generation of safe genetic vaccines. PMID:20851082

  4. A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India

    PubMed Central

    Rao, Pavitra N.; Uplekar, Swapna; Kayal, Sriti; Mallick, Prashant K.; Bandyopadhyay, Nabamita; Kale, Sonal; Singh, Om P.; Mohanty, Akshaya; Mohanty, Sanjib; Wassmer, Samuel C.

    2016-01-01

    A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy. PMID:27008882

  5. A Systematic Assessment of Accuracy in Detecting Somatic Mosaic Variants by Deep Amplicon Sequencing: Application to NF2 Gene

    PubMed Central

    Sestini, Roberta; Candita, Luisa; Capone, Gabriele Lorenzo; Barbetti, Lorenzo; Falconi, Serena; Frusconi, Sabrina; Giotti, Irene; Giuliani, Costanza; Torricelli, Francesca; Benelli, Matteo; Papi, Laura

    2015-01-01

    The accurate detection of low-allelic variants is still challenging, particularly for the identification of somatic mosaicism, where matched control sample is not available. High throughput sequencing, by the simultaneous and independent analysis of thousands of different DNA fragments, might overcome many of the limits of traditional methods, greatly increasing the sensitivity. However, it is necessary to take into account the high number of false positives that may arise due to the lack of matched control samples. Here, we applied deep amplicon sequencing to the analysis of samples with known genotype and variant allele fraction (VAF) followed by a tailored statistical analysis. This method allowed to define a minimum value of VAF for detecting mosaic variants with high accuracy. Then, we exploited the estimated VAF to select candidate alterations in NF2 gene in 34 samples with unknown genotype (30 blood and 4 tumor DNAs), demonstrating the suitability of our method. The strategy we propose optimizes the use of deep amplicon sequencing for the identification of low abundance variants. Moreover, our method can be applied to different high throughput sequencing approaches to estimate the background noise and define the accuracy of the experimental design. PMID:26066488

  6. RiboFR-Seq: a novel approach to linking 16S rRNA amplicon profiles to metagenomes.

    PubMed

    Zhang, Yanming; Ji, Peifeng; Wang, Jinfeng; Zhao, Fangqing

    2016-06-01

    16S rRNA amplicon analysis and shotgun metagenome sequencing are two main culture-independent strategies to explore the genetic landscape of various microbial communities. Recently, numerous studies have employed these two approaches together, but downstream data analyses were performed separately, which always generated incongruent or conflict signals on both taxonomic and functional classifications. Here we propose a novel approach, RiboFR-Seq (Ribosomal RNA gene flanking region sequencing), for capturing both ribosomal RNA variable regions and their flanking protein-coding genes simultaneously. Through extensive testing on clonal bacterial strain, salivary microbiome and bacterial epibionts of marine kelp, we demonstrated that RiboFR-Seq could detect the vast majority of bacteria not only in well-studied microbiomes but also in novel communities with limited reference genomes. Combined with classical amplicon sequencing and shotgun metagenome sequencing, RiboFR-Seq can link the annotations of 16S rRNA and metagenomic contigs to make a consensus classification. By recognizing almost all 16S rRNA copies, the RiboFR-seq approach can effectively reduce the taxonomic abundance bias resulted from 16S rRNA copy number variation. We believe that RiboFR-Seq, which provides an integrated view of 16S rRNA profiles and metagenomes, will help us better understand diverse microbial communities. PMID:26984526

  7. RiboFR-Seq: a novel approach to linking 16S rRNA amplicon profiles to metagenomes

    PubMed Central

    Zhang, Yanming; Ji, Peifeng; Wang, Jinfeng; Zhao, Fangqing

    2016-01-01

    16S rRNA amplicon analysis and shotgun metagenome sequencing are two main culture-independent strategies to explore the genetic landscape of various microbial communities. Recently, numerous studies have employed these two approaches together, but downstream data analyses were performed separately, which always generated incongruent or conflict signals on both taxonomic and functional classifications. Here we propose a novel approach, RiboFR-Seq (Ribosomal RNA gene flanking region sequencing), for capturing both ribosomal RNA variable regions and their flanking protein-coding genes simultaneously. Through extensive testing on clonal bacterial strain, salivary microbiome and bacterial epibionts of marine kelp, we demonstrated that RiboFR-Seq could detect the vast majority of bacteria not only in well-studied microbiomes but also in novel communities with limited reference genomes. Combined with classical amplicon sequencing and shotgun metagenome sequencing, RiboFR-Seq can link the annotations of 16S rRNA and metagenomic contigs to make a consensus classification. By recognizing almost all 16S rRNA copies, the RiboFR-seq approach can effectively reduce the taxonomic abundance bias resulted from 16S rRNA copy number variation. We believe that RiboFR-Seq, which provides an integrated view of 16S rRNA profiles and metagenomes, will help us better understand diverse microbial communities. PMID:26984526

  8. A portable automatic endpoint detection system for amplicons of loop mediated isothermal amplification on microfluidic compact disk platform.

    PubMed

    Uddin, Shah Mukim; Ibrahim, Fatimah; Sayad, Abkar Ahmed; Thiha, Aung; Pei, Koh Xiu; Mohktar, Mas S; Hashim, Uda; Cho, Jongman; Thong, Kwai Lin

    2015-01-01

    In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10(-3) ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment. PMID:25751077

  9. A Portable Automatic Endpoint Detection System for Amplicons of Loop Mediated Isothermal Amplification on Microfluidic Compact Disk Platform

    PubMed Central

    Uddin, Shah Mukim; Ibrahim, Fatimah; Sayad, Abkar Ahmed; Thiha, Aung; Pei, Koh Xiu; Mohktar, Mas S.; Hashim, Uda; Cho, Jongman; Thong, Kwai Lin

    2015-01-01

    In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10−3 ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment. PMID:25751077

  10. A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India.

    PubMed

    Rao, Pavitra N; Uplekar, Swapna; Kayal, Sriti; Mallick, Prashant K; Bandyopadhyay, Nabamita; Kale, Sonal; Singh, Om P; Mohanty, Akshaya; Mohanty, Sanjib; Wassmer, Samuel C; Carlton, Jane M

    2016-06-01

    A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy. PMID:27008882

  11. 'Baby talk' attracts neighbors to new Manassas, Va., birthing center.

    PubMed

    Botvin, Judith D

    2005-01-01

    An dollar 80 million expansion plan recently was launched by Prince William Hospital, Manassas, Va. The cornerstone of the plan is the new Hylton Family Birthing Center. The hospital employed a full range of tactics to promote the opening of the birthing center. These ranged from quarterly newsletters to billboards; from tours and radio spots to a newly revised web site. The campaign is characterized by infant testimonials, gentle humor and bright colors. PMID:15971721

  12. Validation of KENO V.a Comparison with Critical Experiments

    SciTech Connect

    Jordan, W.C.

    1999-01-01

    Section 1 of this report documents the validation of KENO V.a against 258 critical experiments. Experiments considered were primarily high or low enriched uranium systems. The results indicate that the KENO V.a Monte Carlo Criticality Program accurately calculates a broad range of critical experiments. A substantial number of the calculations showed a positive or negative bias in excess of 1 1/2% in k-effective (k{sub eff}). Classes of criticals which show a bias include 3% enriched green blocks, highly enriched uranyl fluoride slab arrays, and highly enriched uranyl nitrate arrays. If these biases are properly taken into account, the KENO V.a code can be used with confidence for the design and criticality safety analysis of uranium-containing systems. Section 2 of this report documents the results of investigation into the cause of the bias observed in Sect. 1. The results of this study indicate that the bias seen in Sect. 1 is caused by code bias, cross-section bias, reporting bias, and modeling bias. There is evidence that many of the experiments used in this validation and in previous validations are not adequately documented. The uncertainty in the experimental parameters overshadows bias caused by the code and cross sections and prohibits code validation to better than about 1% in k{sub eff}.

  13. The HER2 amplicon includes several genes required for the growth and survival of HER2 positive breast cancer cells — A data description

    PubMed Central

    Hongisto, Vesa; Aure, Miriam Ragle; Mäkelä, Rami; Sahlberg, Kristine Kleivi

    2014-01-01

    A large number of breast cancers are characterized by amplification and overexpression of the chromosome segment surrounding the HER2 (ERBB2) oncogene. As the HER2 amplicon at 17q12 contains multiple genes, we have systematically explored the role of the HER2 co-amplified genes in breast cancer cell growth and their relation to trastuzumab resistance. We integrated array comparative genomic hybridization (aCGH) data of the HER2 amplicon from 71 HER2 positive breast tumors and 10 cell lines with systematic functional RNA interference analysis of 23 core amplicon genes with several phenotypic endpoints in a panel of trastuzumab responding and non-responding HER2 positive breast cancer cells. In this Data in Brief we give a detailed description of the experimental procedures and the data analysis methods used in the study (1). PMID:26484103

  14. Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala-activated protein C mediated by factor Va.

    PubMed Central

    Gale, A. J.; Sun, X.; Heeb, M. J.; Griffin, J. H.

    1997-01-01

    The human plasma serine protease, activated protein C (APC), primarily exerts its anticoagulant function by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. A recombinant active site Ser 360 to Ala mutation of protein C was prepared, and the mutant protein was expressed in human 293 kidney cells and purified. The activation peptide of the mutant protein C zymogen was cleaved by a snake venom activator, Protac C, but the "activated" S360A APC did not have amidolytic activity. However, it did exhibit significant anticoagulant activity both in clotting assays and in a purified protein assay system that measured prothrombinase activity. The S360A APC was compared to plasma-derived and wild-type recombinant APC. The anticoagulant activity of the mutant, but not native APC, was resistant to diisopropyl fluorophosphate, whereas all APCs were inhibited by monoclonal antibodies against APC. In contrast to native APC, S360A APC was not inactivated by serine protease inhibitors in plasma and did not bind to the highly reactive mutant protease inhibitor M358R alpha 1 antitrypsin. Since plasma serpins provide the major mechanism for inactivating APC in vivo, this suggests that S360A APC would have a long half-life in vivo, with potential therapeutic advantages. S360A APC rapidly inhibited factor Va in a nonenzymatic manner since it apparently did not proteolyze factor Va. These data suggest that native APC may exhibit rapid nonenzymatic anticoagulant activity followed by enzymatic irreversible proteolysis of factor Va. The results of clotting assays and prothrombinase assays showed that S360A APC could not inhibit the variant Gln 506-FVa compared with normal Arg 506-FVa, suggesting that the active site of S360A APC binds to FVa at or near Arg 506. PMID:9007985

  15. Vascular and biochemical risk factors of vascular dementia after lacunar strokes (S-VaD) and after multiinfarcts in strategic areas (M-VaD).

    PubMed

    Graban, Ałła; Bednarska-Makaruk, Małgorzata; Bochyńska, Anna; Lipczyńska-Łojkowska, Wanda; Ryglewicz, Danuta; Wehr, Hanna

    2009-08-15

    Vascular cognitive impairment is an important cause of cognitive decline in the elderly. Ischemic lesions in the brain have an influence on the natural history of dementia. Vascular dementia can be caused by small-vessels disease (S-VaD) or by large-artery atherosclerosis with vascular lesions in strategic areas of the brain (M-VaD). In both cases changes in white matter are observed. In 60 patients with S-VaD and in 34 with M-VaD the presence of vascular and biochemical risk factors was evaluated and compared to age and sex matched 126 controls without dementia. Coronary artery disease, atrial fibrillation, hypertension and strokes were observed more frequently in both investigated groups. Of biochemical risk factors, hyperhomocysteinemia (associated with low levels of folic acid and vitamin B 12) and low HDL cholesterol levels were found in both forms of VaD. PMID:19339023

  16. Short-Read Assembly of Full-Length 16S Amplicons Reveals Bacterial Diversity in Subsurface Sediments

    PubMed Central

    Miller, Christopher S.; Handley, Kim M.; Wrighton, Kelly C.; Frischkorn, Kyle R.; Thomas, Brian C.; Banfield, Jillian F.

    2013-01-01

    In microbial ecology, a fundamental question relates to how community diversity and composition change in response to perturbation. Most studies have had limited ability to deeply sample community structure (e.g. Sanger-sequenced 16S rRNA libraries), or have had limited taxonomic resolution (e.g. studies based on 16S rRNA hypervariable region sequencing). Here, we combine the higher taxonomic resolution of near-full-length 16S rRNA gene amplicons with the economics and sensitivity of short-read sequencing to assay the abundance and identity of organisms that represent as little as 0.01% of sediment bacterial communities. We used a new version of EMIRGE optimized for large data size to reconstruct near-full-length 16S rRNA genes from amplicons sheared and sequenced with Illumina technology. The approach allowed us to differentiate the community composition among samples acquired before perturbation, after acetate amendment shifted the predominant metabolism to iron reduction, and once sulfate reduction began. Results were highly reproducible across technical replicates, and identified specific taxa that responded to the perturbation. All samples contain very high alpha diversity and abundant organisms from phyla without cultivated representatives. Surprisingly, at the time points measured, there was no strong loss of evenness, despite the selective pressure of acetate amendment and change in the terminal electron accepting process. However, community membership was altered significantly. The method allows for sensitive, accurate profiling of the “long tail” of low abundance organisms that exist in many microbial communities, and can resolve population dynamics in response to environmental change. PMID:23405248

  17. Gene-dosage dependent overexpression at the 13q amplicon identifies DIS3 as candidate oncogene in colorectal cancer progression.

    PubMed

    de Groen, Florence L M; Krijgsman, Oscar; Tijssen, Marianne; Vriend, Lianne E M; Ylstra, Bauke; Hooijberg, Erik; Meijer, Gerrit A; Steenbergen, Renske D M; Carvalho, Beatriz

    2014-04-01

    Colorectal cancer (CRC) development is in most cases marked by the accumulation of genomic alterations including gain of the entire q-arm of chromosome 13. This aberration occurs in 40%-60% of all CRC and is associated with progression from adenoma to carcinoma. To date, little is known about the effect of the 13q amplicon on the expression of the therein located genes and their functional relevance. We therefore aimed to identify candidate genes at the 13q amplicon that contribute to colorectal adenoma to carcinoma progression in a gene dosage-dependent manner. Integrative analysis of whole genome expression and DNA copy number signatures resulted in the identification of 36 genes on 13q of which significant overexpression in carcinomas compared with adenomas was linked to a copy number gain. Five genes showing high levels of overexpression in carcinomas versus adenomas were further tested by quantitative reverse transcription-PCR in two independent sample sets of colorectal tumors (n = 40 and n = 47). DIS3 and LRCH1 revealed significant overexpression in carcinomas compared with adenomas in a 13q gain dependent manner. Silencing of DIS3 affected important tumorigenic characteristics such as viability, migration, and invasion. In conclusion, significant overexpression of DIS3 and LRCH1 associated with adenoma to carcinoma progression is linked to the CRC specific gain of 13q. The functional relevance of this copy number aberration was corroborated for DIS3, thereby identifying this gene as novel candidate oncogene contributing to the 13q-driven adenoma to carcinoma progression. PMID:24478024

  18. Quantification of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) using amplicon-fusion-site polymerase chain reaction (AFS-PCR)

    PubMed Central

    2012-01-01

    The amplification of putative oncogenes is a common finding within the genome of various cancer types. Identification and further targeting of specific junction sites within the sequence of genomic amplicons (amplicon fusion sites, AFS) by PCR (AFS-PCR) is suitable for quantification of minimal residual disease (MRD). This approach has recently been developed and described for MYCN amplified neuroblastomas. To compare AFS-PCR directly to routinely used MRD diagnostic strategies, we mapped the amplified genomic regions (ampGR) of an iAMP21-amplicon in high resolution of a patient with acute lymphoblastic leukemia (ALL). Successfully, we established AFS-PCR covering junction sites between ampGR within the iAMP21-amplicon. Quantification of MRD by AFS-PCR was directly comparable to IgH/TCR based real time quantitative PCR and fluorescence activated cell sorting (FACS) analysis in consecutive bone marrow (BM) specimens. Our data give an additional proof of concept of AFS-PCR for quantification of MRD. The method could be taken into account for ALL patients with genomic amplifications as alternative MRD diagnostic, if no or qualitatively poor Ig/TCR-PCRs are available. PMID:23210797

  19. Cloning and physical mapping of DNA sequences encompassing a region in N-myc amplicons of a human neuroblastoma cell line.

    PubMed Central

    Akiyama, K; Nishi, Y

    1991-01-01

    Cloning and physical mapping of DNA sequences encompassing N-myc amplicons of a human neuroblastoma cell line were done. A number of lambda phage clones within this region were isolated using the probes prepared by the phenol emulsion reassociation technique. Based on the restriction mapping, they were integrated into 8 contigs with sizes of 25-60 kb which, in total, encompassed a 330 kb region. Several amplicons, 100, 420, 480 and 520 kb in size as a Notl fragment, were identified using hexagonal field gel electrophoresis, and the contigs were assigned in these Notl fragments. The region encompassed by the contigs was equivalent to some 60-80% of the amplicons identified as a Notl fragment. In order to compare the amplified regions flanking the N-myc gene among the cell lines, the phage clones to cover the whole contigs were used for hybridization as a probe. The results showed that the portions of the whole contigs ranging 18-45% were also amplified in the cell lines examined. These results allowed us to identified the 'rearranged sites' which were rather evenly distributed, one at every 40 kb, through the contigs. These observations lead to the idea that an amplified DNA domain is constructed after the multiple rearrangements and then increases in number, finally resulting in the formation of subsets of amplicons with sequence homogeneity. Images PMID:1762918

  20. KENO V.a Primer: A Primer for Criticality Calculations with SCALE/KENO V.a Using CSPAN for Input

    SciTech Connect

    Busch, R.D.

    2003-01-17

    The SCALE (Standardized Computer Analyses for Licensing Evaluation) computer software system developed at Oak Ridge National Laboratory (ORNL) is widely used and accepted around the world for criticality safety analyses. The well-known KENO V.a three-dimensional Monte Carlo criticality computer code is the primary criticality safety analysis tool in SCALE. The KENO V.a primer is designed to help a new user understand and use the SCALE/KENO V.a Monte Carlo code for nuclear criticality safety analyses. It assumes that the user has a college education in a technical field. There is no assumption of familiarity with Monte Carlo codes in general or with SCALE/KENO V.a in particular. The primer is designed to teach by example, with each example illustrating two or three features of SCALE/KENO V.a that are useful in criticality analyses. The primer is based on SCALE 4.4a, which includes the Criticality Safety Processor for Analysis (CSPAN) input processor for Windows personal computers (PCs). A second edition of the primer, which uses the new KENO Visual Editor, is currently under development at ORNL and is planned for publication in late 2003. Each example in this first edition of the primer uses CSPAN to provide the framework for data input. Starting with a Quickstart section, the primer gives an overview of the basic requirements for SCALE/KENO V.a input and allows the user to quickly run a simple criticality problem with SCALE/KENO V.a. The sections that follow Quickstart include a list of basic objectives at the beginning that identifies the goal of the section and the individual SCALE/KENO V.a features which are covered in detail in the example problems in that section. Upon completion of the primer, a new user should be comfortable using CSPAN to set up criticality problems in SCALE/KENO V.a.

  1. Mixed-Methods Research in a Complex Multisite VA Health Services Study: Variations in the Implementation and Characteristics of Chiropractic Services in VA.

    PubMed

    Khorsan, Raheleh; Cohen, Angela B; Lisi, Anthony J; Smith, Monica M; Delevan, Deborah; Armstrong, Courtney; Mittman, Brian S

    2013-01-01

    Maximizing the quality and benefits of newly established chiropractic services represents an important policy and practice goal for the US Department of Veterans Affairs' healthcare system. Understanding the implementation process and characteristics of new chiropractic clinics and the determinants and consequences of these processes and characteristics is a critical first step in guiding quality improvement. This paper reports insights and lessons learned regarding the successful application of mixed methods research approaches-insights derived from a study of chiropractic clinic implementation and characteristics, Variations in the Implementation and Characteristics of Chiropractic Services in VA (VICCS). Challenges and solutions are presented in areas ranging from selection and recruitment of sites and participants to the collection and analysis of varied data sources. The VICCS study illustrates the importance of several factors in successful mixed-methods approaches, including (1) the importance of a formal, fully developed logic model to identify and link data sources, variables, and outcomes of interest to the study's analysis plan and its data collection instruments and codebook and (2) ensuring that data collection methods, including mixed-methods, match study aims. Overall, successful application of a mixed-methods approach requires careful planning, frequent trade-offs, and complex coding and analysis. PMID:24489589

  2. Communication between VA providers and sexual and gender minority veterans: a pilot study.

    PubMed

    Sherman, Michelle D; Kauth, Michael R; Shipherd, Jillian C; Street, Richard L

    2014-05-01

    Approximately one million gay and lesbian Americans are veterans, and rates of engagement in the Veterans Affairs (VA) health care system may be increasing for both sexual and gender minority veterans. Very little research has examined the experience of these veterans when receiving care at VA health care facilities. The purpose of this study was to explore the experiences, beliefs, and preferences of lesbian, gay, bisexual, or transgender (LGBT) veterans in their communication with VA health care providers. LGBT veterans (n = 58) participated in focus groups or individual interviews and completed self-report measures at two southern VA hospitals. Approximately 2/3 of veterans report that none of their VA providers have specifically asked about their sexual orientation, and 24% of the veterans indicate that they have not disclosed their orientation to any VA provider. Although some veterans want providers to initiate these discussions, veterans also expressed fears about disclosure and its possible negative consequences. Similarly, LGBT veterans report varied opinions about the appropriateness of routine assessment of minority status. Only 28% of these veterans experience VA as welcoming to them as LGBT veterans. Systematic training is needed for all VA providers about the rationale for assessing sexual and gender orientation. Staff education should include specific skills for initiating these assessments, and ways of responding to veteran concerns about discussing this topic in the VA health care system. PMID:24588107

  3. Molecular characterization of a novel amplicon at 1q21-q22 frequently observed in human sarcomas.

    PubMed Central

    Forus, A.; Berner, J. M.; Meza-Zepeda, L. A.; Saeter, G.; Mischke, D.; Fodstad, O.; Myklebost, O.

    1998-01-01

    In a recent comparative genomic hybridization (CGH) study of a panel of sarcomas, we detected recurrent amplification of 1q21-q22 in soft tissue and bone tumours. Amplification of this region had not previously been associated with sarcoma development, but occasional amplification of CACY/S100A6 and MUC1 in 1q21 had been reported for melanoma and breast carcinoma respectively. Initial screening by Southern blot analysis showed amplification of S100A6, FLG and SPRR3 in several sarcomas and, in a first attempt to characterize the 1q21-q22 amplicon in more detail, we have now investigated the amplification status of these and 11 other markers in the region in 35 sarcoma samples. FLG was the most frequently amplified gene, and the markers located in the same 4.5-Mb region as FLG showed a higher incidence of amplification than the more distal ones. However, for most of the 14 markers, amplification levels were low, and only APOA2 and the anonymous marker D1S3620 showed high-level amplifications (> tenfold increases) in one sample each. We used fluorescence in situ hybridization (FISH) to determine the amplification patterns of two overlapping yeast artificial chromosomes (YACs) covering the region between D1S3620 and FLG (789f2 and 764a1), as well as two more distally located YACs in nine selected samples. Six samples had amplification of the YAC containing D1S3620 and, in three, 764a1 was also included. Five of these tumours showed normal copies of the more distal YACs; thus, it seems likely that an important gene may be located within 789f2, or very close. Two samples had high copy numbers of the most distal YACs. Taken together, FISH and molecular analyses indicate complex amplification patterns in 1q21-q22 with at least two amplicons: one located near D1S3620/789f2 and one more distal. Images Figure 1 Figure 2 Figure 3 PMID:9716033

  4. Parallelization of KENO-Va Monte Carlo code

    NASA Astrophysics Data System (ADS)

    Ramón, Javier; Peña, Jorge

    1995-07-01

    KENO-Va is a code integrated within the SCALE system developed by Oak Ridge that solves the transport equation through the Monte Carlo Method. It is being used at the Consejo de Seguridad Nuclear (CSN) to perform criticality calculations for fuel storage pools and shipping casks. Two parallel versions of the code: one for shared memory machines and other for distributed memory systems using the message-passing interface PVM have been generated. In both versions the neutrons of each generation are tracked in parallel. In order to preserve the reproducibility of the results in both versions, advanced seeds for random numbers were used. The CONVEX C3440 with four processors and shared memory at CSN was used to implement the shared memory version. A FDDI network of 6 HP9000/735 was employed to implement the message-passing version using proprietary PVM. The speedup obtained was 3.6 in both cases.

  5. Development of 30 kVA class fully superconducting generator

    SciTech Connect

    Tsukamoto, O.; Amemiya, N.; Takao, T. . Faculty of Engineering); Akita, S. ); Ohishi, K.; Shimuzu, H.; Tanaka, Y. ); Uchikawa, Y. )

    1992-01-01

    This paper reports that the authors are developing a 4 poles 30 kVA class fully superconducting generator to investigate the characteristics of superconducting armature winding subject to the rotating magnetic field produced by the superconducting rotor and behavior of a superconducting generator connected to an electric power utility grid. A static test of the armature winding have been performed by applying 50 Hz AC current. AC quench currents of the armature windings have reached to 200 Arms after several quenches which was well over the rated current. A static test of the field windings have been also performed to verify its rated performance. In the paper, detailed configurations and electrical test results of the generator are shown.

  6. Targeting the Central Nervous System with Herpes Simplex Virus / Sleeping Beauty Hybrid Amplicon Vectors

    PubMed Central

    de Silva, Suresh; Bowers, William J.

    2014-01-01

    The pursuits of sustainable treatments for diseases and disorders that afflict the central nervous system (CNS) have proven challenging for the field of viral vector-based gene therapy. However, recent advances in viral vector technology coupled with efficient delivery methods have opened up new avenues that show promise at the preclinical testing stage. The development of the Herpes Simplex Virus/Sleeping Beauty (HSV/SB) hybrid vector represents such an advance for devising treatments targeting the CNS with its potential for stably integrating large transgenomic segments of DNA within the genomes of transduced cells. In utero administration of this hybrid vector into the embryonic mouse brain has revealed the capacity for widespread transgene dissemination due to the targeting of a neuronal precursor cell population. This unique feature has provided the means to stably express a transgene throughout the brain for prolonged periods, which is a prerequisite for the treatment of progressive CNS disorders. In this review we provide a comprehensive breakdown of the characteristics of the HSV/SB vector system and how it can be efficiently employed in the derivation of CNS-targeted gene therapeutic strategies. PMID:21711226

  7. 38 CFR 62.32 - Supportive service: Assistance in obtaining VA benefits.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...; and (4) Health care services. (b) Grantees are not permitted to represent participants before VA with... 38 Pensions, Bonuses, and Veterans' Relief 2 2011-07-01 2011-07-01 false Supportive service: Assistance in obtaining VA benefits. 62.32 Section 62.32 Pensions, Bonuses, and Veterans' Relief...

  8. 38 CFR 62.32 - Supportive service: Assistance in obtaining VA benefits.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...; and (4) Health care services. (b) Grantees are not permitted to represent participants before VA with... 38 Pensions, Bonuses, and Veterans' Relief 2 2014-07-01 2014-07-01 false Supportive service: Assistance in obtaining VA benefits. 62.32 Section 62.32 Pensions, Bonuses, and Veterans' Relief...

  9. 38 CFR 62.32 - Supportive service: Assistance in obtaining VA benefits.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...; and (4) Health care services. (b) Grantees are not permitted to represent participants before VA with... 38 Pensions, Bonuses, and Veterans' Relief 2 2013-07-01 2013-07-01 false Supportive service: Assistance in obtaining VA benefits. 62.32 Section 62.32 Pensions, Bonuses, and Veterans' Relief...

  10. 77 FR 21158 - VA Directive 0005 on Scientific Integrity: Availability for Review and Comment

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-09

    ... AFFAIRS VA Directive 0005 on Scientific Integrity: Availability for Review and Comment AGENCY: Office of... (VA) Directive 0005 on Scientific Integrity. The Draft Directive incorporates the principles of scientific integrity contained in the Presidential Memorandum of March 9, 2009, and the Director, Office...

  11. 30 CFR 57.22229 - Weekly testing (I-A, III, and V-A mines).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Weekly testing (I-A, III, and V-A mines). 57.22229 Section 57.22229 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR...-A, III, and V-A mines). (a) The mine atmosphere shall be tested for methane and carbon monoxide...

  12. 30 CFR 57.22229 - Weekly testing (I-A, III, and V-A mines).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Weekly testing (I-A, III, and V-A mines). 57.22229 Section 57.22229 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR...-A, III, and V-A mines). (a) The mine atmosphere shall be tested for methane and carbon monoxide...

  13. 30 CFR 57.22229 - Weekly testing (I-A, III, and V-A mines).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Weekly testing (I-A, III, and V-A mines). 57.22229 Section 57.22229 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR...-A, III, and V-A mines). (a) The mine atmosphere shall be tested for methane and carbon monoxide...

  14. 38 CFR 58.16 - VA Form 10-0144-Certification Regarding Lobbying.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false VA Form 10-0144-Certification Regarding Lobbying. 58.16 Section 58.16 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) FORMS § 58.16 VA Form 10-0144—Certification Regarding Lobbying. ER06JA00.013...

  15. 48 CFR 803.7000 - Display of the VA Hotline poster.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... poster. 803.7000 Section 803.7000 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS... Improper Business Practices 803.7000 Display of the VA Hotline poster. (a) Under the circumstances described in paragraph (b) of this section, a contractor must display prominently a VA Hotline...

  16. 48 CFR 803.7000 - Display of the VA Hotline poster.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... poster. 803.7000 Section 803.7000 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS... Improper Business Practices 803.7000 Display of the VA Hotline poster. (a) Under the circumstances described in paragraph (b) of this section, a contractor must display prominently a VA Hotline...

  17. 48 CFR 803.7000 - Display of the VA Hotline poster.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... poster. 803.7000 Section 803.7000 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS... Improper Business Practices 803.7000 Display of the VA Hotline poster. (a) Under the circumstances described in paragraph (b) of this section, a contractor must display prominently a VA Hotline...

  18. 48 CFR 803.7000 - Display of the VA Hotline poster.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... poster. 803.7000 Section 803.7000 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS... Improper Business Practices 803.7000 Display of the VA Hotline poster. (a) Under the circumstances described in paragraph (b) of this section, a contractor must display prominently a VA Hotline...

  19. 48 CFR 803.7000 - Display of the VA Hotline poster.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... poster. 803.7000 Section 803.7000 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS... Improper Business Practices 803.7000 Display of the VA Hotline poster. (a) Under the circumstances described in paragraph (b) of this section, a contractor must display prominently a VA Hotline...

  20. 75 FR 47461 - Drawbridge Operation Regulation; Elizabeth River, Eastern Branch, Norfolk, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-06

    ... Register (74 FR 52143) and a notice of proposed rulemaking (NPRM) entitled ``Drawbridge Operation Regulations; Elizabeth River, Eastern Branch, Norfolk, VA'' in the Federal Register (74 FR 52158). We received..., Eastern Branch, Norfolk, VA'' in the Federal Register (75 FR 9521) and a supplemental notice of...

  1. 77 FR 48127 - Foreign-Trade Zone 20-Suffolk, VA; Notification of Proposed Production Activity, Usui...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-13

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF COMMERCE Foreign-Trade Zones Board Foreign-Trade Zone 20--Suffolk, VA; Notification of Proposed Production Activity, Usui International Corporation, (Diesel Engine Fuel Lines), Chesapeake, VA The Virginia Port Authority, grantee of FTZ 20, submitted a...

  2. 75 FR 76082 - Agency Information Collection (Credit Underwriting Standards and Procedures for Processing VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-07

    ... collection of information through http://www.Regulations.gov or to VA's OMB Desk Officer, OMB Human Resources..., NW., Washington, DC 20420, (202) 461-7485, FAX (202) 273-0443 or e-mail denise.mclamb@.va.gov. Please... AFFAIRS Agency Information Collection (Credit Underwriting Standards and Procedures for Processing...

  3. 77 FR 70212 - Agency Information Collection Activities Under OMB Review: VA MATIC Enrollment/Change

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-23

    ... or email crystal.rennie@va.gov . Please refer to ``OMB Control No. 2900-0525.'' SUPPLEMENTARY... AFFAIRS Agency Information Collection Activities Under OMB Review: VA MATIC Enrollment/Change AGENCY..., 2012. ADDRESSES: Submit written comments on the collection of information through...

  4. 77 FR 39346 - Proposed Information Collection (Statement of Accredited Representative in Appealed Case, VA Form...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-02

    ... Affairs, 810 Vermont Avenue NW., Washington, DC 20420 or email Sue.Hamlin@mail.va.gov . Please refer to... AFFAIRS Proposed Information Collection (Statement of Accredited Representative in Appealed Case, VA Form... Affairs. ACTION: Notice. SUMMARY: The Board of Veterans' Appeals (BVA), Department of Veterans Affairs...

  5. 78 FR 59099 - Agency Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-25

    ... Affairs, 810 Vermont Avenue NW., Washington, DC 20420, (202) 632-7492 or email crystal.rennie@va.gov... AFFAIRS Agency Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity... through www.Regulations.gov , or to Office of Information and Regulatory Affairs, Office of Management...

  6. 76 FR 24567 - Proposed Information Collection (VA MATIC Authorization); Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-02

    ... AFFAIRS Proposed Information Collection (VA MATIC Authorization); Comment Request AGENCY: Veterans... Administration (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public comment on the...) at http://www.Regulations.gov ; or to Nancy J. Kessinger, Veterans Benefits Administration...

  7. 78 FR 48314 - Drawbridge Operation Regulation; Milford Haven Inlet, Hudgins, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-08

    ... SECURITY Coast Guard 33 CFR Part 117 Drawbridge Operation Regulation; Milford Haven Inlet, Hudgins, VA... Guard has issued a temporary deviation from the operating schedule that governs the VA State Route 223....m. March 31, 2014. ADDRESSES: The docket for this deviation, is available at...

  8. Use of Medicare and DOD data for improving VA race data quality.

    PubMed

    Stroupe, Kevin T; Tarlov, Elizabeth; Zhang, Qiuying; Haywood, Thomas; Owens, Arika; Hynes, Denise M

    2010-01-01

    We evaluated the improvement in Department of Veterans Affairs (VA) race data completeness that could be achieved by linking VA data with data from Medicare and the Department of Defense (DOD) and examined agreement in values across the data sources. After linking VA with Medicare and DOD records for a 10% sample of VA patients, we calculated the percentage for which race could be identified in those sources. To evaluate race agreement, we calculated sensitivities, specificities, positive predictive values (PPVs), negative predictive values, and kappa statistics. Adding Medicare (and DOD) data improved race data completeness from 48% to 76%. Among older patients (≥65 years), adding Medicare data improved data completeness to nearly 100%. Among younger patients (<65 years), combining Medicare and DOD data improved completeness to 75%, 18 percentage points beyond that achieved with Medicare data alone. PPVs for white and African-American categories were 98.6 and 94.7, respectively, in Medicare and 97.0 and 96.5, respectively, in DOD data using VA self-reported race as the gold standard. PPVs for the non-African-American minority groups were lower, ranging from 30.5 to 48.2. Kappa statistics reflected these patterns. Supplementing VA with Medicare and DOD data improves VA race data completeness substantially. More study is needed to understand poor rates of agreement between VA and external sources in identifying non-African-American minority individuals. PMID:21110252

  9. 75 FR 20774 - Establishment of Class E Airspace; Fort A.P. Hill, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-21

    ... final rule with a request for comments in the Federal Register on December 7, 2009 (74 FR 63974), Docket... Federal Aviation Administration 14 CFR Part 71 Establishment of Class E Airspace; Fort A.P. Hill, VA... Register December 7, 2009 that establishes Class E airspace at Fort A.P. Hill, VA. DATES: Effective...

  10. The Impact of VA and Navy Hospital Collaboration on Medical School Education

    ERIC Educational Resources Information Center

    Atre-Vaidya, Nutan; Ross, Arthur, III; Sandu, Ioana C.; Hassan, Tariq

    2009-01-01

    Objective: The U.S. Department of Veterans Affairs (VA) is the largest single provider of medical education in the United States and is often the preferred training site for medical students and residents. However, changing priorities of patients and the marketplace are forcing medical schools and the VA to consider new ways of practicing medicine…

  11. Myosin Va Transports Dense Core Secretory Vesicles in Pancreatic MIN6 β-CellsV⃞

    PubMed Central

    Varadi, Aniko; Tsuboi, Takashi; Rutter, Guy A.

    2005-01-01

    The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic β-cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of vesicles. By contrast, MyoVa immunoreactivity was poorly colocalized with mitochondrial or other markers. Demonstrating an important role for MyoVa in the recruitment of secretory vesicles to the cell surface, a reduction of MyoVa protein levels achieved by RNA interference caused a significant decrease in glucose- or depolarization-stimulated insulin secretion. Similarly, expression of the dominant-negative–acting globular tail domain of MyoVa decreased by ∼50% the number of vesicles docked at the plasma membrane and by 87% the number of depolarization-stimulated exocytotic events detected by total internal reflection fluorescence microscopy. We conclude that MyoVa-driven movements of vesicles along the cortical actin network are essential for the terminal stages of regulated exocytosis in β-cells. PMID:15788565

  12. 38 CFR 26.7 - VA environmental decision making and documents.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... environmental decision making and documents. (a) Relevant environmental documents shall accompany other decision documents as they proceed through the decision-making process. (b) The major decision points for VA actions... observing the provisions of these regulations, VA must act in accordance with CEQ Regulations, 40 CFR...

  13. 38 CFR 26.7 - VA environmental decision making and documents.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... environmental decision making and documents. (a) Relevant environmental documents shall accompany other decision documents as they proceed through the decision-making process. (b) The major decision points for VA actions... observing the provisions of these regulations, VA must act in accordance with CEQ Regulations, 40 CFR...

  14. 38 CFR 26.7 - VA environmental decision making and documents.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... environmental decision making and documents. (a) Relevant environmental documents shall accompany other decision documents as they proceed through the decision-making process. (b) The major decision points for VA actions... observing the provisions of these regulations, VA must act in accordance with CEQ Regulations, 40 CFR...

  15. 38 CFR 26.7 - VA environmental decision making and documents.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... environmental decision making and documents. (a) Relevant environmental documents shall accompany other decision documents as they proceed through the decision-making process. (b) The major decision points for VA actions... observing the provisions of these regulations, VA must act in accordance with CEQ Regulations, 40 CFR...

  16. 38 CFR 17.66 - Notice of noncompliance with VA standards.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... AFFAIRS MEDICAL Community Residential Care § 17.66 Notice of noncompliance with VA standards. If the hearing official determines that an approved community residential care facility does not comply with the... standards must be met in order to avoid revocation of VA approval; (c) The community residential...

  17. 38 CFR 17.66 - Notice of noncompliance with VA standards.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... AFFAIRS MEDICAL Community Residential Care § 17.66 Notice of noncompliance with VA standards. If the hearing official determines that an approved community residential care facility does not comply with the... standards must be met in order to avoid revocation of VA approval; (c) The community residential...

  18. 33 CFR 334.130 - Atlantic Ocean off Wallops Island and Chincoteague Inlet, Va.; danger zone.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 3 2014-07-01 2014-07-01 false Atlantic Ocean off Wallops Island and Chincoteague Inlet, Va.; danger zone. 334.130 Section 334.130 Navigation and Navigable Waters... REGULATIONS § 334.130 Atlantic Ocean off Wallops Island and Chincoteague Inlet, Va.; danger zone. (a) The...

  19. 77 FR 39404 - Safety Zone; Wicomico Community Fireworks Rain Date, Great Wicomico River, Mila, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-03

    ... SECURITY Coast Guard 33 CFR Part 165 RIN 1625-AA00 Safety Zone; Wicomico Community Fireworks Rain Date..., VA in support of the Wicomico Community Rain Date Fireworks event. This action is necessary to.... 165.T05-0425 Safety Zone; Wicomico Community Fireworks Rain Date, Great Wicomico River, Mila, VA....

  20. Making Bullying Prevention a Priority in Finnish Schools: The KiVa Antibullying Program

    ERIC Educational Resources Information Center

    Salmivalli, Christina; Poskiparta, Elisa

    2012-01-01

    The KiVa antibullying program has been widely implemented in Finnish comprehensive schools since 2009. The program is predicated on the idea that a positive change in the behaviors of classmates can reduce the rewards gained by the perpetrators of bullying and consequently their motivation to bully in the first place. KiVa involves both universal…

  1. 77 FR 27120 - Safety Zone; Virginia Beach Oceanfront Air Show, Atlantic Ocean, Virginia Beach, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-09

    ..., Virginia Beach, VA in the Federal Register (76 FR 13519). We received one comment on the proposed rule. No... SECURITY Coast Guard 33 CFR Part 165 RIN 1625-AA00 Safety Zone; Virginia Beach Oceanfront Air Show, Atlantic Ocean, Virginia Beach, VA AGENCY: Coast Guard, DHS. ACTION: Temporary final rule. SUMMARY:...

  2. 78 FR 56151 - Safety Zone, North Atlantic Ocean; Virginia Beach, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-12

    ... Acronyms DHS Department of Homeland Security FR Federal Register NPRM Notice of Proposed Rulemaking A... SECURITY Coast Guard 33 CFR Part 165 RIN 1625-AA00 Safety Zone, North Atlantic Ocean; Virginia Beach, VA... zone on the navigable waters of the North Atlantic Ocean in Virginia Beach, VA to support the...

  3. 78 FR 31840 - Safety Zone; USO Patriotic Festival Air Show, Atlantic Ocean; Virginia Beach, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-28

    ...-9826. SUPPLEMENTARY INFORMATION: Table of Acronyms DHS Department of Homeland Security FR Federal... Ocean; Virginia Beach, VA AGENCY: Coast Guard, DHS. ACTION: Temporary final rule. SUMMARY: The Coast... Beach, VA. This action is necessary to provide for the safety of life on navigable waters during the...

  4. 33 CFR 334.220 - Chesapeake Bay, south of Tangier Island, Va.; naval firing range.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Island, Va.; naval firing range. 334.220 Section 334.220 Navigation and Navigable Waters CORPS OF....220 Chesapeake Bay, south of Tangier Island, Va.; naval firing range. (a) The danger zone. Beginning... especially notified to the contrary. (2) All vessels, other than naval craft, are forbidden to anchor...

  5. 33 CFR 334.220 - Chesapeake Bay, south of Tangier Island, Va.; naval firing range.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Island, Va.; naval firing range. 334.220 Section 334.220 Navigation and Navigable Waters CORPS OF....220 Chesapeake Bay, south of Tangier Island, Va.; naval firing range. (a) The danger zone. Beginning... especially notified to the contrary. (2) All vessels, other than naval craft, are forbidden to anchor...

  6. 33 CFR 334.290 - Elizabeth River, Southern Branch, Va., naval restricted areas.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ..., Va., naval restricted areas. 334.290 Section 334.290 Navigation and Navigable Waters CORPS OF....290 Elizabeth River, Southern Branch, Va., naval restricted areas. (a) The areas—(1) St. Helena Annex Area. Beginning at a point at St. Helena Annex of the Norfolk Naval Shipyard, on the eastern shore...

  7. 33 CFR 334.290 - Elizabeth River, Southern Branch, Va., naval restricted areas.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ..., Va., naval restricted areas. 334.290 Section 334.290 Navigation and Navigable Waters CORPS OF....290 Elizabeth River, Southern Branch, Va., naval restricted areas. (a) The areas—(1) St. Helena Annex Area. Beginning at a point at St. Helena Annex of the Norfolk Naval Shipyard, on the eastern shore...

  8. 38 CFR 21.9510 - Claims, VA's duty to assist, and time limits.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2012-07-01 2012-07-01 false Claims, VA's duty to assist, and time limits. 21.9510 Section 21.9510 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) VOCATIONAL REHABILITATION AND EDUCATION Post-9/11 GI Bill Claims and Applications § 21.9510 Claims, VA's duty to...

  9. 76 FR 34248 - Equestrian Stables at Meadowood Special Recreation Management Area, VA; Information Sharing Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-13

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF THE INTERIOR Equestrian Stables at Meadowood Special Recreation Management Area, VA; Information Sharing Meeting AGENCY... equestrian stables at Meadowood Special Recreation Management Area (SRMA), located in Lorton, VA, and...

  10. 48 CFR 853.236-70 - VA Form 10-6298, Architect-Engineer Fee Proposal.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false VA Form 10-6298, Architect-Engineer Fee Proposal. 853.236-70 Section 853.236-70 Federal Acquisition Regulations System DEPARTMENT OF...-Engineer Fee Proposal. VA Form 10-6298, Architect-Engineer Fee Proposal, shall be used as prescribed in...

  11. 48 CFR 853.236-70 - VA Form 10-6298, Architect-Engineer Fee Proposal.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false VA Form 10-6298, Architect-Engineer Fee Proposal. 853.236-70 Section 853.236-70 Federal Acquisition Regulations System DEPARTMENT OF...-Engineer Fee Proposal. VA Form 10-6298, Architect-Engineer Fee Proposal, shall be used as prescribed in...

  12. 30 CFR 57.22605 - Blasting from the surface (V-A mines).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... applicable requirements of 30 CFR parts 18 through 36. (e) This standard applies only to mines blasting... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Blasting from the surface (V-A mines). 57.22605... Standards for Methane in Metal and Nonmetal Mines Explosives § 57.22605 Blasting from the surface (V-A...

  13. 78 FR 49920 - Drawbridge Operation Regulation; Elizabeth River, Eastern Branch, Norfolk, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-16

    ... Norfolk Southern 5 Railroad Bridge, across the Elizabeth River Eastern Branch, mile 1.1, at Norfolk, VA... operating schedule, the Norfolk Southern 5 Railroad Bridge, mile 1.1, in Norfolk, VA, the draw must open... maintenance. The Norfolk Southern 5 railroad Bridge, at mile 1.1, across the Elizabeth River (Eastern...

  14. 77 FR 3841 - Proposed Information Collection (Survey of Veteran Enrollees (Quality and Efficiency of VA Health...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-25

    ... Care)) Activities Under OMB Review AGENCY: Veterans Health Administration, Department of Veterans... VA Health Care), VA Form 10-21088. OMB Control Number: 2900-0725. Type of Review: Extension of a... promote quality and efficient delivery of health care through the use of health information...

  15. 78 FR 77204 - Proposed Information Collection (VA National Veterans Sports Programs and Special Event Surveys...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-20

    ... AFFAIRS Proposed Information Collection (VA National Veterans Sports Programs and Special Event Surveys... solicits comments on the information needed to evaluate the National Veterans Sports Programs and Special... ``OMB Control No. 2900-NEW (VA National Veterans Sports Programs and Special Event Surveys)'' in...

  16. 33 CFR 80.515 - Cape Henry, VA to Cape Hatteras, NC.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Cape Henry, VA to Cape Hatteras, NC. 80.515 Section 80.515 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Fifth District § 80.515 Cape Henry, VA...

  17. 77 FR 56709 - Proposed Information Collection (VA Subcontracting Report for Service Disabled Veteran-Owned...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-13

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF VETERANS AFFAIRS Proposed Information Collection (VA Subcontracting Report for Service Disabled Veteran-Owned Small... for Service Disabled Veteran-Owned Small Business and Veteran-Owned Small Business Concerns, VA...

  18. 77 FR 70210 - Agency Information Collection (VA Subcontracting Report for Service Disabled Veteran-owned Small...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-23

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF VETERANS AFFAIRS Agency Information Collection (VA Subcontracting Report for Service Disabled Veteran-owned Small....'' SUPPLEMENTARY INFORMATION: Title: VA Subcontracting Report for Service Disabled Veteran-owned Small Business...

  19. 77 FR 30050 - VA National Academic Affiliations Council, Notice of meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-21

    ... AFFAIRS VA National Academic Affiliations Council, Notice of meeting The Department of Veterans Affairs... the National Academic Affiliations Council will be held on June 5-6, 2012, in Suite 878 at 1800 G... affecting partnerships between VA and its academic affiliates. On June 5, the Council will review the...

  20. Evolution of MHC class I genes in the endangered loggerhead sea turtle (Caretta caretta) revealed by 454 amplicon sequencing

    PubMed Central

    2013-01-01

    Background In evolutionary and conservation biology, parasitism is often highlighted as a major selective pressure. To fight against parasites and pathogens, genetic diversity of the immune genes of the major histocompatibility complex (MHC) are particularly important. However, the extensive degree of polymorphism observed in these genes makes it difficult to conduct thorough population screenings. Methods We utilized a genotyping protocol that uses 454 amplicon sequencing to characterize the MHC class I in the endangered loggerhead sea turtle (Caretta caretta) and to investigate their evolution at multiple relevant levels of organization. Results MHC class I genes revealed signatures of trans-species polymorphism across several reptile species. In the studied loggerhead turtle individuals, it results in the maintenance of two ancient allelic lineages. We also found that individuals carrying an intermediate number of MHC class I alleles are larger than those with either a low or high number of alleles. Conclusions Multiple modes of evolution seem to maintain MHC diversity in the loggerhead turtles, with relatively high polymorphism for an endangered species. PMID:23627726

  1. Amplicon-Based Pyrosequencing Reveals High Diversity of Protistan Parasites in Ships' Ballast Water: Implications for Biogeography and Infectious Diseases.

    PubMed

    Pagenkopp Lohan, K M; Fleischer, R C; Carney, K J; Holzer, K K; Ruiz, G M

    2016-04-01

    Ships' ballast water (BW) commonly moves macroorganisms and microorganisms across the world's oceans and along coasts; however, the majority of these microbial transfers have gone undetected. We applied high-throughput sequencing methods to identify microbial eukaryotes, specifically emphasizing the protistan parasites, in ships' BW collected from vessels calling to the Chesapeake Bay (Virginia and Maryland, USA) from European and Eastern Canadian ports. We utilized tagged-amplicon 454 pyrosequencing with two general primer sets, amplifying either the V4 or V9 domain of the small subunit (SSU) of the ribosomal RNA (rRNA) gene complex, from total DNA extracted from water samples collected from the ballast tanks of bulk cargo vessels. We detected a diverse group of protistan taxa, with some known to contain important parasites in marine systems, including Apicomplexa (unidentified apicomplexans, unidentified gregarines, Cryptosporidium spp.), Dinophyta (Blastodinium spp., Euduboscquella sp., unidentified syndinids, Karlodinium spp., Syndinium spp.), Perkinsea (Parvilucifera sp.), Opisthokonta (Ichthyosporea sp., Pseudoperkinsidae, unidentified ichthyosporeans), and Stramenopiles (Labyrinthulomycetes). Further characterization of groups with parasitic taxa, consisting of phylogenetic analyses for four taxa (Cryptosporidium spp., Parvilucifera spp., Labyrinthulomycetes, and Ichthyosporea), revealed that sequences were obtained from both known and novel lineages. This study demonstrates that high-throughput sequencing is a viable and sensitive method for detecting parasitic protists when present and transported in the ballast water of ships. These data also underscore the potential importance of human-aided dispersal in the biogeography of these microbes and emerging diseases in the world's oceans. PMID:26476551

  2. Fungi Sailing the Arctic Ocean: Speciose Communities in North Atlantic Driftwood as Revealed by High-Throughput Amplicon Sequencing.

    PubMed

    Rämä, Teppo; Davey, Marie L; Nordén, Jenni; Halvorsen, Rune; Blaalid, Rakel; Mathiassen, Geir H; Alsos, Inger G; Kauserud, Håvard

    2016-08-01

    High amounts of driftwood sail across the oceans and provide habitat for organisms tolerating the rough and saline environment. Fungi have adapted to the extremely cold and saline conditions which driftwood faces in the high north. For the first time, we applied high-throughput sequencing to fungi residing in driftwood to reveal their taxonomic richness, community composition, and ecology in the North Atlantic. Using pyrosequencing of ITS2 amplicons obtained from 49 marine logs, we found 807 fungal operational taxonomic units (OTUs) based on clustering at 97 % sequence similarity cut-off level. The phylum Ascomycota comprised 74 % of the OTUs and 20 % belonged to Basidiomycota. The richness of basidiomycetes decreased with prolonged submersion in the sea, supporting the general view of ascomycetes being more extremotolerant. However, more than one fourth of the fungal OTUs remained unassigned to any fungal class, emphasising the need for better DNA reference data from the marine habitat. Different fungal communities were detected in coniferous and deciduous logs. Our results highlight that driftwood hosts a considerably higher fungal diversity than currently known. The driftwood fungal community is not a terrestrial relic but a speciose assemblage of fungi adapted to the stressful marine environment and different kinds of wooden substrates found in it. PMID:27147245

  3. Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine.

    PubMed

    Chaitanya, Lakshmi; Ralf, Arwin; van Oven, Mannis; Kupiec, Tomasz; Chang, Joseph; Lagacé, Robert; Kayser, Manfred

    2015-12-01

    Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long-range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications. PMID:26387877

  4. Factors That Affect Large Subunit Ribosomal DNA Amplicon Sequencing Studies of Fungal Communities: Classification Method, Primer Choice, and Error

    PubMed Central

    Porter, Teresita M.; Golding, G. Brian

    2012-01-01

    Nuclear large subunit ribosomal DNA is widely used in fungal phylogenetics and to an increasing extent also amplicon-based environmental sequencing. The relatively short reads produced by next-generation sequencing, however, makes primer choice and sequence error important variables for obtaining accurate taxonomic classifications. In this simulation study we tested the performance of three classification methods: 1) a similarity-based method (BLAST + Metagenomic Analyzer, MEGAN); 2) a composition-based method (Ribosomal Database Project naïve Bayesian classifier, NBC); and, 3) a phylogeny-based method (Statistical Assignment Package, SAP). We also tested the effects of sequence length, primer choice, and sequence error on classification accuracy and perceived community composition. Using a leave-one-out cross validation approach, results for classifications to the genus rank were as follows: BLAST + MEGAN had the lowest error rate and was particularly robust to sequence error; SAP accuracy was highest when long LSU query sequences were classified; and, NBC runs significantly faster than the other tested methods. All methods performed poorly with the shortest 50–100 bp sequences. Increasing simulated sequence error reduced classification accuracy. Community shifts were detected due to sequence error and primer selection even though there was no change in the underlying community composition. Short read datasets from individual primers, as well as pooled datasets, appear to only approximate the true community composition. We hope this work informs investigators of some of the factors that affect the quality and interpretation of their environmental gene surveys. PMID:22558215

  5. Potential applications of next generation DNA sequencing of 16S rRNA gene amplicons in microbial water quality monitoring.

    PubMed

    Vierheilig, J; Savio, D; Ley, R E; Mach, R L; Farnleitner, A H; Reischer, G H

    2015-01-01

    The applicability of next generation DNA sequencing (NGS) methods for water quality assessment has so far not been broadly investigated. This study set out to evaluate the potential of an NGS-based approach in a complex catchment with importance for drinking water abstraction. In this multi-compartment investigation, total bacterial communities in water, faeces, soil, and sediment samples were investigated by 454 pyrosequencing of bacterial 16S rRNA gene amplicons to assess the capabilities of this NGS method for (i) the development and evaluation of environmental molecular diagnostics, (ii) direct screening of the bulk bacterial communities, and (iii) the detection of faecal pollution in water. Results indicate that NGS methods can highlight potential target populations for diagnostics and will prove useful for the evaluation of existing and the development of novel DNA-based detection methods in the field of water microbiology. The used approach allowed unveiling of dominant bacterial populations but failed to detect populations with low abundances such as faecal indicators in surface waters. In combination with metadata, NGS data will also allow the identification of drivers of bacterial community composition during water treatment and distribution, highlighting the power of this approach for monitoring of bacterial regrowth and contamination in technical systems. PMID:26606090

  6. Analysis of black fungal biofilms occurring at domestic water taps. I: compositional analysis using Tag-Encoded FLX Amplicon Pyrosequencing.

    PubMed

    Heinrichs, Guido; Hübner, Iris; Schmidt, Carsten K; de Hoog, G Sybren; Haase, Gerhard

    2013-06-01

    Mass growth of dark fungal biofilms on water taps and associated habitats was observed in various German drinking water distribution systems recently. Customers of affected drinking water systems are anxious about potential and unknown health risks. These environments are known to harbour a fungal flora also comprising a variety of fungal opportunists that are well known to cause superficial mycoses in humans (Exophiala equina, Exophiala lecanii-corni) but are not known to establish dark biofilms so far. To gain profound insight on composition of respective biofilms, a metagenomic approach using Tag-Encoded FLX Amplicon Pyrosequencing (TEFAP) of the ribosomal internal transcribed spacer 2 region in comparison with a classical cultivation approach using Sabouraud agar with chloramphenicol and erythritol-chloramphenicol-agar was performed. E. lecanii-corni was found to be the major component in 10 of 13 biofilms analysed independently of the method used. Alternaria sp., E. equina, Fusarium spp. and Ochroconis spp. were also relatively abundant. As expected, TEFAP usually revealed a higher diversity than the cultivation approaches. For example, opportunistic species like Candida albicans or Exophiala dermatitidis were detected in very low amounts. In conclusion, TEFAP turned out to be a promising and powerful tool for the semi-quantitative analysis of fungal biofilms. Referring to relevant literature, potential biological hazards caused by fungi of the dark biofilms can be regarded as low. PMID:23385952

  7. Empirical analysis on future-cash arbitrage risk with portfolio VaR

    NASA Astrophysics Data System (ADS)

    Chen, Rongda; Li, Cong; Wang, Weijin; Wang, Ze

    2014-03-01

    This paper constructs the positive arbitrage position by alternating the spot index with Chinese Exchange Traded Fund (ETF) portfolio and estimating the arbitrage-free interval of futures with the latest trade data. Then, an improved Delta-normal method was used, which replaces the simple linear correlation coefficient with tail dependence correlation coefficient, to measure VaR (Value-at-risk) of the arbitrage position. Analysis of VaR implies that the risk of future-cash arbitrage is less than that of investing completely in either futures or spot market. Then according to the compositional VaR and the marginal VaR, we should increase the futures position and decrease the spot position appropriately to minimize the VaR, which can minimize risk subject to certain revenues.

  8. VA Health Professional Scholarship and Visual Impairment and Orientation and Mobility Professional Scholarship Programs. Final rule.

    PubMed

    2013-08-20

    The Department of Veterans Affairs (VA) is amending its VA Health Professional Scholarship Program (HPSP) regulations. VA is also establishing regulations for a new program, the Visual Impairment and Orientation and Mobility Professional Scholarship Program (VIOMPSP). These regulations comply with and implement sections 302 and 603 of the Caregivers and Veterans Omnibus Health Services Act of 2010 (the 2010 Act). Section 302 of the 2010 Act established the VIOMPSP, which authorizes VA to provide financial assistance to certain students seeking a degree in visual impairment or orientation or mobility, in order to increase the supply of qualified blind rehabilitation specialists for VA and the United States. Section 603 of the 2010 Act reauthorized and modified HPSP, a program that provides scholarships for education or training in certain health care occupations. PMID:23977714

  9. Identifying Homelessness among Veterans Using VA Administrative Data: Opportunities to Expand Detection Criteria

    PubMed Central

    Peterson, Rachel; Gundlapalli, Adi V.; Metraux, Stephen; Carter, Marjorie E.; Palmer, Miland; Redd, Andrew; Samore, Matthew H.; Fargo, Jamison D.

    2015-01-01

    Researchers at the U.S. Department of Veterans Affairs (VA) have used administrative criteria to identify homelessness among U.S. Veterans. Our objective was to explore the use of these codes in VA health care facilities. We examined VA health records (2002-2012) of Veterans recently separated from the military and identified as homeless using VA conventional identification criteria (ICD-9-CM code V60.0, VA specific codes for homeless services), plus closely allied V60 codes indicating housing instability. Logistic regression analyses examined differences between Veterans who received these codes. Health care services and co-morbidities were analyzed in the 90 days post-identification of homelessness. VA conventional criteria identified 21,021 homeless Veterans from Operations Enduring Freedom, Iraqi Freedom, and New Dawn (rate 2.5%). Adding allied V60 codes increased that to 31,260 (rate 3.3%). While certain demographic differences were noted, Veterans identified as homeless using conventional or allied codes were similar with regards to utilization of homeless, mental health, and substance abuse services, as well as co-morbidities. Differences were noted in the pattern of usage of homelessness-related diagnostic codes in VA facilities nation-wide. Creating an official VA case definition for homelessness, which would include additional ICD-9-CM and other administrative codes for VA homeless services, would likely allow improved identification of homeless and at-risk Veterans. This also presents an opportunity for encouraging uniformity in applying these codes in VA facilities nationwide as well as in other large health care organizations. PMID:26172386

  10. Investigation of Microbial Diversity in Geothermal Hot Springs in Unkeshwar, India, Based on 16S rRNA Amplicon Metagenome Sequencing.

    PubMed

    Mehetre, Gajanan T; Paranjpe, Aditi; Dastager, Syed G; Dharne, Mahesh S

    2016-01-01

    Microbial diversity in geothermal waters of the Unkeshwar hot springs in Maharashtra, India, was studied using 16S rRNA amplicon metagenomic sequencing. Taxonomic analysis revealed the presence of Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, Archeae, and OD1 phyla. Metabolic function prediction analysis indicated a battery of biological information systems indicating rich and novel microbial diversity, with potential biotechnological applications in this niche. PMID:26950332

  11. Using surface-enhanced Raman spectroscopy and electrochemically driven melting to discriminate Yersinia pestis from Y. pseudotuberculosis based on single nucleotide polymorphisms within unpurified polymerase chain reaction amplicons.

    PubMed

    Papadopoulou, Evanthia; Goodchild, Sarah A; Cleary, David W; Weller, Simon A; Gale, Nittaya; Stubberfield, Michael R; Brown, Tom; Bartlett, Philip N

    2015-02-01

    The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples. PMID:25551670

  12. Mapping and characterization of the amplicon near APOA2 in 1q23 in human sarcomas by FISH and array CGH

    PubMed Central

    Kresse, Stine H; Berner, Jeanne-Marie; Meza-Zepeda, Leonardo A; Gregory, Simon G; Kuo, Wen-Lin; Gray, Joe W; Forus, Anne; Myklebost, Ola

    2005-01-01

    Background Amplification of the q21-q23 region on chromosome 1 is frequently found in sarcomas and a variety of other solid tumours. Previous analyses of sarcomas have indicated the presence of at least two separate amplicons within this region, one located in 1q21 and one located near the apolipoprotein A-II (APOA2) gene in 1q23. In this study we have mapped and characterized the amplicon in 1q23 in more detail. Results We have used fluorescence in situ hybridisation (FISH) and microarray-based comparative genomic hybridisation (array CGH) to map and define the borders of the amplicon in 10 sarcomas. A subregion of approximately 800 kb was identified as the core of the amplicon. The amplification patterns of nine possible candidate target genes located to this subregion were determined by Southern blot analysis. The genes activating transcription factor 6 (ATF6) and dual specificity phosphatase 12 (DUSP12) showed the highest level of amplification, and they were also shown to be over-expressed by quantitative real-time reverse transcription PCR (RT-PCR). In general, the level of expression reflected the level of amplification in the different tumours. DUSP12 was expressed significantly higher than ATF6 in a subset of the tumours. In addition, two genes known to be transcriptionally activated by ATF6, glucose-regulated protein 78 kDa and -94 kDa (GRP78 and GRP94), were shown to be over-expressed in the tumours that showed over-expression of ATF6. Conclusion ATF6 and DUSP12 seem to be the most likely candidate target genes for the 1q23 amplification in sarcomas. Both genes have possible roles in promoting cell growth, which makes them interesting candidate targets. PMID:16274472

  13. Development of a control region-based mtDNA SNaPshot™ selection tool, integrated into a mini amplicon sequencing method.

    PubMed

    Weiler, Natalie E C; de Vries, Gerda; Sijen, Titia

    2016-03-01

    Mitochondrial DNA (mtDNA) analysis is regularly applied to forensic DNA samples with limited amounts of nuclear DNA (nDNA), such as hair shafts and bones. Generally, this mtDNA analysis involves examination of the hypervariable control region by Sanger sequencing of amplified products. When samples are severely degraded, small-sized amplicons can be applied and an earlier described mini-mtDNA method by Eichmann et al. [1] that accommodates ten mini amplicons in two multiplexes is found to be a very robust approach. However, in cases with large numbers of samples, like when searching for hairs with an mtDNA profile deviant from that of the victim, the method is time (and cost) consuming. Previously, Chemale et al. [2] described a SNaPshot™-based screening tool for a Brazilian population that uses standard-size amplicons for HVS-I and HVS-II. Here, we describe a similar tool adapted to the full control region and compatible with mini-mtDNA amplicons. Eighteen single nucleotide polymorphisms (SNPs) were selected based on their relative frequencies in a European population. They showed a high discriminatory power in a Dutch population (97.2%). The 18 SNPs are assessed in two SNaPshot™ multiplexes that pair to the two mini-mtDNA amplification multiplexes. Degenerate bases are included to limit allele dropout due to SNPs at primer binding site positions. Three SNPs provide haplogroup information. Reliability testing showed no differences with Sanger sequencing results. Since mini-mtSNaPshot screening uses only a small portion of the same PCR products used for Sanger sequencing, no additional DNA extract is consumed, which is forensically advantageous. PMID:26976467

  14. Combined Amplicon Pyrosequencing Assays Reveal Presence of the Apicomplexan “type-N” (cf. Gemmocystis cylindrus) and Chromera velia on the Great Barrier Reef, Australia

    PubMed Central

    Šlapeta, Jan; Linares, Marjorie C.

    2013-01-01

    Background The coral is predominantly composed of the metabolically dependent coral host and the photosynthetic dinoflagellate Symbiodinium sp. The system as a whole interacts with symbiotic eukaryotes, bacteria and viruses. Gemmocystiscylindrus (cf. “type-N” symbiont) belonging to the obligatory parasitic phylum Apicomplexa (Alveolata) is ubiquitous in the Caribbean coral, but its presence in the Great Barrier Reef coral has yet to be documented. Approaches allowing identification of the healthy community from the pathogenic or saprobic organisms are needed for sustainable coral reef monitoring. Methods & Principal Findings We investigated the diversity of eukaryotes associated with a common reef-building corals from the southern Great Barrier Reef. We used three tag encoded 454 amplicon pyrosequencing assays targeting eukaryote small-subunit rRNA gene to demonstrate the presence of the apicomplexan type-N and a photosynthetic sister species to Apicomplexa - Chromeravelia. Amplicon pyrosequencing revealed presence of the small-subunit rRNA genes of known eukaryotic pathogens (Cryptosporidium and Cryptococcus). We therefore conducted bacterial tag encoded amplicon pyrosequencing assay for small-subunit rRNA gene to support effluent exposure of the coral. Bacteria of faecal origin (Enterobacteriales) formed 41% of total sequences in contrast to 0-2% of the coral-associated bacterial communities with and without C. velia, respectively. Significance This is the first time apicomplexan type-N has been detected in the Great Barrier Reef. Eukaryote tag encoded amplicon pyrosequencing assays demonstrate presence of apicomplexan type-N and C. Velia in total coral DNA. The data highlight the need for combined approaches for eukaryotic diversity studies coupled with bacterial community assessment to achieve a more realistic goals of defining the holobiont community and assessing coral disease. With increasing evidence of Apicomplexa in coral reef environments, it is

  15. Investigation of Microbial Diversity in Geothermal Hot Springs in Unkeshwar, India, Based on 16S rRNA Amplicon Metagenome Sequencing

    PubMed Central

    Mehetre, Gajanan T.; Paranjpe, Aditi; Dastager, Syed G.

    2016-01-01

    Microbial diversity in geothermal waters of the Unkeshwar hot springs in Maharashtra, India, was studied using 16S rRNA amplicon metagenomic sequencing. Taxonomic analysis revealed the presence of Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, Archeae, and OD1 phyla. Metabolic function prediction analysis indicated a battery of biological information systems indicating rich and novel microbial diversity, with potential biotechnological applications in this niche. PMID:26950332

  16. Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers: Comparison with Sanger sequencing.

    PubMed

    Yamamoto, F; Höglund, B; Fernandez-Vina, M; Tyan, D; Rastrou, M; Williams, T; Moonsamy, P; Goodridge, D; Anderson, M; Erlich, H A; Holcomb, C L

    2015-12-01

    Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType HLA genotyping assay to provide very high resolution (VHR) typing, by the addition of 8 primer pairs to the original 14, to genotype 11 HLA loci. These additional amplicons help resolve common and well-documented alleles and exclude commonly found null alleles in genotype ambiguity strings. Simplification of workflow to reduce the initial preparation effort using early pooling of amplicons or the Fluidigm Access Array™ is also described. Performance of the VHR assay was evaluated on 28 well characterized cell lines using Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance was 98.4%; 1.6% had no genotype assignment. Of concordant calls, 53% were unambiguous. To further assess the assay, 59 clinical samples were genotyped and results compared to unambiguous allele assignments obtained by prior sequence-based typing supplemented with SSO and/or SSP. Concordance was 98.7% with 58.2% as unambiguous calls; 1.3% could not be assigned. Our results show that the amplicon-based VHR assay is robust and can replace current Sanger methodology. Together with software enhancements, it has the potential to provide even higher resolution HLA typing. PMID:26037172

  17. Characteristics and VA Health Care Utilization of U.S. Veterans Who Completed Suicide in Oregon between 2000 and 2005

    ERIC Educational Resources Information Center

    Basham, Chandra; Denneson, Lauren M.; Millet, Lisa; Shen, Xun; Duckart, Jonathan; Dobscha, Steven K.

    2011-01-01

    Oregon Violent Death Reporting System data were linked with Veterans Affairs (VA) administrative data to identify and describe veterans who completed suicide in Oregon from 2000 to 2005 (n = 968), and to describe their VA health care utilization in the year prior to death. Twenty-two percent had received health care in the VA system. Of these, 57%…

  18. 33 CFR 334.340 - Chesapeake Bay off Plumtree Island, Hampton, Va.; Air Force precision test area.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Island, Hampton, Va.; Air Force precision test area. 334.340 Section 334.340 Navigation and Navigable... REGULATIONS § 334.340 Chesapeake Bay off Plumtree Island, Hampton, Va.; Air Force precision test area. (a) The... Command, Langley Air Force Base, Va., shall be responsible for publicizing in advance through the...

  19. 33 CFR 334.340 - Chesapeake Bay off Plumtree Island, Hampton, Va.; Air Force precision test area.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Island, Hampton, Va.; Air Force precision test area. 334.340 Section 334.340 Navigation and Navigable... REGULATIONS § 334.340 Chesapeake Bay off Plumtree Island, Hampton, Va.; Air Force precision test area. (a) The... Command, Langley Air Force Base, Va., shall be responsible for publicizing in advance through the...

  20. 33 CFR 334.340 - Chesapeake Bay off Plumtree Island, Hampton, Va.; Air Force precision test area.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Island, Hampton, Va.; Air Force precision test area. 334.340 Section 334.340 Navigation and Navigable... REGULATIONS § 334.340 Chesapeake Bay off Plumtree Island, Hampton, Va.; Air Force precision test area. (a) The... Command, Langley Air Force Base, Va., shall be responsible for publicizing in advance through the...

  1. 33 CFR 334.340 - Chesapeake Bay off Plumtree Island, Hampton, Va.; Air Force precision test area.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Island, Hampton, Va.; Air Force precision test area. 334.340 Section 334.340 Navigation and Navigable... REGULATIONS § 334.340 Chesapeake Bay off Plumtree Island, Hampton, Va.; Air Force precision test area. (a) The... Command, Langley Air Force Base, Va., shall be responsible for publicizing in advance through the...

  2. 33 CFR 334.340 - Chesapeake Bay off Plumtree Island, Hampton, Va.; Air Force precision test area.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Island, Hampton, Va.; Air Force precision test area. 334.340 Section 334.340 Navigation and Navigable... REGULATIONS § 334.340 Chesapeake Bay off Plumtree Island, Hampton, Va.; Air Force precision test area. (a) The... Command, Langley Air Force Base, Va., shall be responsible for publicizing in advance through the...

  3. Flexural Stiffness of Myosin Va Subdomains as Measured from Tethered Particle Motion.

    PubMed

    Michalek, Arthur J; Kennedy, Guy G; Warshaw, David M; Ali, M Yusuf

    2015-01-01

    Myosin Va (MyoVa) is a processive molecular motor involved in intracellular cargo transport on the actin cytoskeleton. The motor's processivity and ability to navigate actin intersections are believed to be governed by the stiffness of various parts of the motor's structure. Specifically, changes in calcium may regulate motor processivity by altering the motor's lever arm stiffness and thus its interhead communication. In order to measure the flexural stiffness of MyoVa subdomains, we use tethered particle microscopy, which relates the Brownian motion of fluorescent quantum dots, which are attached to various single- and double-headed MyoVa constructs bound to actin in rigor, to the motor's flexural stiffness. Based on these measurements, the MyoVa lever arm and coiled-coil rod domain have comparable flexural stiffness (0.034 pN/nm). Upon addition of calcium, the lever arm stiffness is reduced 40% as a result of calmodulins potentially dissociating from the lever arm. In addition, the flexural stiffness of the full-length MyoVa construct is an order of magnitude less stiff than both a single lever arm and the coiled-coil rod. This suggests that the MyoVa lever arm-rod junction provides a flexible hinge that would allow the motor to maneuver cargo through the complex intracellular actin network. PMID:26770194

  4. Homeless and nonhomeless VA service users likely eligible for Medicaid expansion.

    PubMed

    Tsai, Jack; Rosenheck, Robert A

    2014-01-01

    Administrative data on the population of Department of Veterans Affairs (VA) service users in 2010 under the age of 65 (n = 3,841,225) were analyzed to identify the number and characteristics of homeless and nonhomeless VA service users who are likely to be eligible for the Medicaid expansion (LEME) option under the Affordable Care Act. Results showed that, estimating conservatively, about 1.2 million (21%) current VA users are LEME if all states implement the expansion. Homeless service users were twice as likely to be eligible than nonhomeless users (64% vs 30%). VA service users who are LEME, regardless of housing status, were physically healthier than those not LEME but were more likely to have substance use disorders and posttraumatic stress disorder. These findings suggest that many VA service users are LEME, particularly those who are homeless and/or have mental health needs. Cross-system use of VA and Medicaid-funded services may be advantageous for veterans with extensive medical and psychiatric needs but also risks fragmented care. Information and education for VA clinicians and their patients about possible implications of the Affordable Care Act may be important. PMID:25358031

  5. Flexural Stiffness of Myosin Va Subdomains as Measured from Tethered Particle Motion

    PubMed Central

    Michalek, Arthur J.; Kennedy, Guy G.; Warshaw, David M.; Ali, M. Yusuf

    2015-01-01

    Myosin Va (MyoVa) is a processive molecular motor involved in intracellular cargo transport on the actin cytoskeleton. The motor's processivity and ability to navigate actin intersections are believed to be governed by the stiffness of various parts of the motor's structure. Specifically, changes in calcium may regulate motor processivity by altering the motor's lever arm stiffness and thus its interhead communication. In order to measure the flexural stiffness of MyoVa subdomains, we use tethered particle microscopy, which relates the Brownian motion of fluorescent quantum dots, which are attached to various single- and double-headed MyoVa constructs bound to actin in rigor, to the motor's flexural stiffness. Based on these measurements, the MyoVa lever arm and coiled-coil rod domain have comparable flexural stiffness (0.034 pN/nm). Upon addition of calcium, the lever arm stiffness is reduced 40% as a result of calmodulins potentially dissociating from the lever arm. In addition, the flexural stiffness of the full-length MyoVa construct is an order of magnitude less stiff than both a single lever arm and the coiled-coil rod. This suggests that the MyoVa lever arm-rod junction provides a flexible hinge that would allow the motor to maneuver cargo through the complex intracellular actin network. PMID:26770194

  6. Identification and characterization of multiple novel Rab–myosin Va interactions

    PubMed Central

    Lindsay, Andrew J.; Jollivet, Florence; Horgan, Conor P.; Khan, Amir R.; Raposo, Graça; McCaffrey, Mary W.; Goud, Bruno

    2013-01-01

    Myosin Va is a widely expressed actin-based motor protein that binds members of the Rab GTPase family (3A, 8A, 10, 11A, 27A) and is implicated in many intracellular trafficking processes. To our knowledge, myosin Va has not been tested in a systematic screen for interactions with the entire Rab GTPase family. To that end, we report a yeast two-hybrid screen of all human Rabs for myosin Va-binding ability and reveal 10 novel interactions (3B, 3C, 3D, 6A, 6A′, 6B, 11B, 14, 25, 39B), which include interactions with three new Rab subfamilies (Rab6, Rab14, Rab39B). Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems. We demonstrate that myosin Va has three distinct Rab-binding domains on disparate regions of the motor (central stalk, an alternatively spliced exon, and the globular tail). Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes. We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes. PMID:24006491

  7. Uncultured bacterial diversity in a seawater recirculating aquaculture system revealed by 16S rRNA gene amplicon sequencing.

    PubMed

    Lee, Da-Eun; Lee, Jinhwan; Kim, Young-Mog; Myeong, Jeong-In; Kim, Kyoung-Ho

    2016-04-01

    Bacterial diversity in a seawater recirculating aquaculture system (RAS) was investigated using 16S rRNA amplicon sequencing to understand the roles of bacterial communities in the system. The RAS was operated at nine different combinations of temperature (15°C, 20°C, and 25°C) and salinity (20‰, 25‰, and 32.5‰). Samples were collected from five or six RAS tanks (biofilters) for each condition. Fifty samples were analyzed. Proteobacteria and Bacteroidetes were most common (sum of both phyla: 67.2% to 99.4%) and were inversely proportional to each other. Bacteria that were present at an average of ≥ 1% included Actinobacteria (2.9%) Planctomycetes (2.0%), Nitrospirae (1.5%), and Acidobacteria (1.0%); they were preferentially present in packed bed biofilters, mesh biofilters, and maturation biofilters. The three biofilters showed higher diversity than other RAS tanks (aerated biofilters, floating bed biofilters, and fish tanks) from phylum to operational taxonomic unit (OTU) level. Samples were clustered into several groups based on the bacterial communities. Major taxonomic groups related to family Rhodobacteraceae and Flavobacteriaceae were distributed widely in the samples. Several taxonomic groups like [Saprospiraceae], Cytophagaceae, Octadecabacter, and Marivita showed a cluster-oriented distribution. Phaeobacter and Sediminicola-related reads were detected frequently and abundantly at low temperature. Nitrifying bacteria were detected frequently and abundantly in the three biofilters. Phylogenetic analysis of the nitrifying bacteria showed several similar OTUs were observed widely through the biofilters. The diverse bacterial communities and the minor taxonomic groups, except for Proteobacteria and Bacteroidetes, seemed to play important roles and seemed necessary for nitrifying activity in the RAS, especially in packed bed biofilters, mesh biofilters, and maturation biofilters. PMID:27033205

  8. Nuclear species-diagnostic SNP markers mined from 454 amplicon sequencing reveal admixture genomic structure of modern citrus varieties.

    PubMed

    Curk, Franck; Ancillo, Gema; Ollitrault, Frédérique; Perrier, Xavier; Jacquemoud-Collet, Jean-Pierre; Garcia-Lor, Andres; Navarro, Luis; Ollitrault, Patrick

    2015-01-01

    Most cultivated Citrus species originated from interspecific hybridisation between four ancestral taxa (C. reticulata, C. maxima, C. medica, and C. micrantha) with limited further interspecific recombination due to vegetative propagation. This evolution resulted in admixture genomes with frequent interspecific heterozygosity. Moreover, a major part of the phenotypic diversity of edible citrus results from the initial differentiation between these taxa. Deciphering the phylogenomic structure of citrus germplasm is therefore essential for an efficient utilization of citrus biodiversity in breeding schemes. The objective of this work was to develop a set of species-diagnostic single nucleotide polymorphism (SNP) markers for the four Citrus ancestral taxa covering the nine chromosomes, and to use these markers to infer the phylogenomic structure of secondary species and modern cultivars. Species-diagnostic SNPs were mined from 454 amplicon sequencing of 57 gene fragments from 26 genotypes of the four basic taxa. Of the 1,053 SNPs mined from 28,507 kb sequence, 273 were found to be highly diagnostic for a single basic taxon. Species-diagnostic SNP markers (105) were used to analyse the admixture structure of varieties and rootstocks. This revealed C. maxima introgressions in most of the old and in all recent selections of mandarins, and suggested that C. reticulata × C. maxima reticulation and introgression processes were important in edible mandarin domestication. The large range of phylogenomic constitutions between C. reticulata and C. maxima revealed in mandarins, tangelos, tangors, sweet oranges, sour oranges, grapefruits, and orangelos is favourable for genetic association studies based on phylogenomic structures of the germplasm. Inferred admixture structures were in agreement with previous hypotheses regarding the origin of several secondary species and also revealed the probable origin of several acid citrus varieties. The developed species-diagnostic SNP

  9. Redefining the Human Oral Mycobiome with Improved Practices in Amplicon-based Taxonomy: Discovery of Malassezia as a Prominent Commensal

    PubMed Central

    Dupuy, Amanda K.; David, Marika S.; Li, Lu; Heider, Thomas N.; Peterson, Jason D.; Montano, Elizabeth A.; Dongari-Bagtzoglou, Anna; Diaz, Patricia I.; Strausbaugh, Linda D.

    2014-01-01

    Fungi are a large, complex group, increasingly recognized as emerging threats. Their roles as modifiers of health mandate accurate portrayals of fungal communities in humans. As an entry point into the airways and gastrointestinal tract, fungi in the mouth are relevant to several biocompartments. We have revised current practices in sequence-based taxonomy assignments and employed the improvements to address the question of the fungal genera present in the healthy human mouth. The human oral mycobiome was surveyed using massively parallel, high throughput sequencing of internal transcribed spacer 1 (ITS1) amplicons from saliva following robust extraction methods. Taxonomy was assigned by comparison to a curated reference dataset, followed by filtering with an empirically determined BLAST E-value match statistic (10−42). Nomenclature corrections further refined results by conjoining redundant names for a single fungal genus. Following these curation steps, about two-thirds of the initially identified genera were eliminated. In comparison with the one similar metagenomic study and several earlier culture-based ones, our findings change the current conception of the oral mycobiome, especially with the discovery of the high prevalence and abundance of the genus Malassezia. Previously identified as an important pathogen of the skin, and recently reported as the predominant fungal genus at the nostril and backs of the head and ear, this is the first account of Malassezia in the human mouth. Findings from this study were in good agreement with others on the existence of many consensus members of the core mycobiome, and on unique patterns for individual subjects. This research offered a cautionary note about unconditional acceptance of lengthy lists of community members produced by automated assignments, provided a roadmap for enhancing the likely biological relevance of sequence-based fungal surveys, and built the foundation for understanding the role of fungi in health

  10. Nuclear Species-Diagnostic SNP Markers Mined from 454 Amplicon Sequencing Reveal Admixture Genomic Structure of Modern Citrus Varieties

    PubMed Central

    Curk, Franck; Ancillo, Gema; Ollitrault, Frédérique; Perrier, Xavier; Jacquemoud-Collet, Jean-Pierre; Garcia-Lor, Andres; Navarro, Luis; Ollitrault, Patrick

    2015-01-01

    Most cultivated Citrus species originated from interspecific hybridisation between four ancestral taxa (C. reticulata, C. maxima, C. medica, and C. micrantha) with limited further interspecific recombination due to vegetative propagation. This evolution resulted in admixture genomes with frequent interspecific heterozygosity. Moreover, a major part of the phenotypic diversity of edible citrus results from the initial differentiation between these taxa. Deciphering the phylogenomic structure of citrus germplasm is therefore essential for an efficient utilization of citrus biodiversity in breeding schemes. The objective of this work was to develop a set of species-diagnostic single nucleotide polymorphism (SNP) markers for the four Citrus ancestral taxa covering the nine chromosomes, and to use these markers to infer the phylogenomic structure of secondary species and modern cultivars. Species-diagnostic SNPs were mined from 454 amplicon sequencing of 57 gene fragments from 26 genotypes of the four basic taxa. Of the 1,053 SNPs mined from 28,507 kb sequence, 273 were found to be highly diagnostic for a single basic taxon. Species-diagnostic SNP markers (105) were used to analyse the admixture structure of varieties and rootstocks. This revealed C. maxima introgressions in most of the old and in all recent selections of mandarins, and suggested that C. reticulata × C. maxima reticulation and introgression processes were important in edible mandarin domestication. The large range of phylogenomic constitutions between C. reticulata and C. maxima revealed in mandarins, tangelos, tangors, sweet oranges, sour oranges, grapefruits, and orangelos is favourable for genetic association studies based on phylogenomic structures of the germplasm. Inferred admixture structures were in agreement with previous hypotheses regarding the origin of several secondary species and also revealed the probable origin of several acid citrus varieties. The developed species-diagnostic SNP

  11. The Histone Variant H3.3 Is Enriched at Drosophila Amplicon Origins but Does Not Mark Them for Activation

    PubMed Central

    Paranjape, Neha P.; Calvi, Brian R.

    2016-01-01

    Eukaryotic DNA replication begins from multiple origins. The origin recognition complex (ORC) binds origin DNA and scaffolds assembly of a prereplicative complex (pre-RC), which is subsequently activated to initiate DNA replication. In multicellular eukaryotes, origins do not share a strict DNA consensus sequence, and their activity changes in concert with chromatin status during development, but mechanisms are ill-defined. Previous genome-wide analyses in Drosophila and other organisms have revealed a correlation between ORC binding sites and the histone variant H3.3. This correlation suggests that H3.3 may designate origin sites, but this idea has remained untested. To address this question, we examined the enrichment and function of H3.3 at the origins responsible for developmental gene amplification in the somatic follicle cells of the Drosophila ovary. We found that H3.3 is abundant at these amplicon origins. H3.3 levels remained high when replication initiation was blocked, indicating that H3.3 is abundant at the origins before activation of the pre-RC. H3.3 was also enriched at the origins during early oogenesis, raising the possibility that H3.3 bookmarks sites for later amplification. However, flies null mutant for both of the H3.3 genes in Drosophila did not have overt defects in developmental gene amplification or genomic replication, suggesting that H3.3 is not essential for the assembly or activation of the pre-RC at origins. Instead, our results imply that the correlation between H3.3 and ORC sites reflects other chromatin attributes that are important for origin function. PMID:27172191

  12. Bacterial Community Shift in Treated Periodontitis Patients Revealed by Ion Torrent 16S rRNA Gene Amplicon Sequencing

    PubMed Central

    Jünemann, Sebastian; Prior, Karola; Szczepanowski, Rafael; Harks, Inga; Ehmke, Benjamin; Goesmann, Alexander; Stoye, Jens; Harmsen, Dag

    2012-01-01

    Periodontitis, one of the most common diseases in the world, is caused by a mixture of pathogenic bacteria and inflammatory host responses and often treated by antimicrobials as an adjunct to scaling and root planing (SRP). Our study aims to elucidate explorative and descriptive temporal shifts in bacterial communities between patients treated by SRP alone versus SRP plus antibiotics. This is the first metagenomic study using an Ion Torrent Personal Genome Machine (PGM). Eight subgingival plaque samples from four patients with chronic periodontitis, taken before and two months after intervention were analyzed. Amplicons from the V6 hypervariable region of the 16S rRNA gene were generated and sequenced each on a 314 chip. Sequencing reads were clustered into operational taxonomic units (OTUs, 3% distance), described by community metrics, and taxonomically classified. Reads ranging from 599,933 to 650,416 per sample were clustered into 1,648 to 2,659 non-singleton OTUs, respectively. Increased diversity (Shannon and Simpson) in all samples after therapy was observed regardless of the treatment type whereas richness (ACE) showed no correlation. Taxonomic analysis revealed different microbial shifts between both therapy approaches at all taxonomic levels. Most remarkably, the genera Porphyromonas, Tannerella, Treponema, and Filifactor all harboring periodontal pathogenic species were removed almost only in the group treated with SPR and antibiotics. For the species T. forsythia and P. gingivalis results were corroborated by real-time PCR analysis. In the future, hypothesis free metagenomic analysis could be the key in understanding polymicrobial diseases and be used for therapy monitoring. Therefore, as read length continues to increase and cost to decrease, rapid benchtop sequencers like the PGM might finally be used in routine diagnostic. PMID:22870235

  13. Classification of pmoA amplicon pyrosequences using BLAST and the lowest common ancestor method in MEGAN

    PubMed Central

    Dumont, Marc G.; Lüke, Claudia; Deng, Yongcui; Frenzel, Peter

    2013-01-01

    The classification of high-throughput sequencing data of protein-encoding genes is not as well established as for 16S rRNA. The objective of this work was to develop a simple and accurate method of classifying large datasets of pmoA sequences, a common marker for methanotrophic bacteria. A taxonomic system for pmoA was developed based on a phylogenetic analysis of available sequences. The taxonomy incorporates the known diversity of pmoA present in public databases, including both sequences from cultivated and uncultivated organisms. Representative sequences from closely related genes, such as those encoding the bacterial ammonia monooxygenase, were also included in the pmoA taxonomy. In total, 53 low-level taxa (genus-level) are included. Using previously published datasets of high-throughput pmoA amplicon sequence data, we tested two approaches for classifying pmoA: a naïve Bayesian classifier and BLAST. Classification of pmoA sequences based on BLAST analyses was performed using the lowest common ancestor (LCA) algorithm in MEGAN, a software program commonly used for the analysis of metagenomic data. Both the naïve Bayesian and BLAST methods were able to classify pmoA sequences and provided similar classifications; however, the naïve Bayesian classifier was prone to misclassifying contaminant sequences present in the datasets. Another advantage of the BLAST/LCA method was that it provided a user-interpretable output and enabled novelty detection at various levels, from highly divergent pmoA sequences to genus-level novelty. PMID:22558000

  14. Responses of soil microeukaryotic communities to short-term fumigation-incubation revealed by MiSeq amplicon sequencing

    PubMed Central

    Chen, Lin; Xu, Jianming; Feng, Youzhi; Wang, Juntao; Yu, Yongjie; Brookes, Philip C.

    2015-01-01

    In soil microbiology, there is a “paradox” of soil organic carbon (SOC) mineralization, which is that even though chloroform fumigation destroys majority of the soil microbial biomass, SOC mineralization continues at the same rate as in the non-fumigated soil during the incubation period. Soil microeukaryotes as important SOC decomposers, however, their community-level responses to chloroform fumigation are not well understood. Using the 18S rRNA gene amplicon sequencing, we analyzed the composition, diversity, and C-metabolic functions of a grassland soil and an arable soil microeukaryotic community in response to fumigation followed by a 30-day incubation. The grassland and arable soil microeukaryotic communities were dominated by the fungal Ascomycota (80.5–93.1% of the fungal sequences), followed by the protistan Cercozoa and Apicomplexa. In the arable soil fungal community, the predominance of the class Sordariomycetes was replaced by the class Eurotiomycetes after fumigation at days 7 and 30 of the incubation. Fumigation changed the microeukaryotic α-diversity in the grassland soil at days 0 and 7, and β-diversity in the arable soil at days 7 and 30. Network analysis indicated that after fumigation fungi were important groups closely related to other taxa. Most phylotypes (especially Sordariomycetes, Dothideomycetes, Coccidia, and uncultured Chytridiomycota) were inhibited, and only a few were positively stimulated by fumigation. Despite the inhibited Sordariomycetes, the fumigated communities mainly consisted of Eurotiomycetes and Sordariomycetes (21.9 and 36.5% relative frequency, respectively), which are able to produce hydrolytic enzymes associated with SOC mineralization. Our study suggests that fumigation not only decreases biomass size, but modulates the composition and diversity of the soil microeukaryotic communities, which are capable of driving SOC mineralization by release of hydrolytic enzymes during short-term fumigation-incubation. PMID

  15. Screening of male breast cancer and of breast-ovarian cancer families for BRCA2 mutations using large bifluorescent amplicons

    PubMed Central

    Pages, S; Caux, V; Stoppa-Lyonnet, D; Tosi, M

    2001-01-01

    41 breast cancer or breast-ovarian cancer families, including 12 families with at least one affected first-degree male relative, were screened for mutations in the BRCA2 gene. Mutations had not been found in the BRCA1 gene of these families. Chemical cleavage of Mismatch was used to identify nucleotide changes within large PCR products (average size 1.2 kb) that carried strand-specific fluorescent end-labels. 15 amplicons were sufficient to scan 18 exons, including the large exon 11. The remaining 9 small exons were examined by Denaturing Gradient Gel Electrophoresis. The high sensitivity of this approach was documented by the detection, in these 41 patients, of all 9 exonic single nucleotide polymorphisms reported with heterozygosity >0.1. Truncating BRCA2 mutations were found in 7 of the 41 families. 3 of them were in the group of 12 families comprising cases of male breast cancer. Since the methods used here have no bias for particular types of mutations, these data confirm the high proportion of frameshifts among mutations in BRCA2. However, relevant single nucleotide substitutions were also found: one resulting in a stop codon and another one, present in a male patient, was the previously reported change Asp2723His, that affects a highly conserved region of the BRCA2 protein. This study indicates a BRCA2 contribution of 10% (95% CI 2.5–17.5) to our original cohort of 59 breast-ovarian cancer families, whereas the contribution of BRCA1 had been estimated at 46% (95% CI 33–59). © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11207042

  16. Microbial Community Composition and Diversity via 16S rRNA Gene Amplicons: Evaluating the Illumina Platform

    PubMed Central

    Sinclair, Lucas; Osman, Omneya Ahmed; Bertilsson, Stefan; Eiler, Alexander

    2015-01-01

    As new sequencing technologies become cheaper and older ones disappear, laboratories switch vendors and platforms. Validating the new setups is a crucial part of conducting rigorous scientific research. Here we report on the reliability and biases of performing bacterial 16S rRNA gene amplicon paired-end sequencing on the MiSeq Illumina platform. We designed a protocol using 50 barcode pairs to run samples in parallel and coded a pipeline to process the data. Sequencing the same sediment sample in 248 replicates as well as 70 samples from alkaline soda lakes, we evaluated the performance of the method with regards to estimates of alpha and beta diversity. Using different purification and DNA quantification procedures we always found up to 5-fold differences in the yield of sequences between individually barcodes samples. Using either a one-step or a two-step PCR preparation resulted in significantly different estimates in both alpha and beta diversity. Comparing with a previous method based on 454 pyrosequencing, we found that our Illumina protocol performed in a similar manner – with the exception for evenness estimates where correspondence between the methods was low. We further quantified the data loss at every processing step eventually accumulating to 50% of the raw reads. When evaluating different OTU clustering methods, we observed a stark contrast between the results of QIIME with default settings and the more recent UPARSE algorithm when it comes to the number of OTUs generated. Still, overall trends in alpha and beta diversity corresponded highly using both clustering methods. Our procedure performed well considering the precisions of alpha and beta diversity estimates, with insignificant effects of individual barcodes. Comparative analyses suggest that 454 and Illumina sequence data can be combined if the same PCR protocol and bioinformatic workflows are used for describing patterns in richness, beta-diversity and taxonomic composition. PMID:25647581

  17. The Histone Variant H3.3 Is Enriched at Drosophila Amplicon Origins but Does Not Mark Them for Activation.

    PubMed

    Paranjape, Neha P; Calvi, Brian R

    2016-01-01

    Eukaryotic DNA replication begins from multiple origins. The origin recognition complex (ORC) binds origin DNA and scaffolds assembly of a prereplicative complex (pre-RC), which is subsequently activated to initiate DNA replication. In multicellular eukaryotes, origins do not share a strict DNA consensus sequence, and their activity changes in concert with chromatin status during development, but mechanisms are ill-defined. Previous genome-wide analyses in Drosophila and other organisms have revealed a correlation between ORC binding sites and the histone variant H3.3. This correlation suggests that H3.3 may designate origin sites, but this idea has remained untested. To address this question, we examined the enrichment and function of H3.3 at the origins responsible for developmental gene amplification in the somatic follicle cells of the Drosophila ovary. We found that H3.3 is abundant at these amplicon origins. H3.3 levels remained high when replication initiation was blocked, indicating that H3.3 is abundant at the origins before activation of the pre-RC. H3.3 was also enriched at the origins during early oogenesis, raising the possibility that H3.3 bookmarks sites for later amplification. However, flies null mutant for both of the H3.3 genes in Drosophila did not have overt defects in developmental gene amplification or genomic replication, suggesting that H3.3 is not essential for the assembly or activation of the pre-RC at origins. Instead, our results imply that the correlation between H3.3 and ORC sites reflects other chromatin attributes that are important for origin function. PMID:27172191

  18. Effects of inspired CO2, hyperventilation, and time on VA/Q inequality in the dog

    NASA Technical Reports Server (NTRS)

    Tsukimoto, K.; Arcos, J. P.; Schaffartzik, W.; Wagner, P. D.; West, J. B.

    1992-01-01

    In a recent study by Tsukimoto et al. (J. Appl. Physiol. 68: 2488-2493, 1990), CO2 inhalation appeared to reduce the size of the high ventilation-perfusion ratio (VA/Q) mode commonly observed in anesthetized mechanically air-ventilated dogs. In that study, large tidal volumes (VT) were used during CO2 inhalation to preserve normocapnia. To separate the influences of CO2 and high VT on the VA/Q distribution in the present study, we examined the effect of inspired CO2 on the high VA/Q mode using eight mechanically ventilated dogs (4 given CO2, 4 controls). The VA/Q distribution was measured first with normal VT and then with increased VT. In the CO2 group at high VT, data were collected before, during, and after CO2 inhalation. With normal VT, there was no difference in the size of the high VA/Q mode between groups [10.5 +/- 3.5% (SE) of ventilation in the CO2 group, 11.8 +/- 5.2% in the control group]. Unexpectedly, the size of the high VA/Q mode decreased similarly in both groups over time, independently of the inspired PCO2, at a rate similar to the fall in cardiac output over time. The reduction in the high VA/Q mode together with a simultaneous increase in alveolar dead space (estimated by the difference between inert gas dead space and Fowler dead space) suggests that poorly perfused high VA/Q areas became unperfused over time. A possible mechanism is that elevated alveolar pressure and decreased cardiac output eliminate blood flow from corner vessels in nondependent high VA/Q regions.

  19. Electrical Resistivity Study of a Pleistocene Riverbed in Saltville, VA

    NASA Astrophysics Data System (ADS)

    Herman, R. B.; Whisonant, R. C.

    2008-05-01

    A shallow capacitively coupled resistivity survey was performed in Saltville, VA, in an area of suspected buried Pleistocene river deposits. Previous excavations in the sediments beneath the Saltville valley floor had been performed to recover late Pleistocene megafaunal remains and possible Clovis-age human artifacts. These digs encountered a zone, one to two meters deep, of gravel-sized rock fragments, including some boulders up to 75 cm. in diameter. These large clasts are rounded, show some imbrication (shingle-like overlapping indicative of current flow), and have been interpreted as river channel deposits. Carbon 14 dates from the megafaunal bones within and just above the gravel bed yielded dates of 14,500 years BP. Resistivity signals in a number of locations were consistent with cobbles and boulders deposited in a river channel. These signals are generally bowl- shaped areas with large circular (2-d scans) anomalies near the center, and smaller circular anomalies tapering out towards both sides. The bowl-shaped anomalies are within 3 meters of the surface. With several lines imaged in this survey a rough path of the riverbed, along with a number of branchings is traceable in the survey area. An exploratory hole confirmed the presence of a layer of rounded cobbles and boulders 1.3 meters deep beneath one of the survey lines.

  20. Sediment dynamics over multiple time scales in Dyke Marsh Preserve (Potomac River, VA)

    NASA Astrophysics Data System (ADS)

    Palinkas, C. M.; Walters, D.

    2010-12-01

    Tidal freshwater marshes are critical components of fluvial and estuarine ecosystems, yet sediment dynamics within them have not received as much attention as their saltwater counterparts. This study examines sedimentation in Dyke Marsh Preserve, located on the Potomac River (VA), focusing on understanding the spatial variability present over multiple time scales. Bimonthly sediment data were collected using ceramic tiles, and seasonal- and decadal-scale sedimentation was determined via 7Be (half-life 53.3 days) and 210Pb (half-life 22.3 years), respectively. Results were also compared to SET data collected by the National Park Service since 2006. Preliminary data indicate that sites at lower elevations have higher sedimentation rates, likely related to their close proximity to the sediment source. Mass accumulation rates generally decreased with increasing time scale, such that the seasonal rates were greater than the SET-derived accretion rates, which were in turn greater than the decadal-scale rates. However, the bimonthly rates were the lowest observed, probably because the sampling period (May-October 2010) did not include the main depositional period of the year, which would be integrated by the other techniques.

  1. Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap.

    PubMed Central

    Conway, J E; Zolotukhin, S; Muzyczka, N; Hayward, G S; Byrne, B J

    1997-01-01

    Recombinant adeno-associated virus (AAV) type 2 (rAAV) vectors have recently been shown to have great utility as gene transfer agents both in vitro and in vivo. One of the problems associated with the use of rAAV vectors has been the difficulty of large-scale vector production. Low-efficiency plasmid transfection of the rAAV vector and complementing AAV type 2 (AAV-2) functions (rep and cap) followed by superinfection with adenovirus has been the standard approach to rAAV production. The objectives of this study were to demonstrate the ability of a recombinant herpes simplex virus type 1 (HSV-1) amplicon expressing AAV-2 Rep and Cap to support replication and packaging of rAAV vectors. HSV-1 amplicon vectors were constructed which contain the AAV-2 rep and cap genes under control of their native promoters (p5, p19, and p40). An HSV-1 amplicon vector, HSV-RC/KOS or HSV-RC/d27, was generated by supplying helper functions with either wild-type HSV-1 (KOS strain) or the ICP27-deleted mutant of HSV-1, d27-1, respectively. Replication of the amplicon stocks is not inhibited by the presence of AAV-2 Rep proteins, which highlights important differences between HSV-1 and adenovirus replication and the mechanism of providing helper function for productive AAV infection. Coinfection of rAAV and HSV-RC/KOS resulted in the replication and amplification of rAAV genomes. Similarly, rescue and replication of rAAV genomes occurred when rAAV vector plasmids were transfected into cells followed by HSV-RC/KOS infection and when two rAAV proviral cell lines were infected with HSV-RC/KOS or HSV-RC/d27. Production of infectious rAAV by rescue from two rAAV proviral cell lines has also been achieved with HSV-RC/KOS and HSV-RC/d27. The particle titer of rAAV produced with HSV-RC/d27 is equal to that achieved by supplying rep and cap by transfection followed by adenovirus superinfection. Importantly, no detectable wild-type AAV-2 is generated with this approach. These results demonstrate

  2. [A clinical experience of V-A bypass using a new antithrombotic coating material].

    PubMed

    Kawahito, K; Ino, T; Ide, H; Adachi, H; Mizuhara, A; Yamaguchi, A

    1992-07-01

    To perform V-A bypass with minimal systemically administered heparin, we used a equipment coated by fluorine-acryl-styrene-urethane-silicone graft copolymer. A 67 year old woman developed right heart failure after CABG was treated for 25 hours on V-A bypass without oxygenator by using this new antithrombotic coating material. During V-A bypass, hemodynamics were stable. Not coagulative nor hemolytic disorder was observed. And she did not suffer from thrombotic nor hemorrhagic complications. Scanning electron microscopy of coated equipment demonstrated only minor deposits on the surface, and morphologic study of platelet was almost normal. By using this new antithrombotic material it is possible to perform V-A bypass with minimal heparinization, thus avoiding the risk of major coagulation complications. PMID:1506705

  3. 75 FR 52989 - Specialty Minerals, Inc., Franklin, VA; Notice of Negative Determination Regarding Application...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-30

    ... (75 FR 38142). The workers produced precipitated calcium carbonate used in the production of paper... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF LABOR Employment and Training Administration Specialty Minerals, Inc., Franklin, VA; Notice of...

  4. 77 FR 24268 - Agency Information Collection (Dependents' Application for VA Educational Benefits) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-23

    ... Assistance (DEA) and Post-9/11 GI Bill Marine Gunnery Sergeant John David Fry Scholarship (Fry Scholarship) benefits. VA will use the information collected to determine whether a claimant qualifies for DEA or...

  5. 78 FR 66266 - Drawbridge Operation Regulation; Atlantic Intracoastal Waterway (AIWW), Chesapeake, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-05

    ... Chesapeake, VA. This deviation is necessary to facilitate maintenance work on the rotary span limit switches... 33 CFR 117.997(e), to facilitate maintenance of the moveable spans on the structure. The...

  6. 78 FR 12600 - VA Homeless Providers Grant and Per Diem Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-25

    ... and dealing with mental health issues, including substance use. Other related VA programs that offer... professions, Health records, Homeless, Mental health programs, Reporting and recordkeeping requirements... a diagnosis from a licensed mental health professional, with at least one documented...

  7. 38 CFR 17.96 - Medication prescribed by non-VA physicians.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... AFFAIRS MEDICAL Outpatient Treatment § 17.96 Medication prescribed by non-VA physicians. Any prescription... the Mexican Border Period, World War I, World War II, the Korean Conflict, or the Vietnam Era...

  8. 77 FR 72816 - Foreign-Trade Zone 20-Suffolk, VA; Authorization of Production Activity; Usui International...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-06

    ... International Corporation (Diesel Engine Fuel Lines); Chesapeake, VA On June 28, 2012, the Virginia Port... 400), including notice in the Federal Register inviting public comment (77 FR 48127-48128,...

  9. 77 FR 74279 - Agency Information Collection (VA/DOD Joint Disability Evaluation Board Claim): Activity under...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-13

    ... President Bush's Interagency Task Force on Returning Global War on Terror Heroes, VA and the Department of Defense (DOD) have agreed to develop a joint process in which Global War on Terror (GWOT) service...

  10. 75 FR 9277 - Proposed Information Collection (VA National Rehabilitation Special Events, Event Registration...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-01

    ... AFFAIRS Proposed Information Collection (VA National Rehabilitation Special Events, Event Registration Applications); Comment Request AGENCY: Office of National Programs and Special Events, Department of Veterans Affairs. ACTION: Notice. SUMMARY: The Office of National Programs and Special Events (NPSE), Department...

  11. 38 CFR 3.1706 - Burial allowance for a veteran who died while hospitalized by VA.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... within a State, or to the border limits of the United States. (2) VA may pay the plot or interment allowance for burial in a veterans cemetery under § 3.1707, Plot or interment allowance. (Authority: 38...

  12. 76 FR 70345 - Drawbridge Operation Regulations; Elizabeth River, Eastern Branch, Norfolk, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-14

    ... operation of the Norfolk Southern 5 Railroad Bridge, mile 1.1, Norfolk, VA. Under this temporary deviation... for train crossings or periodic maintenance. The Norfolk Southern 5 Bridge, at mile 1.1, across...

  13. 78 FR 55777 - Proposed Information Collection (VA, National Veterans Sports Programs and Special Events, Event...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-11

    ... AFFAIRS Proposed Information Collection (VA, National Veterans Sports Programs and Special Events, Event Registration Applications); Comment Request AGENCY: Office of National Veterans Sports Programs and Special Events, Department of Veterans Affairs. ACTION: Notice. SUMMARY: The Office of National Veterans...

  14. 79. Credit JTL. Detail of 2000 kVA generator (unit 2) ...

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

    79. Credit JTL. Detail of 2000 kVA generator (unit 2) looking southwest. Note the elevated neutral on upper part of generator (added later). - Battle Creek Hydroelectric System, Battle Creek & Tributaries, Red Bluff, Tehama County, CA

  15. 38 CFR 17.52 - Hospital care and medical services in non-VA facilities.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2012-07-01 2012-07-01 false Hospital care and medical services in non-VA facilities. 17.52 Section 17.52 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS MEDICAL Use of Public Or Private Hospitals § 17.52 Hospital care and medical services in non-VA facilities. (a) When...

  16. 46 CFR 7.55 - Cape Henry, VA to Cape Fear, NC.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 1 2014-10-01 2014-10-01 false Cape Henry, VA to Cape Fear, NC. 7.55 Section 7.55 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PROCEDURES APPLICABLE TO THE PUBLIC BOUNDARY LINES Atlantic Coast § 7.55 Cape Henry, VA to Cape Fear, NC. (a) A line drawn from Rudee Inlet Jetty Light “2”...

  17. 46 CFR 7.55 - Cape Henry, VA to Cape Fear, NC.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 1 2013-10-01 2013-10-01 false Cape Henry, VA to Cape Fear, NC. 7.55 Section 7.55 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PROCEDURES APPLICABLE TO THE PUBLIC BOUNDARY LINES Atlantic Coast § 7.55 Cape Henry, VA to Cape Fear, NC. (a) A line drawn from Rudee Inlet Jetty Light “2”...

  18. 46 CFR 7.55 - Cape Henry, VA to Cape Fear, NC.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Cape Henry, VA to Cape Fear, NC. 7.55 Section 7.55 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PROCEDURES APPLICABLE TO THE PUBLIC BOUNDARY LINES Atlantic Coast § 7.55 Cape Henry, VA to Cape Fear, NC. (a) A line drawn from Rudee Inlet Jetty Light “2”...

  19. 46 CFR 7.45 - Cape Henlopen, DE to Cape Charles, VA.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Cape Henlopen, DE to Cape Charles, VA. 7.45 Section 7.45 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PROCEDURES APPLICABLE TO THE PUBLIC BOUNDARY LINES Atlantic Coast § 7.45 Cape Henlopen, DE to Cape Charles, VA. (a) A line drawn from the easternmost extremity of Indian River Inlet North...

  20. 30 CFR 57.22240 - Actions at 2.0 percent methane (V-A mines).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Actions at 2.0 percent methane (V-A mines). 57... MINES Safety Standards for Methane in Metal and Nonmetal Mines Ventilation § 57.22240 Actions at 2.0 percent methane (V-A mines). If methane reaches 2.0 percent in the mine atmosphere, all persons other...

  1. Exploring the Gastrointestinal “Nemabiome”: Deep Amplicon Sequencing to Quantify the Species Composition of Parasitic Nematode Communities

    PubMed Central

    Avramenko, Russell W.; Redman, Elizabeth M.; Lewis, Roy; Yazwinski, Thomas A.; Wasmuth, James D.; Gilleard, John S.

    2015-01-01

    Parasitic helminth infections have a considerable impact on global human health as well as animal welfare and production. Although co-infection with multiple parasite species within a host is common, there is a dearth of tools with which to study the composition of these complex parasite communities. Helminth species vary in their pathogenicity, epidemiology and drug sensitivity and the interactions that occur between co-infecting species and their hosts are poorly understood. We describe the first application of deep amplicon sequencing to study parasitic nematode communities as well as introduce the concept of the gastro-intestinal “nemabiome”. The approach is analogous to 16S rDNA deep sequencing used to explore microbial communities, but utilizes the nematode ITS-2 rDNA locus instead. Gastro-intestinal parasites of cattle were used to develop the concept, as this host has many well-defined gastro-intestinal nematode species that commonly occur as complex co-infections. Further, the availability of pure mono-parasite populations from experimentally infected cattle allowed us to prepare mock parasite communities to determine, and correct for, species representation biases in the sequence data. We demonstrate that, once these biases have been corrected, accurate relative quantitation of gastro-intestinal parasitic nematode communities in cattle fecal samples can be achieved. We have validated the accuracy of the method applied to field-samples by comparing the results of detailed morphological examination of L3 larvae populations with those of the sequencing assay. The results illustrate the insights that can be gained into the species composition of parasite communities, using grazing cattle in the mid-west USA as an example. However, both the technical approach and the concept of the ‘nemabiome’ have a wide range of potential applications in human and veterinary medicine. These include investigations of host-parasite and parasite-parasite interactions

  2. Valerenic acid derivatives as novel subunit-selective GABAA receptor ligands –in vitro and in vivo characterization

    PubMed Central

    Khom, S; Strommer, B; Ramharter, J; Schwarz, T; Schwarzer, C; Erker, T; Ecker, GF; Mulzer, J; Hering, S

    2010-01-01

    BACKGROUND AND PURPOSE Subunit-specific modulators of γ-aminobutyric acid (GABA) type A (GABAA) receptors can help to assess the physiological function of receptors with different subunit composition and also provide the basis for the development of new drugs. Valerenic acid (VA) was recently identified as a β2/3 subunit-specific modulator of GABAA receptors with anxiolytic potential. The aim of the present study was to generate VA derivatives as novel GABAA receptor modulators and to gain insight into the structure–activity relation of this molecule. EXPERIMENTAL APPROACH The carboxyl group of VA was substituted by an uncharged amide or amides with different chain length. Modulation of GABAA receptors composed of different subunit compositions by the VA derivatives was studied in Xenopus oocytes by means of the two-microelectrode voltage-clamp technique. Half-maximal stimulation of GABA-induced chloride currents (IGABA) through GABAA receptors (EC50) and efficacies (maximal stimulation of IGABA) were estimated. Anxiolytic activity of the VA derivatives was studied in mice, applying the elevated plus maze test. KEY RESULTS Valerenic acid amide (VA-A) displayed the highest efficacy (more than twofold greater IGABA enhancement than VA) and highest potency (EC50= 13.7 ± 2.3 µM) on α1β3 receptors. Higher efficacy and potency of VA-A were also observed on α1β2γ2s and α3β3γ2s receptors. Anxiolytic effects were most pronounced for VA-A. CONCLUSIONS AND IMPLICATIONS Valerenic acid derivatives with higher efficacy and affinity can be generated. Greater in vitro action of the amide derivative correlated with a more pronounced anxiolytic effect in vivo. The data give further confidence in targeting β3 subunit containing GABAA receptors for development of anxiolytics. PMID:20718740

  3. Partnership to improve quality care for veterans: the VA Nursing Academy.

    PubMed

    Harper, Doreen C; Selleck, Cynthia S; Eagerton, Gregory; Froelich, Kimberly

    2015-01-01

    More than 22 million living veterans reside in the United States. In fact, understanding military culture and the experiences of these veterans is important to their ongoing health care and the unique challenges faced by many. The Veterans Affairs (VA) Nursing Academy began in 2007 to fund pilot partnerships between schools of nursing and local VA health care facilities to better serve our veteran population. Fifteen academic/service partnerships were selected for funding between 2007 and 2009 with the goals of expanding faculty and professional development, increasing nursing student enrollment, providing opportunities for educational and practice innovations, and increasing the recruitment and retention of VA nurses. This article details critical components of the partnership developed between the Birmingham VA Medical Center and the University of Alabama at Birmingham School of Nursing, a VA Nursing Academy partnership funded in the 2009 cohort. Site-specific goals of the partnership are described along with a discussion of the framework used to develop the Birmingham VA Nursing Academy, which includes relationship building, engagement, governance, evaluation of outcomes, and sustainability. The logic model developed for the partnership is included, and the interim outputs and outcomes of this practice-academic partnership are detailed, a number of which can be replicated by VAs and schools of nursing across the country. PMID:25601246

  4. VaRank: a simple and powerful tool for ranking genetic variants

    PubMed Central

    Geoffroy, Véronique; Pizot, Cécile; Redin, Claire; Piton, Amélie; Vasli, Nasim; Stoetzel, Corinne; Blavier, André; Laporte, Jocelyn

    2015-01-01

    Background. Most genetic disorders are caused by single nucleotide variations (SNVs) or small insertion/deletions (indels). High throughput sequencing has broadened the catalogue of human variation, including common polymorphisms, rare variations or disease causing mutations. However, identifying one variation among hundreds or thousands of others is still a complex task for biologists, geneticists and clinicians. Results. We have developed VaRank, a command-line tool for the ranking of genetic variants detected by high-throughput sequencing. VaRank scores and prioritizes variants annotated either by Alamut Batch or SnpEff. A barcode allows users to quickly view the presence/absence of variants (with homozygote/heterozygote status) in analyzed samples. VaRank supports the commonly used VCF input format for variants analysis thus allowing it to be easily integrated into NGS bioinformatics analysis pipelines. VaRank has been successfully applied to disease-gene identification as well as to molecular diagnostics setup for several hundred patients. Conclusions. VaRank is implemented in Tcl/Tk, a scripting language which is platform-independent but has been tested only on Unix environment. The source code is available under the GNU GPL, and together with sample data and detailed documentation can be downloaded from http://www.lbgi.fr/VaRank/. PMID:25780760

  5. Myosin Va bound to phagosomes binds to F-actin and delays microtubule-dependent motility.

    PubMed

    Al-Haddad, A; Shonn, M A; Redlich, B; Blocker, A; Burkhardt, J K; Yu, H; Hammer, J A; Weiss, D G; Steffen, W; Griffiths, G; Kuznetsov, S A

    2001-09-01

    We established a light microscopy-based assay that reconstitutes the binding of phagosomes purified from mouse macrophages to preassembled F-actin in vitro. Both endogenous myosin Va from mouse macrophages and exogenous myosin Va from chicken brain stimulated the phagosome-F-actin interaction. Myosin Va association with phagosomes correlated with their ability to bind F-actin in an ATP-regulated manner and antibodies to myosin Va specifically blocked the ATP-sensitive phagosome binding to F-actin. The uptake and retrograde transport of phagosomes from the periphery to the center of cells in bone marrow macrophages was observed in both normal mice and mice homozygous for the dilute-lethal spontaneous mutation (myosin Va null). However, in dilute-lethal macrophages the accumulation of phagosomes in the perinuclear region occurred twofold faster than in normal macrophages. Motion analysis revealed saltatory phagosome movement with temporarily reversed direction in normal macrophages, whereas almost no reversals in direction were observed in dilute-lethal macrophages. These observations demonstrate that myosin Va mediates phagosome binding to F-actin, resulting in a delay in microtubule-dependent retrograde phagosome movement toward the cell center. We propose an "antagonistic/cooperative mechanism" to explain the saltatory phagosome movement toward the cell center in normal macrophages. PMID:11553713

  6. US Veterans' Use Of VA Mental Health Services And Disability Compensation Increased From 2001 To 2010.

    PubMed

    Tsai, Jack; Rosenheck, Robert A

    2016-06-01

    There has been concern about the capacity of the Department of Veterans Affairs (VA) health care system to provide care for veterans returning from war zones in the Middle East and Afghanistan. We used two nationally representative surveys of US veterans in 2001 and 2010 to examine changes in the veteran population and veterans' use of health care services after a decade of war. The population was older and more diverse in 2010 than in 2001. In both years, veterans who served in the World War II era or earlier were more likely to have been exposed to combat-related trauma than veterans of more recent service eras. In 2010 veterans who served in the Persian Gulf War era (1990 through the wars in Iraq and Afghanistan) were more likely to have used VA mental health services and to have received VA disability compensation than veterans of previous service eras. Compared to veterans in 2001, those in 2010 were two times more likely to have used any VA health services and were more likely to have received VA disability compensation. These findings highlight changes in the veteran population over time that are important to consider in planning for future VA services. PMID:27269011

  7. VaRank: a simple and powerful tool for ranking genetic variants.

    PubMed

    Geoffroy, Véronique; Pizot, Cécile; Redin, Claire; Piton, Amélie; Vasli, Nasim; Stoetzel, Corinne; Blavier, André; Laporte, Jocelyn; Muller, Jean

    2015-01-01

    Background. Most genetic disorders are caused by single nucleotide variations (SNVs) or small insertion/deletions (indels). High throughput sequencing has broadened the catalogue of human variation, including common polymorphisms, rare variations or disease causing mutations. However, identifying one variation among hundreds or thousands of others is still a complex task for biologists, geneticists and clinicians. Results. We have developed VaRank, a command-line tool for the ranking of genetic variants detected by high-throughput sequencing. VaRank scores and prioritizes variants annotated either by Alamut Batch or SnpEff. A barcode allows users to quickly view the presence/absence of variants (with homozygote/heterozygote status) in analyzed samples. VaRank supports the commonly used VCF input format for variants analysis thus allowing it to be easily integrated into NGS bioinformatics analysis pipelines. VaRank has been successfully applied to disease-gene identification as well as to molecular diagnostics setup for several hundred patients. Conclusions. VaRank is implemented in Tcl/Tk, a scripting language which is platform-independent but has been tested only on Unix environment. The source code is available under the GNU GPL, and together with sample data and detailed documentation can be downloaded from http://www.lbgi.fr/VaRank/. PMID:25780760

  8. Semi-nonparametric VaR forecasts for hedge funds during the recent crisis

    NASA Astrophysics Data System (ADS)

    Del Brio, Esther B.; Mora-Valencia, Andrés; Perote, Javier

    2014-05-01

    The need to provide accurate value-at-risk (VaR) forecasting measures has triggered an important literature in econophysics. Although these accurate VaR models and methodologies are particularly demanded for hedge fund managers, there exist few articles specifically devoted to implement new techniques in hedge fund returns VaR forecasting. This article advances in these issues by comparing the performance of risk measures based on parametric distributions (the normal, Student’s t and skewed-t), semi-nonparametric (SNP) methodologies based on Gram-Charlier (GC) series and the extreme value theory (EVT) approach. Our results show that normal-, Student’s t- and Skewed t- based methodologies fail to forecast hedge fund VaR, whilst SNP and EVT approaches accurately success on it. We extend these results to the multivariate framework by providing an explicit formula for the GC copula and its density that encompasses the Gaussian copula and accounts for non-linear dependences. We show that the VaR obtained by the meta GC accurately captures portfolio risk and outperforms regulatory VaR estimates obtained through the meta Gaussian and Student’s t distributions.

  9. U.S. Dept. Veterans Affairs (VA) SMEC-bio Reporting for Leadership Decision Support

    PubMed Central

    Gamage, Shantini D.; Simbartl, Loretta A.; Kralovic, Stephen M.; Wallace, Katherine S.; Roselle, Gary A.

    2013-01-01

    Objective To assess Reports sent from the United States VA Subject Matter Expertise Center for Biological Events (SMEC-bio) – a proof-of-concept decision support initiative – to the VA Integrated Operations Center (VA IOC). Introduction VA is the U.S. federal agency responsible for providing services to America’s Veterans. Within VA, VHA is the organization responsible for administration of health care services. VHA, with 152 Medical Centers and over 900 outpatient clinics located throughout the U.S. and territories, provided care to over 5 million patients in 2011. After the 2009 H1N1 influenza pandemic, OSP, which oversees VA senior level briefing of preparedness issues, conceptualized and initiated SMEC-bio as a protocol-based mechanism to incorporate timely VHA subject matter expertise into leadership decision making via the VA IOC. Previous work has examined collection and integration of data from VA and interagency sources for trend and predictive analyses (1). This current work is an initial assessment of SMEC-bio reporting, which has been in development for the past year and functions on an ad hoc basis for decision support; needs and gaps can be assessed toward a formalized communication plan with the VA IOC. Methods In May, 2011, SMEC-bio designed a Report template. All SMEC-bio Reports submitted to the VA IOC using the template were assessed based on reason for the Report, timing, data sources used, and outcome. A gap analysis was conducted to identify areas for further improvement. Results Eight SMEC-bio Reports were produced since the template was initiated in May, 2011. The reasons for reporting fell into the following categories: 1) briefings of interagency protocol activations [e.g. National Biosurveillance Integration System (NBIS) protocol]; 2) Requests for Information (RFIs) from the VA IOC regarding specific biological events (e.g. 2012 H3N2v influenza associated with swine at fairs); 3) RFIs from the VA IOC on general infectious diseases

  10. Contribution of Amino Acid Region 659−663 of Factor Va Heavy Chain to the Activity of Factor Xa within Prothrombinase†,‡

    PubMed Central

    2010-01-01

    Factor Va, the cofactor of prothrombinase, is composed of heavy and light chains associated noncovalently in the presence of divalent metal ions. The COOH-terminal region of the heavy chain contains acidic amino acid clusters that are important for cofactor activity. In this work, we have investigated the role of amino acid region 659−663, which contains five consecutive acidic amino acid residues, by site-directed mutagenesis. We have generated factor V molecules in which all residues were mutated to either lysine (factor V5K) or alanine (factor V5A). We have also constructed a mutant molecule with this region deleted (factor VΔ659−663). The recombinant molecules along with wild-type factor V (factor VWT) were transiently expressed in mammalian cells, purified, and assessed for cofactor activity. Two-stage clotting assays revealed that the mutant molecules had reduced clotting activities compared to that of factor VaWT. Kinetic analyses of prothrombinase assembled with the mutant molecules demonstrated diminished kcat values, while the affinity of all mutant molecules for factor Xa was similar to that for factor VaWT. Gel electrophoresis analyses of plasma-derived and recombinant mutant prothrombin activation demonstrated delayed cleavage of prothrombin at both Arg320 and Arg271 by prothrombinase assembled with the mutant molecules, resulting in meizothrombin lingering throughout the activation process. These results were confirmed after analysis of the cleavage of FPR-meizothrombin. Our findings provide new insights into the structural contribution of the acidic COOH-terminal region of factor Va heavy chain to factor Xa activity within prothrombinase and demonstrate that amino acid region 659−663 from the heavy chain of the cofactor contributes to the regulation of the rate of cleavage of prothrombin by prothrombinase. PMID:20722419

  11. VA Education Benefits: VA Should Strengthen Its Efforts to Help Veterans Make Informed Education Choices. Report to Congressional Requesters. GAO-14-324

    ERIC Educational Resources Information Center

    Emrey-Arras, Melissa

    2014-01-01

    In fiscal year 2013, VA provided over $12 billion in benefits for veterans' postsecondary education; however, questions have been raised as to whether some schools are receiving these funds as a result of inappropriate recruiting practices. GAO was asked to examine issues related to schools' recruitment of veterans. This report examines (1) how…

  12. Characterization of Clostridium perfringens Spores That Lack SpoVA Proteins and Dipicolinic Acid▿

    PubMed Central

    Paredes-Sabja, Daniel; Setlow, Barbara; Setlow, Peter; Sarker, Mahfuzur R.

    2008-01-01

    Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca2+ and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca2+ and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of l-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca2+ and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 103- to 104-fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective α/β-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full

  13. VA/Q distribution during heavy exercise and recovery in humans: implications for pulmonary edema

    NASA Technical Reports Server (NTRS)

    Schaffartzik, W.; Poole, D. C.; Derion, T.; Tsukimoto, K.; Hogan, M. C.; Arcos, J. P.; Bebout, D. E.; Wagner, P. D.

    1992-01-01

    Ventilation-perfusion (VA/Q) inequality has been shown to increase with exercise. Potential mechanisms for this increase include nonuniform pulmonary vasoconstriction, ventilatory time constant inequality, reduced large airway gas mixing, and development of interstitial pulmonary edema. We hypothesized that persistence of VA/Q mismatch after ventilation and cardiac output subside during recovery would be consistent with edema; however, rapid resolution would suggest mechanisms related to changes in ventilation and blood flow per se. Thirteen healthy males performed near-maximal cycle ergometry at an inspiratory PO2 of 91 Torr (because hypoxia accentuates VA/Q mismatch on exercise). Cardiorespiratory variables and inert gas elimination patterns were measured at rest, during exercise, and between 2 and 30 min of recovery. Two profiles of VA/Q distribution behavior emerged during heavy exercise: in group 1 an increase in VA/Q mismatch (log SDQ of 0.35 +/- 0.02 at rest and 0.44 +/- 0.02 at exercise; P less than 0.05, n = 7) and in group 2 no change in VA/Q mismatch (n = 6). There were no differences in anthropometric data, work rate, O2 uptake, or ventilation during heavy exercise between groups. Group 1 demonstrated significantly greater VA/Q inequality, lower vital capacity, and higher forced expiratory flow at 25-75% of forced vital capacity for the first 20 min during recovery than group 2. Cardiac index was higher in group 1 both during heavy exercise and 4 and 6 min postexercise. However, both ventilation and cardiac output returned toward baseline values more rapidly than did VA/Q relationships. Arterial pH was lower in group 1 during exercise and recovery. We conclude that greater VA/Q inequality in group 1 and its persistence during recovery are consistent with the hypothesis that edema occurs and contributes to the increase in VA/Q inequality during exercise. This is supported by observation of greater blood flows and acidosis and, presumably therefore

  14. Topiramate Protects Pericytes from Glucotoxicity: Role for Mitochondrial CA VA in Cerebromicrovascular Disease in Diabetes

    PubMed Central

    Patrick, Ping; Price, Tulin O.; Diogo, Ana L.; Sheibani, Nader; Banks, William A.; Shah, Gul N.

    2015-01-01

    Hyperglycemia in diabetes mellitus causes oxidative stress and pericyte depletion from the microvasculature of the brain thus leading to the Blood-Brain Barrier (BBB) disruption. The compromised BBB exposes the brain to circulating substances, resulting in neurotoxicity and neuronal cell death. The decline in pericyte numbers in diabetic mouse brain and pericyte apoptosis in high glucose cultures are caused by excess superoxide produced during enhanced respiration (mitochondrial oxidative metabolism of glucose). Superoxide is precursor to all Reactive Oxygen Species (ROS) which, in turn, cause oxidative stress. The rate of respiration and thus the ROS production is regulated by mitochondrial carbonic anhydrases (mCA) VA and VB, the two isoforms expressed in the mitochondria. Inhibition of both mCA: decreases the oxidative stress and restores the pericyte numbers in diabetic brain; and reduces high glucose-induced respiration, ROS, oxidative stress, and apoptosis in cultured brain pericytes. However, the individual role of the two isoforms has not been established. To investigate the contribution of mCA VA in ROS production and apoptosis, a mCA VA overexpressing brain pericyte cell line was engineered. These cells were exposed to high glucose and analyzed for the changes in ROS and apoptosis. Overexpression of mCA VA significantly increased pericyte ROS and apoptosis. Inhibition of mCA VA with topiramate prevented increases both in glucose-induced ROS and pericyte death. These results demonstrate, for the first time, that mCA VA regulates the rate of pericyte respiration. These findings identify mCA VA as a novel and specific therapeutic target to protect the cerebromicrovascular bed in diabetes. PMID:26167540

  15. Interaction of blood coagulation factor Va with phospholipid vesicles examined by using lipophilic photoreagents

    SciTech Connect

    Krieg, U.C.; Isaacs, B.S.; Yemul, S.S.; Esmon, C.T.; Bayley, H.; Johnson, A.E.

    1987-01-13

    Two different lipophilic photoreagents, (/sup 3/H)adamantane diazirine and 3-(trifluoromethyl)-3-(m-(/sup 125/I)iodophenyl)diazirine (TID), have been utilized to examine the interactions of blood coagulation factor Va with calcium, prothrombin, factor Xa, and, in particular, phospholipid vesicles. With each of these structurally dissimilar reagents, the extent of photolabeling of factor Va was greater when the protein was bound to a membrane surface than when it was free in solution. Specifically, the covalent photoreaction with Vl, the smaller subunit of factor Va, was 2-fold higher in the presence of phosphatidylcholine/phosphatidylserine (PC/PS, 3:1) vesicles, to which factor Va binds, than in the presence of 100% PC vesicles, to which the protein does not bind. However, the magnitude of the PC/PS-dependent photolabeling was much less than has been observed previously with integral membrane proteins. It therefore appears that the binding of factor Va to the membrane surface exposes Vl to the lipid core of the bilayer, but that only a small portion of the Vl polypeptide is exposed to, or embedded in, the bilayer core. Addition of either prothrombin or active-site-blocked factor Xa to PC/PS-bound factor Va had little effect on the photolabeling of Vl with TID, but reduced substantially the covalent labeling of Vh, the larger subunit of factor Va. This indicates that prothrombin and factor Xa each cover nonpolar surfaces on Vh when the macromolecules associate on the PC/PS surface. It therefore seems likely that the formation of the prothrombinase complex involves a direct interaction between Vh and factor Xa and between Vh and prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Streptococcal R plasmid pIP501: endonuclease site map, resistance determinant location, and construction of novel derivatives.

    PubMed Central

    Evans, R P; Macrina, F L

    1983-01-01

    The streptococcal resistance plasmid pIP501 (30 kilobase pairs [kb]) encodes resistance to chloramphenicol (Cmr) and erythromycin (Emr) and is capable of conjugative transfer among numerous streptococcal species. By using a streptococcal host-vector recombinant DNA system, the Cmr and Emr determinants of pIP501 were localized to 6.3-kb HindIII and 2.1-kb HindIII-AvaI fragments, respectively. pIP501 was lost at a frequency of 22% in Streptococcus sanguis cells grown at 42 degrees C but was stable in cells grown at 37 degrees C (less than 1% frequency of loss). Sequences from a cryptic multicopy plasmid, pVA380-1, were substituted for the pIP501 Emr determinant in vitro, and the resulting recombinant plasmid, designated pVA797, was recovered in transformed S. sanguis cells. The replication of pVA797 was governed by the pVA380-1 sequences based on temperature-stable replication and incompatibility with pVA380-1-derived replicons. The self-ligation of partially cleaved HindIII pIP501 DNA fragments allowed the localization of a pIP501 region involved in autonomous plasmid replication. A small pIP501 derivative (pVA798) obtained from this experiment had a greatly increased copy number but was unstably inherited. Our data indicate that the sequences encoding the resistance determinants and some of the plasmid replication machinery are relatively clustered on the pIP501 molecule. The properties of pVA797 and pVA798 indicate that these molecules will enhance current streptococcal genetic systems from the standpoint of conjugative mobilization (pVA797) and gene amplification (pVA798). PMID:6304011

  17. Within-host competition between Borrelia afzelii ospC strains in wild hosts as revealed by massively parallel amplicon sequencing.

    PubMed

    Strandh, Maria; Råberg, Lars

    2015-08-19

    Infections frequently consist of more than one strain of a given pathogen. Experiments have shown that co-infecting strains often compete, so that the infection intensity of each strain in mixed infections is lower than in single strain infections. Such within-host competition can have important epidemiological and evolutionary consequences. However, the extent of competition has rarely been investigated in wild, naturally infected hosts, where there is noise in the form of varying inoculation doses, asynchronous infections and host heterogeneity, which can potentially alleviate or eliminate competition. Here, we investigated the extent of competition between Borrelia afzelii strains (as determined by ospC genotype) in three host species sampled in the wild. For this purpose, we developed a protocol for 454 amplicon sequencing of ospC, which allows both detection and quantification of each individual strain in an infection. Each host individual was infected with one to six ospC strains. The infection intensity of each strain was lower in mixed infections than in single ones, showing that there was competition. Rank-abundance plots revealed that there was typically one dominant strain, but that the evenness of the relative infection intensity of the different strains in an infection increased with the multiplicity of infection. We conclude that within-host competition can play an important role under natural conditions despite many potential sources of noise, and that quantification by next-generation amplicon sequencing offers new possibilities to dissect within-host interactions in naturally infected hosts. PMID:26150659

  18. Allele-specific extension allows base-pair neutral homozygotes to be discriminated by high-resolution melting of small amplicons.

    PubMed

    Cai, Yanning; Yuan, Yanpeng; Lin, Qingling; Chan, Piu

    2010-11-01

    Not all single-nucleotide polymorphisms (SNPs) can be determined using high-resolution melting (HRM) of small amplicons, especially class 3 and 4 SNPs. This is due mainly to the small shift in the melting temperature (Tm) between two types of homozygote. Choosing rs1869458 (a class 4 SNP) as a sample, we developed a modified small amplicon HRM assay. An allele-specific extension (ASE) primer, which ended at an SNP site and matched only one of the alleles, was added to the reaction as well as additional thermal steps for ASE. Following asymmetric polymerase chain reaction and melting curve analysis, heterozygotes were easily identified. Two types of homozygote were also distinguishable, indicating that extension primers 11 to 13 bases in length worked efficiently in an allele-specific way. Modification of the limiting amplification primer with locked nucleic acid increased the Tm difference between extension and amplification peaks and facilitated subsequent genotyping. In addition, 194 human genomic DNA samples were genotyped with the developed assay and by direct sequencing, with the different methods providing identical genotyping results. In conclusion, ASE-HRM is a simple, inexpensive, closed-tube genotyping method that can be used to examine all types of SNP. PMID:20599636

  19. Within-host competition between Borrelia afzelii ospC strains in wild hosts as revealed by massively parallel amplicon sequencing

    PubMed Central

    Strandh, Maria; Råberg, Lars

    2015-01-01

    Infections frequently consist of more than one strain of a given pathogen. Experiments have shown that co-infecting strains often compete, so that the infection intensity of each strain in mixed infections is lower than in single strain infections. Such within-host competition can have important epidemiological and evolutionary consequences. However, the extent of competition has rarely been investigated in wild, naturally infected hosts, where there is noise in the form of varying inoculation doses, asynchronous infections and host heterogeneity, which can potentially alleviate or eliminate competition. Here, we investigated the extent of competition between Borrelia afzelii strains (as determined by ospC genotype) in three host species sampled in the wild. For this purpose, we developed a protocol for 454 amplicon sequencing of ospC, which allows both detection and quantification of each individual strain in an infection. Each host individual was infected with one to six ospC strains. The infection intensity of each strain was lower in mixed infections than in single ones, showing that there was competition. Rank-abundance plots revealed that there was typically one dominant strain, but that the evenness of the relative infection intensity of the different strains in an infection increased with the multiplicity of infection. We conclude that within-host competition can play an important role under natural conditions despite many potential sources of noise, and that quantification by next-generation amplicon sequencing offers new possibilities to dissect within-host interactions in naturally infected hosts. PMID:26150659

  20. A New Method for Rapid Screening of End-Point PCR Products: Application to Single Genome Amplified HIV and SIV Envelope Amplicons.

    PubMed

    Houzet, Laurent; Deleage, Claire; Satie, Anne-Pascale; Merlande, Laetitia; Mahe, Dominique; Dejucq-Rainsford, Nathalie

    2015-01-01

    PCR is the most widely applied technique for large scale screening of bacterial clones, mouse genotypes, virus genomes etc. A drawback of large PCR screening is that amplicon analysis is usually performed using gel electrophoresis, a step that is very labor intensive, tedious and chemical waste generating. Single genome amplification (SGA) is used to characterize the diversity and evolutionary dynamics of virus populations within infected hosts. SGA is based on the isolation of single template molecule using limiting dilution followed by nested PCR amplification and requires the analysis of hundreds of reactions per sample, making large scale SGA studies very challenging. Here we present a novel approach entitled Long Amplicon Melt Profiling (LAMP) based on the analysis of the melting profile of the PCR reactions using SYBR Green and/or EvaGreen fluorescent dyes. The LAMP method represents an attractive alternative to gel electrophoresis and enables the quick discrimination of positive reactions. We validate LAMP for SIV and HIV env-SGA, in 96- and 384-well plate formats. Because the melt profiling allows the screening of several thousands of PCR reactions in a cost-effective, rapid and robust way, we believe it will greatly facilitate any large scale PCR screening. PMID:26053379

  1. Improving Memory Subsystem Performance Using ViVA: Virtual Vector Architecture

    SciTech Connect

    Gebis, Joseph; Oliker, Leonid; Shalf, John; Williams, Samuel; Yelick, Katherine

    2009-01-12

    The disparity between microprocessor clock frequencies and memory latency is a primary reason why many demanding applications run well below peak achievable performance. Software controlled scratchpad memories, such as the Cell local store, attempt to ameliorate this discrepancy by enabling precise control over memory movement; however, scratchpad technology confronts the programmer and compiler with an unfamiliar and difficult programming model. In this work, we present the Virtual Vector Architecture (ViVA), which combines the memory semantics of vector computers with a software-controlled scratchpad memory in order to provide a more effective and practical approach to latency hiding. ViVA requires minimal changes to the core design and could thus be easily integrated with conventional processor cores. To validate our approach, we implemented ViVA on the Mambo cycle-accurate full system simulator, which was carefully calibrated to match the performance on our underlying PowerPC Apple G5 architecture. Results show that ViVA is able to deliver significant performance benefits over scalar techniques for a variety of memory access patterns as well as two important memory-bound compact kernels, corner turn and sparse matrix-vector multiplication -- achieving 2x-13x improvement compared the scalar version. Overall, our preliminary ViVA exploration points to a promising approach for improving application performance on leading microprocessors with minimal design and complexity costs, in a power efficient manner.

  2. 48 CFR 852.219-72 - Evaluation factor for participation in the VA mentor-protégé program.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... participation in the VA mentor-protégé program. 852.219-72 Section 852.219-72 Federal Acquisition Regulations... Texts of Provisions and Clauses 852.219-72 Evaluation factor for participation in the VA mentor-protégé... the VA Mentor-Protégé Program (DEC2009) This solicitation contains an evaluation factor or...

  3. 48 CFR 852.219-72 - Evaluation factor for participation in the VA mentor-protégé program.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... participation in the VA mentor-protégé program. 852.219-72 Section 852.219-72 Federal Acquisition Regulations... Texts of Provisions and Clauses 852.219-72 Evaluation factor for participation in the VA mentor-protégé... the VA Mentor-Protégé Program (DEC2009) This solicitation contains an evaluation factor or...

  4. 48 CFR 852.219-72 - Evaluation factor for participation in the VA mentor-protégé program.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... participation in the VA mentor-protégé program. 852.219-72 Section 852.219-72 Federal Acquisition Regulations... Texts of Provisions and Clauses 852.219-72 Evaluation factor for participation in the VA mentor-protégé... the VA Mentor-Protégé Program (DEC2009) This solicitation contains an evaluation factor or...

  5. 48 CFR 852.219-72 - Evaluation factor for participation in the VA mentor-protégé program.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... participation in the VA mentor-protégé program. 852.219-72 Section 852.219-72 Federal Acquisition Regulations... Texts of Provisions and Clauses 852.219-72 Evaluation factor for participation in the VA mentor-protégé... the VA Mentor-Protégé Program (DEC2009) This solicitation contains an evaluation factor or...

  6. 48 CFR 852.219-72 - Evaluation factor for participation in the VA mentor-protégé program.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... participation in the VA mentor-protégé program. 852.219-72 Section 852.219-72 Federal Acquisition Regulations... Texts of Provisions and Clauses 852.219-72 Evaluation factor for participation in the VA mentor-protégé... the VA Mentor-Protégé Program (DEC2009) This solicitation contains an evaluation factor or...

  7. Hi-Plex targeted sequencing is effective using DNA derived from archival dried blood spots.

    PubMed

    Nguyen-Dumont, T; Mahmoodi, M; Hammet, F; Tran, T; Tsimiklis, H; Giles, G G; Hopper, J L; Southey, M C; Park, D J

    2015-02-01

    Many genetic epidemiology resources have collected dried blood spots (predominantly as Guthrie Cards) as an economical and efficient means of archiving sources of DNA, conferring great value to genetic screening methods that are compatible with this medium. We applied Hi-Plex to screen the breast cancer predisposition gene PALB2 in 93 Guthrie Card-derived DNA specimens previously characterized for PALB2 genetic variants via DNA derived from lymphoblastoid cell lines, whole blood, and buffy coat. Of the 93 archival Guthrie Card-derived DNAs, 92 (99%) were processed successfully and sequenced using approximately half of a MiSeq run. From these 92 DNAs, all 59 known variants were detected and no false-positive variant calls were yielded. Fully 98.13% of amplicons (5417/5520) were represented within 15-fold of the median coverage (2786 reads), and 99.98% of amplicons (5519/5520) were represented at a depth of 10 read-pairs or greater. With Hi-Plex, we show for the first time that a High-Plex amplicon-based massively parallel sequencing (MPS) system can be applied effectively to DNA prepared from dried blood spot archival specimens and, as such, can dramatically increase the scopes of both method and resource. PMID:25447460

  8. Hi-Plex targeted sequencing is effective using DNA derived from archival dried blood spots

    PubMed Central

    Nguyen-Dumont, T; Mahmoodi, M; Hammet, F; Tran, T; Tsimiklis, H; Giles, GG; Hopper, JL; Southey, MC; Park, DJ

    2014-01-01

    Many genetic epidemiology resources have collected dried blood spots (predominantly as Guthrie Cards) as an economical and efficient means of archiving sources of DNA, conferring great value to genetic screening methods that are compatible with this medium. We applied Hi-Plex to screen the breast cancer predisposition gene PALB2 in 93 Guthrie Card-derived DNA specimens previously characterised for PALB2 genetic variants via DNA derived from lymphoblastoid cell lines, whole blood and buffy coat. 92 of the 93 archival Guthrie Card-derived DNAs (99%) were processed successfully and sequenced using approximately half of a MiSeq run. From these 92, all 59 known variants were detected and no false-positive variant calls were yielded. 98.13% of amplicons (5417/5520) were represented within 15-fold of the median coverage (2786 reads) and 99.98% of amplicons (5519/5520) were represented at a depth of 10 read-pairs or greater. With Hi-Plex, we show for the first time that a high-plex amplicon based MPS system can be applied effectively to DNA prepared from dried blood spot archival specimens and, as such, dramatically increase the scopes of both method and resource. PMID:25447460

  9. Performance-based budgeting in the public sector: an illustration from the VA health care system.

    PubMed

    Yaisawarng, Suthathip; Burgess, James F

    2006-03-01

    This paper estimates frontier cost functions for US Department of Veterans Affairs (VA) hospitals in FY2000 that are consistent with economic theory and explicitly account for cost differences across patients' risk, level of access to care, quality of care, and hospital-specific characteristics. Results indicate that on average VA hospitals in FY2000 operate at efficiency levels of 94%, as compared to previous studies on US private sector hospitals that average closer to 90% efficient. Using these cost frontiers, management systems potentially could be implemented to enhance the equitable allocation of the VA medical care global budget and systematically distribute funds across hospitals and networks. The paper also provides recommendations to improve the efficiency of delivering health care services applicable to public sector organizations. PMID:16331724

  10. The VA's use of DICOM to integrate image data seamlessly into the online patient record.

    PubMed Central

    Kuzmak, P. M.; Dayhoff, R. E.

    1999-01-01

    The US Department of Veterans Affairs (VA) is using the Digital Imaging and Communications in Medicine (DICOM) standard to integrate image data objects from multiple systems for use across the healthcare enterprise. DICOM uses a structured representation of image data and a communication mechanism that allows the VA to easily acquire radiology images and store them directly into the online patient record. Images can then be displayed on low-cost clinician's workstations throughout the medical center. High-resolution diagnostic quality multi-monitor VistA workstations with specialized viewing software can be used for reading radiology images. Various image and study specific items from the DICOM data object are essential for the correct display of images. The VA's DICOM capabilities are now used to interface seven different commercial Picture Archiving and Communication Systems (PACS) and over twenty different radiology image acquisition modalities. PMID:10566327

  11. VA Disability Compensation and Money Spent on Substance Use Among Homeless Veterans: A Controversial Association

    PubMed Central

    Tsai, Jack; Rosenheck, Robert A.

    2015-01-01

    Objective There has long been concern that public support payments are used to support addictive behaviors. This study examined the amount of money homeless veterans spend on alcohol and drugs and the association between public support income, including VA disability compensation, and expenditures on alcohol and drugs. Methods Data were from 1,160 veterans from 19 sites on entry into the Housing and Urban Development–Veterans Affairs Supportive Housing program. Descriptive statistics and nonparametric analyses were conducted. Results About 33% of veterans reported spending money on alcohol and 22% reported spending money on drugs in the past month. No significant association was found between public support income, VA disability compensation, and money spent on alcohol and drugs. Conclusions A substantial proportion of homeless veterans spend some income on alcohol and drugs, but disability income, including VA compensation, does not seem to be related to substance use or money spent on addictive substances. PMID:25726979

  12. Conversion of Input Data between KENO V.a and MCNP File Formats, Version 5L.

    Energy Science and Technology Software Center (ESTSC)

    2007-10-31

    Version 00 The KENO2MCNP program was written to convert KENO V.a input files to MCNP Format. This program currently only works with KENO Va geometries and will not work with geometries that contain more than a single array. A C++ graphical user interface was created that was linked to Fortran routines from KENO V.a that read the material library and Fortran routines from the MCNP Visual Editor that generate the MCNP input file. Either SCALEmore » 5.0 or SCALE 5.1 cross section files will work with this release. This version of KENO2MCNP was tested with CCC-730/MCNP5 1.40 and with CCC-725/SCALE5.0 and CCC-732/SCALE 5.1. Note that this distribution does not include either MCNP or SCALE, which are available separately through either RSICC or the NEA Data Bank.« less

  13. Effects of the KiVa antibullying program on cyberbullying and cybervictimization frequency among Finnish youth.

    PubMed

    Williford, Anne; Elledge, L Christian; Boulton, Aaron J; DePaolis, Kathryn J; Little, Todd D; Salmivalli, Christina

    2013-01-01

    Cyberbullying among school-aged children has received increased attention in recent literature. However, no empirical evidence currently exists on whether existing school-based antibullying programs are effective in targeting the unique aspects of cyberbullying. To address this important gap, the present study investigates the unique effects of the KiVa Antibullying Program on the frequency of cyberbullying and cybervictimization among elementary and middle school youth. Using data from a group randomized controlled trial, multilevel ordinal regression analyses were used to examine differences in the frequencies of cyberbullying and cybervictimization between intervention (N = 9,914) and control students (N = 8,498). The effects of age and gender on frequencies of cyber behaviors were also assessed across conditions. Results revealed a significant intervention effect on the frequency of cybervictimization; KiVa students reported lower frequencies of cybervictimization at posttest than students in a control condition. The effect of condition on the perpetration of cyberbullying was moderated by age. When student age was below the sample mean, KiVa students reported lower frequencies of cyberbullying than students in the control condition. We also found evidence of classroom level variation in cyberbullying and cybervictimization, suggesting cyberbullying is in part a classroom-level phenomenon. KiVa appears to be an efficacious program to address cyber forms of bullying and victimization. We discuss several unique aspects of KiVa that may account for the significant intervention effects. Results suggest that KiVa is an intervention option for schools concerned with reducing cyberbullying behavior and its deleterious effects on children's adjustment. PMID:23659182

  14. Myosin-Va and dynamic actin oppose microtubules to drive long-range organelle transport.

    PubMed

    Evans, Richard D; Robinson, Christopher; Briggs, Deborah A; Tooth, David J; Ramalho, Jose S; Cantero, Marta; Montoliu, Lluis; Patel, Shyamal; Sviderskaya, Elena V; Hume, Alistair N

    2014-08-01

    In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the "highways and local roads" model for transport along microtubule and actin tracks. The "cooperative capture" model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning. PMID:25065759

  15. Motor coupling through lipid membranes enhances transport velocities for ensembles of myosin Va

    PubMed Central

    Nelson, Shane R.; Trybus, Kathleen M.; Warshaw, David M.

    2014-01-01

    Myosin Va is an actin-based molecular motor responsible for transport and positioning of a wide array of intracellular cargoes. Although myosin Va motors have been well characterized at the single-molecule level, physiological transport is carried out by ensembles of motors. Studies that explore the behavior of ensembles of molecular motors have used nonphysiological cargoes such as DNA linkers or glass beads, which do not reproduce one key aspect of vesicular systems—the fluid intermotor coupling of biological lipid membranes. Using a system of defined synthetic lipid vesicles (100- to 650-nm diameter) composed of either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (fluid at room temperature) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (gel at room temperature) with a range of surface densities of myosin Va motors (32–125 motors per μm2), we demonstrate that the velocity of vesicle transport by ensembles of myosin Va is sensitive to properties of the cargo. Gel-state DPPC vesicles bound with multiple motors travel at velocities equal to or less than vesicles with a single myosin Va (∼450 nm/s), whereas surprisingly, ensembles of myosin Va are able to transport fluid-state DOPC vesicles at velocities significantly faster (>700 nm/s) than a single motor. To explain these data, we developed a Monte Carlo simulation that suggests that these reductions in velocity can be attributed to two distinct mechanisms of intermotor interference (i.e., load-dependent modulation of stepping kinetics and binding-site exclusion), whereas faster transport velocities are consistent with a model wherein the normal stepping behavior of the myosin is supplemented by the preferential detachment of the trailing motor from the actin track. PMID:25201964

  16. Physical stability of solid dispersions with respect to thermodynamic solubility of tadalafil in PVP-VA.

    PubMed

    Wlodarski, K; Sawicki, W; Kozyra, A; Tajber, L

    2015-10-01

    The aim of this paper was to evaluate physical stability of solid dispersions in respect to the drug, tadalafil (Td), in vinylpyrrolidone and vinyl acetate block copolymer (PVP-VA). Nine solid dispersions of Td in PVP-VA (Td/PVP-VA) varied in terms of quantitative composition (1:9-9:1, w/w) were successfully produced by spray-drying. Their amorphous nature, supersaturated character and molecular level of mixing (a solid solution structure) were subsequently confirmed using DSC, PXRD, SEM and calculation of Hansen total solubility parameters. Due to thermal degradation of both components before the melting point of Td (302.3°C), an approach based on the drug crystallization from the supersaturated solid dispersion was selected to calculate the solubility of Td in the polymer. Annealing of the Td/PVP-VA solid dispersion (1:1, w/w) at selected temperatures above its Tg resulted in different stable solid dispersions. According to the Gordon-Taylor equation their new Tgs gave the information about the quantitative composition which corresponded to the thermodynamic solubility of Td in PVP-VA at given temperatures of annealing. The obtained relationship was fitted to the exponential function, with the calculated solubility of Td of 20.5% at 25°C. This value was in accordance with the results of hot stage polarizing light microscopy as well as stability tests carried out at 80°C and 0% RH, in which Td solid dispersions containing 10-20% of the drug were the only systems that did not crystallize within two months. A thermal analysis protocol utilizing a fast heating rate was shown to generate Td solubility data complementing the solid dispersion method. The Flory-Huggins model applied for the Td/PVP-VA system yielded the solubility value of 0.1% at 25°C, showing the lack of applicability in this case. PMID:26247119

  17. Comparison of Friction Characteristics on TN and VA Mode Alignment Films with Friction Force Microscopy

    NASA Astrophysics Data System (ADS)

    Kwak, Musun; Chung, Hanrok; Kwon, Hyukmin; Kim, Jehyun; Han, Daekyung; Yi, Yoonseon; Lee, Sangmun; Lee, Chulgu; Cha, Sooyoul

    Using frictional force microscopy (FFM), the friction surface characteristics were compared between twisted nematic (TN) mode and vertical alignment (VA) mode alignment films (AFs). The friction asymmetry was detected depending on temperature conditions on TN mode AF, but not on VA mode AF. The difference between two modes was explained by leaning intermolecular repulsion caused by the pre-tilt angle uniformity and the density of side chain. No level difference according to temperature conditions appeared when the pre-tilt angle were measured after liquid crystal (LC) injection.

  18. Improving PTSD/substance abuse treatment in the VA: a survey of providers.

    PubMed

    Najavits, Lisa M; Norman, Sonya B; Kivlahan, Daniel; Kosten, Thomas R

    2010-01-01

    We surveyed 205 Veterans Affairs (VA) staff on treatment of posttraumatic stress disorder (PTSD), substance use disorder (SUD), and the combination (PTSD/SUD). The survey was anonymous and VA-wide. PTSD/SUD was perceived as more difficult to treat than either disorder alone; gratification in the work was stronger than difficulty (for PTSD, SUD, and PTSD/SUD); and difficulty and gratification appeared separate constructs. Respondents endorsed views that represent expert treatment for the comorbidity; however, there was also endorsement of "myths." Thus, there is a need for more training, policy clarifications, service integration, and adaptations for veterans returning from Iraq and Afghanistan. Limitations are described. PMID:20525033

  19. Influence of PVP/VA copolymer composition on drug-polymer solubility.

    PubMed

    Rask, Malte Bille; Knopp, Matthias Manne; Olesen, Niels Erik; Holm, René; Rades, Thomas

    2016-03-31

    In this study, the influence of copolymer composition on drug-polymer solubility was investigated. The solubility of the model drug celecoxib (CCX) in various polyvinylpyrrolidone/vinyl acetate (PVP/VA) copolymer compositions (70/30, 60/40, 50/50 and 30/70 w/w) and the pure homopolymers polyvinylpyrrolidone (PVP) and polyvinyl acetate (PVA) was predicted at 25 °C using a thermal analysis method based on the recrystallization of a supersaturated amorphous dispersion (recrystallization method). These solubilities were compared with a prediction based on the solubility of CCX in the liquid monomeric precursors of PVP/VA, N-vinylpyrrolidone (NVP) and vinyl acetate (VA), using the Flory-Huggins lattice theory (liquid monomer solubility approach). The solubilities predicted from the liquid monomer solubility approach increased linearly with increasing VP/VA ratio from 0.03-0.60 w/w. Even though the solubilities predicted from the recrystallization method also increased with increasing VP/VA ratio from 0.02-0.40 w/w, the predicted solubility seemed to approach a plateau at high VP/VA ratios. Increasing positive deviations from the Gordon-Taylor equation with increasing VP/VA ratio indicated strong interactions between CCX and the VP repeat unit, which was in accordance with the relatively high solubilities predicted using both methods. As the solubility plateau may be a consequence of steric hindrance caused by the size differences between CCX and the VP repeat units, it is likely that a CCX molecule interacting with a VP repeat unit hinders another CCX molecule from binding to the neighboring repeat units in the polymer chain. Therefore, it is possible that replacing these neighboring hygroscopic VP repeat units with hydrophobic VA repeat units, could increase the physical stability of an amorphous solid dispersion without compromising the drug-polymer solubility. This knowledge could be used advantageously in future development of amorphous drug delivery systems as

  20. Contribution of Amino Acid Region 334−335 from Factor Va Heavy Chain to the Catalytic Efficiency of Prothrombinase†

    PubMed Central

    2008-01-01

    We have demonstrated that amino acids E323, Y324, E330, and V331 from the factor Va heavy chain are required for the interaction of the cofactor with factor Xa and optimum rates of prothrombin cleavage. We have also shown that amino acid region 332−336 contains residues that are important for cofactor function. Using overlapping peptides, we identified amino acids D334 and Y335 as contributors to cofactor activity. We constructed recombinant factor V molecules with the mutations D334 → K and Y335 → F (factor VKF) and D334 → A and Y335 → A (factor VAA). Kinetic studies showed that while factor VaKF and factor VaAA had a KD for factor Xa similar to the KD observed for wild-type factor Va (factor VaWT), the clotting activities of the mutant molecules were impaired and the kcat of prothrombinase assembled with factor VaKF and factor VaAA was reduced. The second-order rate constant of prothrombinase assembled with factor VaKF or factor VaAA for prothrombin activation was ∼10-fold lower than the second-order rate constant for the same reaction catalyzed by prothrombinase assembled with factor VaWT. We also created quadruple mutants combining mutations in the amino acid region 334–335 with mutations at the previously identified amino acids that are important for factor Xa binding (i.e., E323Y324 and E330V331). Prothrombinase assembled with the quadruple mutant molecules displayed a second-order rate constant up to 400-fold lower than the values obtained with prothrombinase assembled with factor VaWT. The data demonstrate that amino acid region 334–335 is required for the rearrangement of enzyme and substrate necessary for efficient catalysis of prothrombin by prothrombinase. PMID:18537263

  1. Cooperative Regulation of the Activity of Factor Xa within Prothrombinase by Discrete Amino Acid Regions from Factor Va Heavy Chain†

    PubMed Central

    2008-01-01

    The prothrombinase complex catalyzes the activation of prothrombin to α-thrombin. We have repetitively shown that amino acid region 695DYDY698 from the COOH terminus of the heavy chain of factor Va regulates the rate of cleavage of prothrombin at Arg271 by prothrombinase. We have also recently demonstrated that amino acid region 334DY335 is required for the optimal activity of prothrombinase. To assess the effect of these six amino acid residues on cofactor activity, we created recombinant factor Va molecules combining mutations at amino acid regions 334–335 and 695−698 as follows: factor V3K (334DY335 → KF and 695DYDY698 → KFKF), factor VKF/4A (334DY335 → KF and 695DYDY698 → AAAA), and factor V6A (334DY335 → AA and 695DYDY698 → AAAA). The recombinant factor V molecules were expressed and purified to homogeneity. Factor Va3K, factor VaK4/4A, and factor Va6A had reduced affinity for factor Xa, when compared to the affinity of the wild-type molecule (factor VaWt) for the enzyme. Prothrombinase assembled with saturating concentrations of factor Va3K had a 6-fold reduced second-order rate constant for prothrombin activation compared to the value obtained with prothrombinase assembled with factor VaWt, while prothrombinase assembled with saturating concentrations of factor VaKF/4A and factor Va6A had approximately 1.5-fold reduced second-order rate constants. Overall, the data demonstrate that amino acid region 334–335 together with amino acid region 695−698 from factor Va heavy chain are part of a cooperative mechanism within prothrombinase regulating cleavage and activation of prothrombin by factor Xa. PMID:18991406

  2. Myosin Va Participates in Acrosomal Formation and Nuclear Morphogenesis during Spermatogenesis of Chinese Mitten Crab Eriocheir sinensis

    PubMed Central

    Sun, Xiao; He, Ying; Hou, Lin; Yang, Wan-Xi

    2010-01-01

    Background The Chinese mitten crab Eriocheir sinensis belongs to the Class Crustacea, Decapoda, Brachyura. The spermatozoon of this species is of aflagellated type, it has a spherical acrosome surrounded by the cup-shaped nucleus, which are unique to brachyurans. For the past several decades, studies on the spermatogenesis of the mitten crab mainly focus on the morphology. Compared with the extensive study of molecular mechanism of spermatogenesis in mammals, relatively less information is available in crustacean species. Myosin Va, a member of Class V myosin, has been implicated in acrosome biogenesis and vesicle transport during spermatogenesis in mammals. In the present study we demonstrate the expression and cellular localization of myosin Va during spermatogenesis in E. sinensis. Methodology/Principal Findings Western blot demonstrated that myosin Va is expressed during spermatogenesis. Immunocytochemical and ultrastructural analyses showed that myosin Va mainly localizes in the cytoplasm in spermatocytes. At the early stage of spermiogenesis, myosin Va binds to the endoplasmic reticulum vesicle (EV) and proacrosomal granule (PG). Subsequently, myosin Va localizes within the proacrosomal vesicle (PV) formed by PG and EV fusion and locates in the membrane complex (MC) at the mid spermatid stage. At the late spermatid stage, myosin Va is associated with the shaping nucleus and mitochondria. In mature spermatozoon, myosin Va predominates in acrosomal tubule (AT) and nucleus. Conclusions/Significance Our study demonstrates that myosin Va may be involved in acrosome biogenesis and nuclear morphogenesis during spermatogenesis in E. sinensis. Considering the distribution and molecular characteristics of myosin Va, we also propose a hypothesis of AT formation in this species. It is the first time to uncover the role of myosin Va in crustacean spermatogenesis. PMID:20856877

  3. 16S rRNA Amplicon Sequencing Demonstrates that Indoor-Reared Bumblebees (Bombus terrestris) Harbor a Core Subset of Bacteria Normally Associated with the Wild Host

    PubMed Central

    Meeus, Ivan; Parmentier, Laurian; Billiet, Annelies; Maebe, Kevin; Van Nieuwerburgh, Filip; Deforce, Dieter; Wäckers, Felix; Vandamme, Peter; Smagghe, Guy

    2015-01-01

    A MiSeq multiplexed 16S rRNA amplicon sequencing of the gut microbiota of wild and indoor-reared Bombus terrestris (bumblebees) confirmed the presence of a core set of bacteria, which consisted of Neisseriaceae (Snodgrassella), Orbaceae (Gilliamella), Lactobacillaceae (Lactobacillus), and Bifidobacteriaceae (Bifidobacterium). In wild B. terrestris we detected several non-core bacteria having a more variable prevalence. Although Enterobacteriaceae are unreported by non next-generation sequencing studies, it can become a dominant gut resident. Furthermore the presence of some non-core lactobacilli were associated with the relative abundance of bifidobacteria. This association was not observed in indoor-reared bumblebees lacking the non-core bacteria, but having a more standardized microbiota compared to their wild counterparts. The impact of the bottleneck microbiota of indoor-reared bumblebees when they are used in the field for pollination purpose is discussed. PMID:25923917

  4. Bacterial tag encoded FLX titanium amplicon pyrosequencing (bTEFAP) based assessment of prokaryotic diversity in metagenome of Lonar soda lake, India

    PubMed Central

    Dudhagara, Pravin; Ghelani, Anjana; Patel, Rajesh; Chaudhari, Rajesh; Bhatt, Shreyas

    2015-01-01

    Bacterial diversity and archaeal diversity in metagenome of the Lonar soda lake sediment were assessed by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). Metagenome comprised 5093 sequences with 2,531,282 bp and 53 ± 2% G + C content. Metagenome sequence data are available at NCBI under the Bioproject database with accession no. PRJNA218849. Metagenome sequence represented the presence of 83.1% bacterial and 10.5% archaeal origin. A total of 14 different bacteria demonstrating 57 species were recorded with dominating species like Coxiella burnetii (17%), Fibrobacter intestinalis (12%) and Candidatus Cloacamonas acidaminovorans (11%). Occurrence of two archaeal phyla representing 24 species, among them Methanosaeta harundinacea (35%), Methanoculleus chikugoensis (12%) and Methanolinea tarda (11%) were dominating species. Significant presence of 11% sequences as an unclassified indicated the possibilities for unknown novel prokaryotes from the metagenome. PMID:26484168

  5. Pyrosequencing of 16S rRNA gene amplicons to study the microbiota in the gastrointestinal tract of carp (Cyprinus carpio L.)

    PubMed Central

    2011-01-01

    The microbes in the gastrointestinal (GI) tract are of high importance for the health of the host. In this study, Roche 454 pyrosequencing was applied to a pooled set of different 16S rRNA gene amplicons obtained from GI content of common carp (Cyprinus carpio) to make an inventory of the diversity of the microbiota in the GI tract. Compared to other studies, our culture-independent investigation reveals an impressive diversity of the microbial flora of the carp GI tract. The major group of obtained sequences belonged to the phylum Fusobacteria. Bacteroidetes, Planctomycetes and Gammaproteobacteria were other well represented groups of micro-organisms. Verrucomicrobiae, Clostridia and Bacilli (the latter two belonging to the phylum Firmicutes) had fewer representatives among the analyzed sequences. Many of these bacteria might be of high physiological relevance for carp as these groups have been implicated in vitamin production, nitrogen cycling and (cellulose) fermentation. PMID:22093413

  6. 33 CFR 334.330 - Atlantic Ocean and connecting waters in vicinity of Myrtle Island, Va.; Air Force practice...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 3 2010-07-01 2010-07-01 false Atlantic Ocean and connecting waters in vicinity of Myrtle Island, Va.; Air Force practice bombing, rocket firing, and gunnery range... waters in vicinity of Myrtle Island, Va.; Air Force practice bombing, rocket firing, and gunnery...

  7. 33 CFR 334.330 - Atlantic Ocean and connecting waters in vicinity of Myrtle Island, Va.; Air Force practice...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 3 2014-07-01 2014-07-01 false Atlantic Ocean and connecting waters in vicinity of Myrtle Island, Va.; Air Force practice bombing, rocket firing, and gunnery range... waters in vicinity of Myrtle Island, Va.; Air Force practice bombing, rocket firing, and gunnery...

  8. 33 CFR 334.280 - James River between the entrance to Skiffes Creek and Mulberry Point, Va.; army training and...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 3 2011-07-01 2011-07-01 false James River between the entrance to Skiffes Creek and Mulberry Point, Va.; army training and small craft testing area. 334.280 Section... Creek and Mulberry Point, Va.; army training and small craft testing area. (a) The restricted...

  9. 33 CFR 334.280 - James River between the entrance to Skiffes Creek and Mulberry Point, Va.; army training and...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 3 2012-07-01 2012-07-01 false James River between the entrance to Skiffes Creek and Mulberry Point, Va.; army training and small craft testing area. 334.280 Section... Creek and Mulberry Point, Va.; army training and small craft testing area. (a) The restricted...

  10. 33 CFR 334.280 - James River between the entrance to Skiffes Creek and Mulberry Point, Va.; army training and...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 3 2013-07-01 2013-07-01 false James River between the entrance to Skiffes Creek and Mulberry Point, Va.; army training and small craft testing area. 334.280 Section... Creek and Mulberry Point, Va.; army training and small craft testing area. (a) The restricted...

  11. 33 CFR 334.280 - James River between the entrance to Skiffes Creek and Mulberry Point, Va.; army training and...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 3 2010-07-01 2010-07-01 false James River between the entrance to Skiffes Creek and Mulberry Point, Va.; army training and small craft testing area. 334.280 Section... Creek and Mulberry Point, Va.; army training and small craft testing area. (a) The restricted...

  12. 33 CFR 334.280 - James River between the entrance to Skiffes Creek and Mulberry Point, Va.; army training and...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 3 2014-07-01 2014-07-01 false James River between the entrance to Skiffes Creek and Mulberry Point, Va.; army training and small craft testing area. 334.280 Section... Creek and Mulberry Point, Va.; army training and small craft testing area. (a) The restricted...

  13. 77 FR 23615 - Payment or Reimbursement for Emergency Services for Nonservice-Connected Conditions in Non-VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-20

    ... Sec. 17.1004(f). In a document published in the Federal Register on May 26, 2011 (76 FR 30598), VA... FR 79071), VA published an entirely separate final rule that added new paragraphs (c) and (d) to Sec...'s administration.'' We thank the commenter for taking the time to ] comment and for the...

  14. 75 FR 61621 - Charges Billed to Third Parties for Prescription Drugs Furnished by VA to a Veteran for a...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-06

    ...), prior to the effective date of this document, VA billed $51 for each prescription filled (see 70 FR 66866, Nov. 3, 2005). In a document published in the Federal Register on July 9, 2009 (74 FR 32819), we... the Outpatient Pharmacy Prescription file or the Drug file at each VA facility (74 FR 32820)....

  15. 75 FR 30915 - Computer Matching Program Between the Department of Veterans Affairs (VA) and the Department of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-02

    ... Management and Budget (OMB) (54 FR 25818), as amended by OMB Circular A-130, 65 FR 77677 (2000). A copy of...--VA, published in the Federal Register at 74 FR 29275 (June 19, 2009), and last amended at 75 FR... AFFAIRS Computer Matching Program Between the Department of Veterans Affairs (VA) and the Department...

  16. 76 FR 44086 - Agency Information Collection (Notice of Waiver of VA Compensation or Pension To Receive Military...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-22

    ... the collection of information through http://www.Regulations.gov or to VA's OMB Desk Officer, OMB... AFFAIRS Agency Information Collection (Notice of Waiver of VA Compensation or Pension To Receive Military... Vermont Avenue, NW., Washington, DC 20420, (202) 461-7485, fax (202) 461-0966 or e-mail...

  17. 30 CFR 57.22218 - Seals and stoppings (III, V-A, and V-B mines).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Seals and stoppings (III, V-A, and V-B mines... NONMETAL MINES Safety Standards for Methane in Metal and Nonmetal Mines Ventilation § 57.22218 Seals and stoppings (III, V-A, and V-B mines). (a) All seals, and those stoppings that separate main intake from...

  18. 30 CFR 57.22218 - Seals and stoppings (III, V-A, and V-B mines).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Seals and stoppings (III, V-A, and V-B mines... NONMETAL MINES Safety Standards for Methane in Metal and Nonmetal Mines Ventilation § 57.22218 Seals and stoppings (III, V-A, and V-B mines). (a) All seals, and those stoppings that separate main intake from...

  19. 30 CFR 57.22218 - Seals and stoppings (III, V-A, and V-B mines).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Seals and stoppings (III, V-A, and V-B mines... NONMETAL MINES Safety Standards for Methane in Metal and Nonmetal Mines Ventilation § 57.22218 Seals and stoppings (III, V-A, and V-B mines). (a) All seals, and those stoppings that separate main intake from...

  20. 30 CFR 57.22218 - Seals and stoppings (III, V-A, and V-B mines).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Seals and stoppings (III, V-A, and V-B mines... NONMETAL MINES Safety Standards for Methane in Metal and Nonmetal Mines Ventilation § 57.22218 Seals and stoppings (III, V-A, and V-B mines). (a) All seals, and those stoppings that separate main intake from...

  1. 30 CFR 57.22218 - Seals and stoppings (III, V-A, and V-B mines).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Seals and stoppings (III, V-A, and V-B mines... NONMETAL MINES Safety Standards for Methane in Metal and Nonmetal Mines Ventilation § 57.22218 Seals and stoppings (III, V-A, and V-B mines). (a) All seals, and those stoppings that separate main intake from...

  2. 33 CFR 165.501 - Chesapeake Bay entrance and Hampton Roads, VA and adjacent waters-Regulated Navigation Area.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Hampton Roads, VA and adjacent waters-Regulated Navigation Area. 165.501 Section 165.501 Navigation and... Areas Fifth Coast Guard District § 165.501 Chesapeake Bay entrance and Hampton Roads, VA and adjacent... Sector Hampton Roads. Designated representative of the Captain of the Port means a person, including...

  3. 33 CFR 165.501 - Chesapeake Bay entrance and Hampton Roads, VA and adjacent waters-Regulated Navigation Area.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Hampton Roads, VA and adjacent waters-Regulated Navigation Area. 165.501 Section 165.501 Navigation and... Areas Fifth Coast Guard District § 165.501 Chesapeake Bay entrance and Hampton Roads, VA and adjacent... Sector Hampton Roads. Designated representative of the Captain of the Port means a person, including...

  4. 33 CFR 165.501 - Chesapeake Bay entrance and Hampton Roads, VA and adjacent waters-Regulated Navigation Area.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Hampton Roads, VA and adjacent waters-Regulated Navigation Area. 165.501 Section 165.501 Navigation and... Areas Fifth Coast Guard District § 165.501 Chesapeake Bay entrance and Hampton Roads, VA and adjacent... Sector Hampton Roads. Designated representative of the Captain of the Port means a person, including...

  5. 33 CFR 165.501 - Chesapeake Bay entrance and Hampton Roads, VA and adjacent waters-Regulated Navigation Area.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Hampton Roads, VA and adjacent waters-Regulated Navigation Area. 165.501 Section 165.501 Navigation and... Areas Fifth Coast Guard District § 165.501 Chesapeake Bay entrance and Hampton Roads, VA and adjacent... Sector Hampton Roads. Designated representative of the Captain of the Port means a person, including...

  6. 33 CFR 165.501 - Chesapeake Bay entrance and Hampton Roads, VA and adjacent waters-Regulated Navigation Area.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Hampton Roads, VA and adjacent waters-Regulated Navigation Area. 165.501 Section 165.501 Navigation and... Areas Fifth Coast Guard District § 165.501 Chesapeake Bay entrance and Hampton Roads, VA and adjacent... Sector Hampton Roads. Designated representative of the Captain of the Port means a person, including...

  7. Identification and mapping of adult plant stripe rust resistance in soft red winter wheat VA00W-38

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Virginia Tech experimental wheat (Triticum aestivum L.) line VA00W-38 has adult plant resistance to race PST-100, which was the predominant race of stripe rust (Puccinia striiformis f. sp. tritici) in the United States from 2003 to 2006. Resistance was characterized in a cross between VA00W-38 a...

  8. 77 FR 64388 - Agency Information Collection (Former POW Medical History), VA Form 10-0048 Activities Under OMB...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-19

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF VETERANS AFFAIRS Agency Information Collection (Former POW Medical History), VA Form 10-0048 Activities Under OMB....'' SUPPLEMENTARY INFORMATION: Title: Former POW Medical History, VA Form 10-0048. OMB Control Number:...

  9. 76 FR 67257 - Computer Matching Program Between the Department of Veterans Affairs (VA) and the Department of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-31

    ... education benefit payments under the Post-9/11 GI Bill. These benefit records are contained in a VA system... Rehabilitation and Employment Records--VA, first published in the Federal Register at 74 FR 9294 (March 3, 1976), and last amended at 74 FR 29275 (June 19, 2009), with other amendments as cited therein. DATES:...

  10. 38 CFR 58.15 - VA Form 10-0143-Department of Veterans Affairs Certification Regarding Drug-Free Workplace...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false VA Form 10-0143-Department of Veterans Affairs Certification Regarding Drug-Free Workplace Requirements for Grantees Other Than Individuals. 58.15 Section 58.15 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) FORMS § 58.15 VA Form...

  11. 38 CFR 58.15 - VA Form 10-0143-Department of Veterans Affairs Certification Regarding Drug-Free Workplace...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2011-07-01 2011-07-01 false VA Form 10-0143-Department of Veterans Affairs Certification Regarding Drug-Free Workplace Requirements for Grantees Other Than Individuals. 58.15 Section 58.15 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) FORMS § 58.15 VA Form...

  12. 75 FR 24510 - Drug and Drug-Related Supply Promotion by Pharmaceutical Company Sales Representatives at VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-05

    ... control the promotion of drugs and drug-related supplies at VA facilities and the business relationships... standard of permissible business practice at VA facilities. It would also facilitate mutually beneficial... terms to be inclusive of all items typically promoted by pharmaceutical sales representatives....

  13. The Quantification of Representative Sequences pipeline for amplicon sequencing: case study on within-population ITS1 sequence variation in a microparasite infecting Daphnia.

    PubMed

    González-Tortuero, E; Rusek, J; Petrusek, A; Gießler, S; Lyras, D; Grath, S; Castro-Monzón, F; Wolinska, J

    2015-11-01

    Next generation sequencing (NGS) platforms are replacing traditional molecular biology protocols like cloning and Sanger sequencing. However, accuracy of NGS platforms has rarely been measured when quantifying relative frequencies of genotypes or taxa within populations. Here we developed a new bioinformatic pipeline (QRS) that pools similar sequence variants and estimates their frequencies in NGS data sets from populations or communities. We tested whether the estimated frequency of representative sequences, generated by 454 amplicon sequencing, differs significantly from that obtained by Sanger sequencing of cloned PCR products. This was performed by analysing sequence variation of the highly variable first internal transcribed spacer (ITS1) of the ichthyosporean Caullerya mesnili, a microparasite of cladocerans of the genus Daphnia. This analysis also serves as a case example of the usage of this pipeline to study within-population variation. Additionally, a public Illumina data set was used to validate the pipeline on community-level data. Overall, there was a good correspondence in absolute frequencies of C. mesnili ITS1 sequences obtained from Sanger and 454 platforms. Furthermore, analyses of molecular variance (amova) revealed that population structure of C. mesnili differs across lakes and years independently of the sequencing platform. Our results support not only the usefulness of amplicon sequencing data for studies of within-population structure but also the successful application of the QRS pipeline on Illumina-generated data. The QRS pipeline is freely available together with its documentation under GNU Public Licence version 3 at http://code.google.com/p/quantification-representative-sequences. PMID:25728529

  14. A new amplicon based approach of whole mitogenome sequencing for phylogenetic and phylogeographic analysis: An example of East African white-eyes (Aves, Zosteropidae).

    PubMed

    Meimberg, Harald; Schachtler, Christina; Curto, Manuel; Husemann, Martin; Habel, Jan Christian

    2016-09-01

    Classical Sanger sequencing is still frequently used to generate sequence data for phylogenetic and phylogeographic inference. In this contribution we present a novel approach to genotype whole mitogenomic haplotypes using Illumina MiSeq reads from indexed amplicons. Our new approach reduces preparation time by multiplexing loci within a single or few PCR reactions and by plate format library construction. The use of paired-end reads allows covering amplicons of about 0.5kb and thus no nebulisation and assembly are necessary. We tested the power and effectiveness of this technique by analysing the mitogenomic diversity of East African white-eye bird species (Zosteropidae), a taxonomically highly diverse and complex species flock found in various ecosystems spread across major parts of Africa. We compare the newly generated mitogenomic data set with published data of three mitochondrial genes for a similar set of populations and taxa. The comparison demonstrates that our new procedure represents a cost effective use of NGS for medium throughput phylogenetic analyses. Using this method, we were able to increase the amount of phylogenetic information significantly, while reducing the costs and effort in the laboratory. The mitogenomic data show a higher resolution than previous studies providing higher support and new insights in the relationships of Zosterops species. Our data suggest to split Z. poliogaster into four distinct species, three of which had previously been proposed: Z. silvanus, Z. mbulensis, Z. kikyuensis and Z. kulalensis. Our approach allows the genotyping of whole mitogenomes for a large number of individuals and thus allows more reliable reconstruction of phylogenetic and phylogeographic relationships - also for non-model organisms. PMID:27233440

  15. 78 FR 39595 - Safety Zone, Fifth Coast Guard District Firework Display, Pagan River; Smithfield, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-02

    ... SECURITY Coast Guard 33 CFR Part 165 RIN 1625-AA00 Safety Zone, Fifth Coast Guard District Firework Display, Pagan River; Smithfield, VA AGENCY: Coast Guard, DHS. ACTION: Temporary Final Rule. SUMMARY: The Coast... the Fifth Coast Guard District. This regulation applies only to one recurring fireworks event, held...

  16. 48 CFR 852.219-9 - VA Small business subcontracting plan minimum requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false VA Small business subcontracting plan minimum requirements. 852.219-9 Section 852.219-9 Federal Acquisition Regulations System... database. The contractor shall annually submit a listing of service-disabled veteran-owned small...

  17. 76 FR 9636 - Franklin Financial Corporation, Inc., Glen Allen, VA; Approval of Conversion Application

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-18

    ... Office of Thrift Supervision Franklin Financial Corporation, Inc., Glen Allen, VA; Approval of Conversion Application Notice is hereby given that on February 11, 2011, the Office of Thrift Supervision approved the..., NE., Atlanta, Georgia 30309. Dated: February 11, 2011. By the Office of Thrift Supervision. Sandra...

  18. 38 CFR 63.10 - Selection of non-VA community-based providers.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... VETERANS AFFAIRS (CONTINUED) HEALTH CARE FOR HOMELESS VETERANS (HCHV) PROGRAM § 63.10 Selection of non-VA... in 48 CFR chapters 1 and 8. Such contracts will be awarded only after the quality, effectiveness and... 38 Pensions, Bonuses, and Veterans' Relief 2 2014-07-01 2014-07-01 false Selection of...

  19. 38 CFR 63.10 - Selection of non-VA community-based providers.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... VETERANS AFFAIRS (CONTINUED) HEALTH CARE FOR HOMELESS VETERANS (HCHV) PROGRAM § 63.10 Selection of non-VA... in 48 CFR chapters 1 and 8. Such contracts will be awarded only after the quality, effectiveness and... 38 Pensions, Bonuses, and Veterans' Relief 2 2013-07-01 2013-07-01 false Selection of...

  20. 30 CFR 57.22229 - Weekly testing (I-A, III, and V-A mines).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Weekly testing (I-A, III, and V-A mines). 57.22229 Section 57.22229 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Safety Standards for Methane in Metal...

  1. 30 CFR 57.22229 - Weekly testing (I-A, III, and V-A mines).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Weekly testing (I-A, III, and V-A mines). 57.22229 Section 57.22229 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Safety Standards for Methane in Metal...

  2. 78 FR 28947 - Fund Availability Under VA's Homeless Providers Grant and Per Diem Program (VANS)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-16

    ... veteran participants for currently operational Grant and Per Diem grantee projects funded under VA's... Grant and Per Diem Program for current operational Grant and Per Diem grantees seeking to purchase a van... operational grant project number may be awarded. However, in the one allowable application, if the grantee...

  3. Belowground-system development of VA mycorrhiza in a restored tallgrass prairie community

    SciTech Connect

    Cook, B.D.; Jastrow, J.D.; Miller, R.M.; McGraw, A.C.

    1987-01-01

    This study was conducted to determine the relationship between root and VA mycorrhizal fungus development in a tallgrass prairie restoration chronosequence. Emphasis is placed on characterizing the relationship of root length, colonized root length, and percentage of root length occupied by the mycorrhizal fungus by differing root size classes. Mycorrhizal fungus composition and populations along the restoration chronosequence was determined. 3 refs., 1 fig.

  4. 77 FR 3844 - Proposed Information Collection (Dependents' Application for VA Educational Benefits) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-25

    ... Survivors' and Dependents' Educational Assistance and Fry Scholarship benefits. DATES: Written comments and... Sergeant John David Fry Scholarship (Fry Scholarship) benefits. VA will use the information collected to determine whether a claimant qualifies for DEA or Fry Scholarship benefits and if the program of...

  5. 75 FR 9557 - Drawbridge Operation Regulation; Elizabeth River, Eastern Branch, Norfolk, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-03

    ... Register (74 FR 52143) and a notice of proposed rulemaking (NPRM) entitled ``Drawbridge Operation Regulations; Elizabeth River, Eastern Branch, Norfolk, VA, in the Federal Register (74 FR 52158). We received... dockets in the January 17, 2008, issue of the Federal Register (73 FR 3316). Public Meeting We do not...

  6. 78 FR 48608 - Drawbridge Operation Regulation; Atlantic Intracoastal Waterway (AIWW), Chesapeake, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-09

    ... operation of the I64 Bridge across the Atlantic Intracoastal Waterway, South Branch of the Elizabeth River, mile 7.1, at Chesapeake, VA. This deviation is necessary to facilitate maintenance work on the moveable... route for vessels transiting this section on the South Branch of the Elizabeth River; however,...

  7. The Effect of Increased Travel Reimbursement Rates on Health Care Utilization in the VA

    ERIC Educational Resources Information Center

    Nelson, Richard E.; Hicken, Bret; West, Alan; Rupper, Randall

    2012-01-01

    Purpose: The reimbursement rate that eligible veterans receive for travel to Department of Veterans Affairs (VA) facilities increased from 11 to 28.5 cents per mile on February 1, 2008. We examined the effect of this policy change on utilization of outpatient, inpatient, and pharmacy services, stratifying veterans based on distance from a VA…

  8. Release of information from Department of Veterans Affairs records--VA. Proposed rule.

    PubMed

    1998-09-10

    This document proposes to amend Department of Veterans Affairs (VA) regulations governing the confidentiality and release of VA records subject to the Privacy Act, the Freedom of Information Act (FOIA) (including the Electronic Freedom of Information Act Amendments of 1996), and the veterans' records confidentiality statute. The proposed rule sets forth a mechanism for the public to obtain information from the VA. The proposed rule is intended to maximize public availability of VA records to the extent permitted by law and considerations such a personal privacy or law enforcement. Essentially these provisions consist of restatements of statute, interpretations of statute, interpretations of case law, interpretations of Executive Orders, and clarification. The proposed amendments also would implement the Electronic Freedom of Information Act Amendments of 1996, court decisions and Executive Branch guidance issued since the regulations were originally published. Further, this document proposes to delegate authority to the Assistant General Counsel for Professional Staff Group IV for making final Departmental decisions on appeals under the Freedom of Information Act, the Privacy Act, and 38 U.S.C. 5701 and 5705. This would simplify decision making by allowing the highest level individual with direct responsibility for decision making to issue decisions. PMID:10182831

  9. 77 FR 20716 - Drawbridge Operation Regulation; Blackwater River, South Quay, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-06

    ..., are part of docket USCG-2011-0943 and are available by going to http://www.regulations.gov , inserting... Register (76 FR 76634). We received no comments on the proposed rule. No public meeting was requested, and... Quay, VA AGENCY: Coast Guard, DHS. ACTION: Final rule. SUMMARY: The Coast Guard is changing...

  10. 75 FR 34641 - Safety Zone; Private Fireworks, Wilson Creek, Gloucester, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-18

    ... are available online by going to http://www.regulations.gov , inserting USCG-2010-0257 in the... Federal Register (75 FR 22545). We received no comments on the proposed rule. No public meeting was..., VA AGENCY: Coast Guard, DHS. ACTION: Temporary final rule. SUMMARY: The Coast Guard is establishing...

  11. 77 FR 63734 - Security Zone; James River, Kingsmill Resort, Williamsburg, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-17

    ... mentioned in this preamble as being available in the docket, go to http://www.regulations.gov , type the...: Table of Acronyms DHS Department of Homeland Security FR Federal Register NPRM Notice of Proposed..., Williamsburg, VA AGENCY: Coast Guard, DHS. ACTION: Temporary final rule. SUMMARY: The U.S. Coast Guard...

  12. Implementing the KiVa Antibullying Program: Recognition of Stable Victims

    ERIC Educational Resources Information Center

    Haataja, Anne; Sainio, Miia; Turtonen, Mira; Salmivalli, Christina

    2016-01-01

    Teachers do not always recognise students who are victimised by their peers. In this study, we examined the recognition of stable victims in 76 schools beginning to implement the KiVa antibullying programme. We focused on 348 victims (9-15 years) who reported victimisation at the pretest and still at wave 2, after five months of programme…

  13. For Children Only? Effects of the KiVa Antibullying Program on Teachers

    ERIC Educational Resources Information Center

    Ahtola, Annarilla; Haataja, Anne; Karna, Antti; Poskiparta, Elisa; Salmivalli, Christina

    2012-01-01

    The effects of an antibullying program, KiVa, on Grade 1-3 teachers' perceptions of bullying were investigated. Data were gathered from 128 teachers in 33 intervention schools and 110 teachers in 29 control schools. Two-level regression models showed that, at the end of the intervention year, self-evaluated competence to tackle bullying was at a…

  14. A Large-Scale Evaluation of the KiVa Antibullying Program: Grades 4-6

    ERIC Educational Resources Information Center

    Karna, Antti; Voeten, Marinus; Little, Todd D.; Poskiparta, Elisa; Kaljonen, Anne; Salmivalli, Christina

    2011-01-01

    This study demonstrates the effectiveness of the KiVa antibullying program using a large sample of 8,237 youth from Grades 4-6 (10-12 years). Altogether, 78 schools were randomly assigned to intervention (39 schools, 4,207 students) and control conditions (39 schools, 4,030 students). Multilevel regression analyses revealed that after 9 months of…

  15. 77 FR 35308 - Proposed Amendment of Restricted Area R-6601; Fort A.P. Hill, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-13

    .... 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 73.66 (Amended) 2. Sec. 73.66 is amended as... Federal Aviation Administration 14 CFR Part 73 Proposed Amendment of Restricted Area R-6601; Fort A.P... restricted area R-6601, Fort A.P. Hill, VA. The U. S. Army requested this action to provide the...

  16. 76 FR 2148 - Specialty Minerals, Inc., Franklin, VA; Notice of Revised Determination on Reconsideration

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-12

    ... on July 1, 2010 (75 FR 38142). On August 19, 2010, the Department issued a Notice of Negative... was published in the Federal Register on August 30, 2010 (75 FR 52989). The workers produced... Employment and Training Administration Specialty Minerals, Inc., Franklin, VA; Notice of...

  17. 78 FR 51067 - VA Health Professional Scholarship and Visual Impairment and Orientation and Mobility...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-20

    ... Mandates Reform Act of 1995 requires, at 2 U.S.C. 1532, that agencies prepare an assessment of anticipated... with and implement sections 302 and 603 of the Caregivers and Veterans Omnibus Health Services Act of 2010 (the 2010 Act). Section 302 of the 2010 Act established the VIOMPSP, which authorizes VA...

  18. 77 FR 75918 - VA Health Professional Scholarship and Visual Impairment and Orientation and Mobility...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-26

    ... Veterans Omnibus Health Services Act of 2010 (the 2010 Act). Section 302 of the 2010 Act established the... rehabilitation specialists for VA and the United States. Section 603 of the 2010 Act reauthorized and modified... 603 of the 2010 Act, Public Law 111-163, which amended the statutory authority for this...

  19. 78 FR 56271 - FY 2014-2020 Draft VA Strategic Plan

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-12

    ...; enhancing the quality of and access to benefits and services through integration within VA and with our... focus from improvements within a service or benefit delivery program to coordination and integration... defining success by Veterans' outcomes; enhancing the quality of and access to benefits and...

  20. 75 FR 41577 - VBA/VHA Musculoskeletal Forum: Improving VA's Disability Evaluation Criteria

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-16

    ... AFFAIRS VBA/VHA Musculoskeletal Forum: Improving VA's Disability Evaluation Criteria AGENCY: Department of... system. See 38 CFR 4.40-4.73. Specifically, diagnostic code descriptors and evaluation criteria will be... Coordinator, Compensation and Pension Service, Department of Veterans Affairs, 810 Vermont Avenue,...