Science.gov

Sample records for amplicon va derived

  1. A geminiviral amplicon (VA) derived from Tomato leaf curl virus (ToLCV) can replicate in a wide variety of plant species and also acts as a VIGS vector

    PubMed Central

    Pandey, Prerna; Choudhury, Nirupam R; Mukherjee, Sunil K

    2009-01-01

    Background The Tomato leaf curl virus (ToLCV) belongs to the genus begomoviridae of the family Geminiviridae. The 2.7 kb DNA genome of the virus encodes all the information required for viral DNA replication, transcription and transmission across the plant cells. However, all of the genome sequences are not required for viral DNA replication. We attempted to reveal the minimal essential region required for DNA replication and stable maintenance. The phenomenon of Virus Induced Gene Silencing (VIGS) has recently been observed with several geminiviruses. We investigated whether the minimal replicating region was also capable of producing siRNAs in planta and a VIGS vector could be constructed using the same minimal sequences. Results We have constructed vectors containing various truncated portions of the Tomato leaf curl virus (ToLCV) genome and established that a segment spanning from common region (CR) to AC3 (ORF coding for a replication enhancer) was the minimal portion which could efficiently replicate in a variety of both monocot and dicot plants. A viral amplicon (VA) vector was constructed using this region that produced siRNAs from various sites of the vector, in a temporal manner in plants, and hence can be used as a VIGS vector. The tomato endogene PCNA was silenced using this vector. Introduction of a mutation in the ORF AC2 (a silencing suppressor) increased the silencing efficiency of the newly constructed vector several folds. Conclusion Our study reveals that the vector is capable of replicating in diverse plant species and is highly efficient in silencing endogenes like PCNA of the host plant, thus acting as a VIGS vector. We observed that the geminiviral ORF AC2 functioned as a silencing suppressor and a null mutation in this ORF increased the efficiency of silencing several fold. This is the first report of construction of improved VIGS vector by mutation of the resident silencing suppressor gene. The present study opens up the possibility of

  2. Herpes simplex virus type 1-derived recombinant and amplicon vectors.

    PubMed

    Fraefel, Cornel; Marconi, Peggy; Epstein, Alberto L

    2011-01-01

    Herpes simplex virus type 1 (HSV-1) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153 kbp double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes (1) the two approaches most commonly used to prepare recombinant vectors through homologous recombination, either in eukaryotic cells or in bacteria, and (2) the two methodologies currently used to generate helper-free amplicon vectors, either using a bacterial artificial chromosome (BAC)-based approach or a Cre/loxP site-specific recombination strategy.

  3. Evaluating sequence-derived mtDNA length heteroplasmy by amplicon size analysis

    PubMed Central

    Berger, C.; Hatzer-Grubwieser, P.; Hohoff, C.; Parson, W.

    2011-01-01

    Length heteroplasmy (LH) in mitochondrial (mt)DNA is usually observed in homopolymeric tracts and manifest as mixture of various length variants. The generally used difference-coded annotation to report mtDNA haplotypes does not express the degree of LH variation present in a sample, even more so, it is sometimes difficult to establish which length variants are present and clearly distinguishable from background noise. It has therefore become routine practice for some researchers to call the dominant type, the “major molecule”, which represents the LH variant that is most abundant in a DNA extract. In the majority of cases a clear single dominant variant can be identified. However, in some samples this interpretation is difficult, i.e. when (almost) equally quantitative LH variants are present or when multiple sequencing primers result in the presentation of different dominant types. To better understand those cases we designed amplicon sizing assays for the five most relevant LH regions in the mtDNA control region (around ntps 16,189, 310, 460, 573, and the AC-repeat between 514 and 524) to determine the ratio of the LH variants by fluorescence based amplicon sizing assays. For difficult LH constellations derived by Sanger sequencing (with Big Dye terminators) these assays mostly gave clear and unambiguous results. In the vast majority of cases we found agreement between the results of the sequence and amplicon analyses and propose this alternative method in difficult cases. PMID:21067985

  4. Quantitative assessment of short amplicons in FFPE-derived long-chain RNA

    PubMed Central

    Kong, Hui; Zhu, Mengou; Cui, Fengyun; Wang, Shuyang; Gao, Xue; Lu, Shaohua; Wu, Ying; Zhu, Hongguang

    2014-01-01

    Formalin-fixed paraffin-embedded (FFPE) tissues are important resources for molecular medical research. However, long-chain RNA analysis is restricted in FFPE tissues due to high levels of degradation. To explore the possibility of long RNA quantification in FFPE tissues, we selected 14 target RNAs (8 mRNAs and 6 long noncoding RNAs) from literatures, and designed short (~60 bp) and long (~200 bp) amplicons for each of them. Colorectal carcinomas with adjacent normal tissues were subjected to quantitative reverse-transcription PCR (quantitative RT-PCR) in 3 cohorts, including 18 snap-frozen and 83 FFPE tissues. We found that short amplicons were amplified more efficiently than long amplicons both in snap-frozen (P = 0.0006) and FFPE (P = 0.0152) tissues. Nonetheless, comparison of colorectal carcinomas with their adjacent normal tissues demonstrated that the consistency of fold-change trends in a single short amplicon between snap-frozen and FFPE tissues was only 36%. Therefore, we innovatively performed quantitative RT-PCR with 3 non-overlapping short amplicons for 14 target RNAs in FFPE tissues. All target RNAs showed a concordance of 100% of fold-change trends in at least two short amplicons, which offers sufficient information for accurate quantification of target RNAs. Our findings demonstrated the possibility of long-chain RNA analysis with 3 non-overlapping short amplicons in standardized-preserved FFPE tissues. PMID:25430878

  5. Comprehensive Cloning of Patient-derived 9022-bp Amplicons of Hepatitis C Virus

    PubMed Central

    Lu, Yang; Xu, Yanjuan; Di Bisceglie, Adrian M.; Fan, Xiaofeng

    2013-01-01

    The instability of recombinant clones accommodating large or full-length viral genomes is frequently a technical challenge in RNA virus research. In an attempt to establish a rapid plasmid-based reverse genetics system that utilizes long RT-PCR technique (LRP), similar difficulty was encountered in the cloning of 9022-bp LRP amplicon. All HCV genotype 1a strains used for LRP cloning showed a remarkable difference in terms of cloning stability. Subsequent analysis revealed the predictive value of phylogenetic positions in determining the cloning stability. Putative E. coli promoters on the HCV genome might be responsible for such cloning difference. An exhaustive exploration, testing nearly one hundred cloning protocols, did not reveal a general approach that can achieve stable cloning for all HCV 1a strains. The selection of appropriate strains, guided by phylogenetic analysis, appears to be necessary prior to the construction of infectious HCV 1a clones. These observations are not only valuable for potentially establishing an HCV 1a cell culture model but also have general implications for other RNA viruses due to concern about cloning instability. PMID:23602804

  6. High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley.

    PubMed

    Hofinger, Bernhard J; Jing, Hai-Chun; Hammond-Kosack, Kim E; Kanyuka, Kostya

    2009-09-01

    An original method has been established for the identification of novel alleles of eukaryotic translation initiation factor 4E (eIF4E) gene, which is required for resistance to agronomically important bymoviruses, in barley germplasm. This method involves scanning for sequence variations in cDNA-derived PCR amplicons using High-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM is a simple, cost-effective, rapid and high-throughput assay, which so far has only been widely used in clinical pathology for molecular diagnostic of diseases and patient genotyping. Application of HRM allowed significant reduction in the amount of expensive Sanger sequencing required for allele mining in plants. The method described here involved an investigation of total cDNA rather than genomic DNA, thus permitting the analyses of shorter (up to 300-bp) and fewer overlapping amplicons to cover the coding sequence. This strategy further reduced the allele mining costs. The sensitivity and accuracy of HRM for predicting genotypes carrying a wide range of nucleotide polymorphisms in eIF4E approached 100%. Results of the current study are promising and suggest that this method could also potentially be applied to the discovery of superior alleles controlling other important traits in barley as well in other model and crop plant species.

  7. High-throughput identification and quantification of Candida species using high resolution derivative melt analysis of panfungal amplicons.

    PubMed

    Mandviwala, Tasneem; Shinde, Rupali; Kalra, Apoorv; Sobel, Jack D; Akins, Robert A

    2010-01-01

    Fungal infections pose unique challenges to molecular diagnostics; fungal molecular diagnostics consequently lags behind bacterial and viral counterparts. Nevertheless, fungal infections are often life-threatening, and early detection and identification of species is crucial to successful intervention. A high throughput PCR-based method is needed that is independent of culture, is sensitive to the level of one fungal cell per milliliter of blood or other tissue types, and is capable of detecting species and resistance mutations. We introduce the use of high resolution melt analysis, in combination with more sensitive, inclusive, and appropriately positioned panfungal primers, to address these needs. PCR-based amplification of the variable internal transcribed regions of the rDNA genes generates an amplicon whose sequence melts with a shape that is characteristic and therefore diagnostic of the species. Simple analysis of the differences between test and reference melt curves generates a single number that calls the species. Early indications suggest that high resolution melt analysis can distinguish all eight major species of Candida of clinical significance without interference from excess human DNA. Candida species, including mixed and novel species, can be identified directly in vaginal samples. This tool can potentially detect, count, and identify fungi in hundreds of samples per day without further manipulation, costs, or delays, offering a major step forward in fungal molecular diagnostics.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  9. Herpes Virus Amplicon Vectors

    PubMed Central

    de Silva, Suresh; Bowers, William J.

    2009-01-01

    Since its emergence onto the gene therapy scene nearly 25 years ago, the replication-defective Herpes Simplex Virus Type-1 (HSV-1) amplicon has gained significance as a versatile gene transfer platform due to its extensive transgene capacity, widespread cellular tropism, minimal immunogenicity, and its amenability to genetic manipulation. Herein, we detail the recent advances made with respect to the design of the HSV amplicon, its numerous in vitro and in vivo applications, and the current impediments this virus-based gene transfer platform faces as it navigates a challenging path towards future clinical testing. PMID:19956558

  10. 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

  11. HSV-1 amplicon vectors: a promising and versatile tool for gene delivery.

    PubMed

    Cuchet, Delphine; Potel, Corinne; Thomas, Joélle; Epstein, Alberto L

    2007-07-01

    Amplicons are defective and non-integrative vectors derived from herpes simplex virus type 1. They carry no virus genes in the vector genome and are, therefore, not toxic to the infected cells or pathogenic for the transduced organisms, making these vectors safe. In addition, the large transgenic capacity of amplicons, which allow delivery of < or = 150 Kbp of foreign DNA, make these vectors one of the most powerful, interesting and versatile gene delivery platforms. Here, the authors present recent technological developments that have significantly improved and extended the use of amplicons, both in cultured cells and in living organisms. In addition, this review illustrates the many possible applications that are presently being developed with amplicons and discuss the many difficulties still pending to be solved in order to achieve stable and physiologically regulated transgenic expression.

  12. Association of Myosin Va and Schwann cells-derived RNA in mammal myelinated axons, analyzed by immunocytochemistry and confocal FRET microscopy.

    PubMed

    Canclini, Lucía; Wallrabe, Horst; Di Paolo, Andrés; Kun, Alejandra; Calliari, Aldo; Sotelo-Silveira, José Roberto; Sotelo, José Roberto

    2014-03-15

    Evidence from multiple sources supports the hypothesis that Schwann cells in the peripheral nervous system transfer messenger RNA and ribosomes to the axons they ensheath. Several technical and methodological difficulties exist for investigators to unravel this process in myelinated axons - a complex two-cell unit. We present an experimental design to demonstrate that newly synthesized RNA is transferred from Schwann cells to axons in association with Myosin Va. The use of quantitative confocal FRET microscopy to track newly-synthesized RNA and determine the molecular association with Myosin Va, is described in detail.

  13. HSV-1 amplicon peptide display vector.

    PubMed

    Spear, Matthew A; Schuback, Deborah; Miyata, Kenichi; Grandi, Paola; Sun, Fang; Yoo, Linda; Nguyen, Anh; Brandt, Curtis R; Breakefield, Xandra O

    2003-01-01

    There are significant uses for expressing foreign peptide epitopes in viral surface attachment proteins in terms of investigating viral targeting, biology, and immunology. HSV-1 attachment, followed by fusion and entry, is mediated in large part by the binding of viral surface glycoproteins to cell surface receptors, primarily through heparan sulfate (HS) glycosaminoglycan residues. We constructed a HSV-1 amplicon plasmid (pCONGA) carrying the gC primary attachment protein gene with unique restriction sites flanking the HS binding domain (HSBD) (residues 33-176) to allow rapid, high efficiency substitution with foreign peptide domains. To test this system, a His tag with an additional unique restriction site (for selection and assay digests) was recombined into the pCONGA HSBD site to create pCONGAH. Infection of pCONGAH transfected Vero cells with HSV-1 helper virus (gCdelta2-3 or hrR3) produced His-modified gC as demonstrated by western blot analysis with co-localization of anti-gC and anti-His tag antibodies to a protein of appropriate molecular weight (50 kd). As CONGA and CONGAH amplicons carry a GFP transgene and the gCdelta2-3 and hrR3 viruses carry a lacZ transgene, vector stocks produced from 1 x 10(5) Vero cells could be titered for competent vector on cell monolayers and were demonstrated to contain 2 x 10(5) amplicon vector transducing units (t.u.)/ml and 1 x 10(7) virus t.u./ml. As the amplicon plasmids also contain the neomycin resistance gene (neo(r)), long term vector producer cell lines were created using G418 selection. This amplicon system provides means to rapidly and efficiently generate HSV-1 amplicon and viral vector expressing surface attachment proteins modified with different peptide epitopes for investigational and therapeutic uses, with the advantages of an amplicon plasmid that can be used with interchangeable helper virus vectors, is designed specifically for easy manipulation, and carries GFP and neo(r) transgenes for marker and

  14. Cooperative Epigenetic Modulation by Cancer Amplicon Genes

    PubMed Central

    Rui, Lixin; Tolga Emre, N. C.; Kruhlak, Michael J.; Chung, Hye-Jung; Steidl, Christian; Slack, Graham; Wright, George W.; Lenz, Georg; Ngo, Vu N.; Shaffer, Arthur L.; Xu, Weihong; Zhao, Hong; Yang, Yandan; Lamy, Laurence; Davis, R. Eric; Xiao, Wenming; Powell, John; Maloney, David; Thomas, Craig J.; Möller, Peter; Rosenwald, Andreas; Ott, German; Muller-Hermelink, Hans Konrad; Savage, Kerry; Connors, Joseph M.; Rimsza, Lisa M.; Campo, Elias; Jaffe, Elaine S.; Delabie, Jan; Smeland, Erlend B.; Weisenburger, Dennis D.; Chan, Wing C.; Gascoyne, Randy D.; Levens, David; Staudt, Louis M.

    2010-01-01

    Chromosome band 9p24 is frequently amplified in primary mediastinal B-cell lymphoma (PMBL) and Hodgkin lymphoma (HL). To identify oncogenes in this amplicon, we screened an RNA interference library targeting amplicon genes and thereby identified JAK2 and the histone demethylase JMJD2C as essential genes in these lymphomas. Inhibition of JAK2 and JMJD2C cooperated in killing these lymphomas by decreasing tyrosine 41 phosphorylation and increasing lysine 9 trimethylation of histone H3, promoting heterochromatin formation. MYC, a major target of JAK2-mediated histone phosphorylation, was silenced following JAK2 and JMJD2C inhibition, with a corresponding increase in repressive chromatin. Hence, JAK2 and JMJD2C cooperatively remodel the PMBL and HL epigenome, offering a mechanistic rationale for the development of JAK2 and JMJD2C inhibitors in these diseases. PMID:21156283

  15. A quantitative SMRT cell sequencing method for ribosomal amplicons.

    PubMed

    Jones, Bethan M; Kustka, Adam B

    2017-04-01

    Advances in sequencing technologies continue to provide unprecedented opportunities to characterize microbial communities. For example, the Pacific Biosciences Single Molecule Real-Time (SMRT) platform has emerged as a unique approach harnessing DNA polymerase activity to sequence template molecules, enabling long reads at low costs. With the aim to simultaneously classify and enumerate in situ microbial populations, we developed a quantitative SMRT (qSMRT) approach that involves the addition of exogenous standards to quantify ribosomal amplicons derived from environmental samples. The V7-9 regions of 18S SSU rDNA were targeted and quantified from protistan community samples collected in the Ross Sea during the Austral summer of 2011. We used three standards of different length and optimized conditions to obtain accurate quantitative retrieval across the range of expected amplicon sizes, a necessary criterion for analyzing taxonomically diverse 18S rDNA molecules from natural environments. The ability to concurrently identify and quantify microorganisms in their natural environment makes qSMRT a powerful, rapid and cost-effective approach for defining ecosystem diversity and function.

  16. Sequence-specific electrochemical detection of asymmetric PCR amplicons of traditional Chinese medicinal plant DNA.

    PubMed

    Lee, Thomas M H; Hsing, I-Ming

    2002-10-01

    In this study, an electrochemistry-based approach to detect nucleic acid amplification products of Chinese herbal genes is reported. Using asymmetric polymerase chain reaction and electrochemical techniques, single-stranded target amplicons are produced from trace amounts of DNA sample and sequence-specific electrochemical detection based on the direct hybridization of the crude amplicon mix and immobilized DNA probe can be achieved. Electrochemically active intercalator Hoechst 33258 is bound to the double-stranded duplex formed by the target amplicon hybridized with the 5'-thiol-derivated DNA probe (16-mer) on the gold electrode surface. The electrochemical current signal of the hybridization event is measured by linear sweep voltammetry, the response of which can be used to differentiate the sequence complementarities of the target amplicons. To improve the reproducibility and sensitivity of the current signal, issues such as electrode surface cleaning, probe immobilization, and target hybridization are addressed. Factors affecting hybridization efficiency including the length and binding region of the target amplicon are discussed. Using our approach, differentiation of Chinese herbal species Fritillaria (F. thunbergii and F. cirrhosa) based on the 16-mer unique sequences in the spacer region of the 5S-rRNA is demonstrated. The ability to detect PCR products using a nonoptical electrochemical detection technique is an important step toward the realization of portable biomicrodevices for on-spot bacterial and viral detections.

  17. 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

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

    PubMed

    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.

  19. Early STAT1 activation after systemic delivery of HSV amplicon vectors suppresses transcription of the vector-encoded transgene.

    PubMed

    Suzuki, Masataka; Chiocca, E Antonio; Saeki, Yoshinaga

    2007-11-01

    The herpes simplex virus (HSV) amplicon vector is a powerful gene delivery vehicle that can accommodate up to 150 kilobase of exogenous DNA. However, amplicon-mediated transgene expression is often transient outside the nervous system. In order to define the role of host immune responses in silencing amplicon-encoded transgenes, we evaluated the kinetics of cytokine-/chemokine-expression after tail-vein injection of a luciferase-encoding amplicon into mice. Type I interferons (IFNs) were induced earliest, within an hour after injection, and several other cytokines/chemokines were subsequently upregulated in the livers of wild-type (WT) mice. When the same experiment was performed in signal transducers and activators of transcription 1 (STAT1)-knockout (KO) mice, the levels of type I IFN expression were significantly lower and chemokine induction was almost non-existent. Importantly, STAT1-KO mice exhibited significantly higher and more sustained luciferase activity than did the WT mice, which is attributable to increased transcriptional activity rather than increased copy numbers of luciferase-encoding vector DNA. Further studies using primary cultured fibroblasts derived from WT and STAT1-KO mice revealed the significance of STAT1 signaling in transcriptional silencing of the amplicon-encoded transgene in vitro. Our results indicate that type I IFNs induced by systemic delivery of HSV amplicon vectors initiate a cascade of immune responses and suppress transgene expression at the transcriptional level by activation of STAT1.

  20. 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.

  1. Determining VA physician requirements through empirically based models.

    PubMed Central

    Lipscomb, J; Kilpatrick, K E; Lee, K L; Pieper, K S

    1995-01-01

    OBJECTIVE: As part of a project to estimate physician requirements for the Department of Veterans Affairs, the Institute of Medicine (IOM) developed and tested empirically based models of physician staffing, by specialty, that could be applied to each VA facility. DATA SOURCE/STUDY SETTING. These analyses used selected data on all patient encounters and all facilities in VA's management information systems for FY 1989. STUDY DESIGN. Production functions (PFs), with patient workload dependent on physicians, other providers, and nonpersonnel factors, were estimated for each of 14 patient care areas in a VA medical center. Inverse production functions (IPFs), with physician staffing levels dependent on workload and other factors, were estimated for each of 11 specialty groupings. These models provide complementary approaches to deriving VA physician requirements for patient care and medical education. DATA COLLECTION/EXTRACTION METHODS. All data were assembled by VA and put in analyzable SAS data sets containing FY 1989 workload and staffing variables used in the PFs and IPFs. All statistical analyses reported here were conducted by the IOM. PRINCIPAL FINDINGS. Existing VA data can be used to develop statistically strong, clinically plausible, empirically based models for calculating physician requirements, by specialty. These models can (1) compare current physician staffing in a given setting with systemwide norms and (2) yield estimates of future staffing requirements conditional on future workload. CONCLUSIONS. Empirically based models can play an important role in determining VA physician staffing requirements. VA should test, evaluate, and revise these models on an ongoing basis. PMID:7860320

  2. Comprehensive genome sequence analysis of a breast cancer amplicon.

    PubMed

    Collins, C; Volik, S; Kowbel, D; Ginzinger, D; Ylstra, B; Cloutier, T; Hawkins, T; Predki, P; Martin, C; Wernick, M; Kuo, W L; Alberts, A; Gray, J W

    2001-06-01

    Gene amplification occurs in most solid tumors and is associated with poor prognosis. Amplification of 20q13.2 is common to several tumor types including breast cancer. The 1 Mb of sequence spanning the 20q13.2 breast cancer amplicon is one of the most exhaustively studied segments of the human genome. These studies have included amplicon mapping by comparative genomic hybridization (CGH), fluorescent in-situ hybridization (FISH), array-CGH, quantitative microsatellite analysis (QUMA), and functional genomic studies. Together these studies revealed a complex amplicon structure suggesting the presence of at least two driver genes in some tumors. One of these, ZNF217, is capable of immortalizing human mammary epithelial cells (HMEC) when overexpressed. In addition, we now report the sequencing of this region in human and mouse, and on quantitative expression studies in tumors. Amplicon localization now is straightforward and the availability of human and mouse genomic sequence facilitates their functional analysis. However, comprehensive annotation of megabase-scale regions requires integration of vast amounts of information. We present a system for integrative analysis and demonstrate its utility on 1.2 Mb of sequence spanning the 20q13.2 breast cancer amplicon and 865 kb of syntenic murine sequence. We integrate tumor genome copy number measurements with exhaustive genome landscape mapping, showing that amplicon boundaries are associated with maxima in repetitive element density and a region of evolutionary instability. This integration of comprehensive sequence annotation, quantitative expression analysis, and tumor amplicon boundaries provide evidence for an additional driver gene prefoldin 4 (PFDN4), coregulated genes, conserved noncoding regions, and associate repetitive elements with regions of genomic instability at this locus.

  3. 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

  4. 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.

  5. Reproducibility and quantitation of amplicon sequencing-based detection

    PubMed Central

    Zhou, Jizhong; Wu, Liyou; Deng, Ye; Zhi, Xiaoyang; Jiang, Yi-Huei; Tu, Qichao; Xie, Jianping; Van Nostrand, Joy D; He, Zhili; Yang, Yunfeng

    2011-01-01

    To determine the reproducibility and quantitation of the amplicon sequencing-based detection approach for analyzing microbial community structure, a total of 24 microbial communities from a long-term global change experimental site were examined. Genomic DNA obtained from each community was used to amplify 16S rRNA genes with two or three barcode tags as technical replicates in the presence of a small quantity (0.1% wt/wt) of genomic DNA from Shewanella oneidensis MR-1 as the control. The technical reproducibility of the amplicon sequencing-based detection approach is quite low, with an average operational taxonomic unit (OTU) overlap of 17.2%±2.3% between two technical replicates, and 8.2%±2.3% among three technical replicates, which is most likely due to problems associated with random sampling processes. Such variations in technical replicates could have substantial effects on estimating β-diversity but less on α-diversity. A high variation was also observed in the control across different samples (for example, 66.7-fold for the forward primer), suggesting that the amplicon sequencing-based detection approach could not be quantitative. In addition, various strategies were examined to improve the comparability of amplicon sequencing data, such as increasing biological replicates, and removing singleton sequences and less-representative OTUs across biological replicates. Finally, as expected, various statistical analyses with preprocessed experimental data revealed clear differences in the composition and structure of microbial communities between warming and non-warming, or between clipping and non-clipping. Taken together, these results suggest that amplicon sequencing-based detection is useful in analyzing microbial community structure even though it is not reproducible and quantitative. However, great caution should be taken in experimental design and data interpretation when the amplicon sequencing-based detection approach is used for quantitative

  6. Detection of immobilized amplicons by ELISA-like techniques.

    PubMed

    Oroskar, A A; Rasmussen, S E; Rasmussen, H N; Rasmussen, S R; Sullivan, B M; Johansson, A

    1996-09-01

    The NucleoLink surface is a physically modified, thermostable, optically clear resin. It allows the covalent binding of 5'-phosphorylated oligonucleotides. Target DNA amplification by polymerase chain reaction (PCR) is accomplished by asymmetric amplification on the covalently immobilized primer that develops into immobilized amplicons. A DNA fragment of bovine leukemia virus is used as a model system for the detection of immobilized amplicons by ELISA-like techniques. Covalently bound oligonucleotides are also utilized as capture probe in the hybridization-based signal amplification for detection of an infectious organism.

  7. Greening America's Capitals - Richmond, VA

    EPA Pesticide Factsheets

    Report from the Greening America's Capitals project in Richmond, VA, to help the city develop design options to protect pedestrians, bicyclists, transit users, and drivers; improve stormwater management; and spur revitalization.

  8. VA Health Care Facilities Locator

    MedlinePlus

    ... VA » Locations » Find Locations Locations Find Locations The javascript used here is for validation purpose only. Your browser doesn't seem to support javascript or has it disabled. This site is a ...

  9. A fungal mock community control for amplicon sequencing experiments

    USDA-ARS?s Scientific Manuscript database

    The field of microbial ecology has been profoundly advanced by the ability to profile the composition of complex microbial communities by means of high throughput amplicon sequencing of marker genes amplified directly from environmental genomic DNA extracts. However, it has become increasingly clear...

  10. Herpes simplex virus type 1-based amplicon vectors for fundamental research in neurosciences and gene therapy of neurological diseases.

    PubMed

    Jerusalinsky, Diana; Baez, María Verónica; Epstein, Alberto Luis

    2012-01-01

    Somatic manipulation of the nervous system without the involvement of the germinal line appears as a powerful counterpart of the transgenic strategy. The use of viral vectors to produce specific, transient and localized knockout, knockdown, ectopic expression or overexpression of a gene, leads to the possibility of analyzing both in vitro and in vivo molecular basis of neural function. In this approach, viral particles engineered to carry transgenic sequences are delivered into discrete brain regions, to transduce cells that will express the transgenic products. Amplicons are replication-incompetent helper-dependent vectors derived from herpes simplex virus type 1 (HSV-1), with several advantages that potentiate their use in neurosciences: (1) minimal toxicity: amplicons do not encode any virus proteins, are neither toxic for the infected cells nor pathogenic for the inoculated animals and elicit low levels of adaptive immune responses; (2) extensive transgene capacity to carry up to 150-kb of foreign DNA; i.e., entire genes with regulatory sequences could be delivered; (3) widespread cellular tropism: amplicons can experimentally infect several cell types including glial cells, though naturally the virus infects mainly neurons and epithelial cells; (4) since the viral genome does not integrate into cellular chromosomes there is low probability to induce insertional mutagenesis. Recent investigations on gene transfer into the brain using these vectors, have focused on gene therapy of inherited genetic diseases affecting the nervous system, such as ataxias, or on neurodegenerative disorders using experimental models of Parkinson's or Alzheimer's disease. Another group of studies used amplicons to investigate complex neural functions such as neuroplasticity, anxiety, learning and memory. In this short review, we summarize recent data supporting the potential of HSV-1 based amplicon vector model for gene delivery and modulation of gene expression in primary cultures

  11. DADA2: High resolution sample inference from Illumina amplicon data

    PubMed Central

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

    2016-01-01

    We present DADA2, a software package that models and corrects Illumina-sequenced amplicon errors. DADA2 infers sample sequences exactly, without coarse-graining into OTUs, and resolves differences of as little as one 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

  12. 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.

  13. Virion-Associated Cofactor High-Mobility Group DNA-Binding Protein-1 Facilitates Transposition from the Herpes Simplex Virus/Sleeping Beauty Amplicon Vector Platform

    PubMed Central

    de Silva, Suresh; Lotta, Louis T.; Burris, Clark A.

    2010-01-01

    Abstract 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

  14. 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

  15. 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

    2016-07-12

    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.

  16. MT-Toolbox: improved amplicon sequencing using molecule tags.

    PubMed

    Yourstone, Scott M; Lundberg, Derek S; Dangl, Jeffery L; Jones, Corbin D

    2014-08-22

    Short oligonucleotides can be used as markers to tag and track DNA sequences. For example, barcoding techniques (i.e. Multiplex Identifiers or Indexing) use short oligonucleotides to distinguish between reads from different DNA samples pooled for high-throughput sequencing. A similar technique called molecule tagging uses the same principles but is applied to individual DNA template molecules. Each template molecule is tagged with a unique oligonucleotide prior to polymerase chain reaction. The resulting amplicon sequences can be traced back to their original templates by their oligonucleotide tag. Consensus building from sequences sharing the same tag enables inference of original template molecules thereby reducing effects of sequencing error and polymerase chain reaction bias. Several independent groups have developed similar protocols for molecule tagging; however, user-friendly software for build consensus sequences from molecule tagged reads is not readily available or is highly specific for a particular protocol. MT-Toolbox recognizes oligonucleotide tags in amplicons and infers the correct template sequence. On a set of molecule tagged test reads, MT-Toolbox generates sequences having on average 0.00047 errors per base. MT-Toolbox includes a graphical user interface, command line interface, and options for speed and accuracy maximization. It can be run in serial on a standard personal computer or in parallel on a Load Sharing Facility based cluster system. An optional plugin provides features for common 16S metagenome profiling analysis such as chimera filtering, building operational taxonomic units, contaminant removal, and taxonomy assignments. MT-Toolbox provides an accessible, user-friendly environment for analysis of molecule tagged reads thereby reducing technical errors and polymerase chain reaction bias. These improvements reduce noise and allow for greater precision in single amplicon sequencing experiments.

  17. Genes Essential for Nod Factor Production and Nodulation Are Located on a Symbiotic Amplicon (AMPRtrCFN299pc60) in Rhizobium tropici

    PubMed Central

    Mavingui, Patrick; Laeremans, Toon; Flores, Margarita; Romero, David; Martínez-Romero, Esperanza; Palacios, Rafael

    1998-01-01

    Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli. Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1. Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless A. tumefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym. PMID:9603874

  18. Genomic amplicons target vesicle recycling in breast cancer

    PubMed Central

    Mills, Gordon B.; Jurisica, Igor; Yarden, Yosef; Norman, Jim C.

    2009-01-01

    Aberrant endocytosis, vesicle targeting, and receptor recycling represent emerging hallmarks of cancer. In this issue of the JCI, Zhang and colleagues demonstrate that RAB-coupling protein (RCP; also known as RAB11FIP1) is a “driver” of the 8p11–12 amplicon in human breast cancer and mouse xenograft models of mammary carcinogenesis (see the related article beginning on page 2171). Their finding that RAB GTPase function enables genomic amplification to confer aggressiveness to mammary tumors adds significantly to the body of evidence supporting pivotal roles for receptor trafficking in the proliferation and metastasis of cancer. PMID:19620778

  19. A universal procedure for primer labelling of amplicons.

    PubMed Central

    Neilan, B A; Wilton, A N; Jacobs, D

    1997-01-01

    Detection and visualisation of nucleic acids is integral to genome analyses. Exponential amplification procedures have provided the means for the manipulation of nucleic acid sequences, which were otherwise inaccessible. We describe the development and application of a universal method for the labelling of any PCR product using a single end-labelled primer. Amplification was performed in a single reaction with the resulting amplicon labelled to a high specific activity. The method was adapted to a wide range of PCRs and significantly reduced the expense of such analyses. PMID:9207046

  20. 78 FR 76061 - Authorization for Non-VA Medical Services

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-16

    ... AFFAIRS 38 CFR Part 17 RIN 2900-AO46 Authorization for Non-VA Medical Services AGENCY: Department of... VA's regulations regarding payment by VA for medical services under VA's statutory authority for non-VA medical care. In the Federal Register on November 28, 2012, VA proposed to remove an outdated...

  1. Lower Chesapeake Bay, VA, USA

    NASA Image and Video Library

    1973-06-22

    SL2-16-174 (22 June 1973) --- Norfolk and the lower Chesapeake Bay, VA (37.5N, 75.5W) at the interface of the Atlantic Ocean can be seen to be a mixture of complex currents. Outgoing tides from the bay generate considerable turbulence as they encounter coastal currents and can be observed by the sediment plumes stirred up as a result of current dynamics. Smooth flowing water has less sediment and appears darker. Turbulent water has lots of sediment and appears lighter in color. Photo credit: NASA

  2. VA-academic partnerships: challenges and rewards for new VA mental health investigators.

    PubMed

    Ayers, Catherine; Arch, Joanna

    2013-12-01

    This study presents the perspectives of academic-VA partners who have recently completed a randomized clinical trial within a VA outpatient clinic. The authors reflect on the challenges and rewards of implementing academic-VA community clinical research partnerships with the aim of assisting new VA investigators and VA collaborators. Staff resistance, time demands, processing delays, and unforeseen barriers represent challenges. However, they are balanced by numerous rewards, including establishment of a research clinic, innovative staff training, and advancement of effectiveness knowledge in community settings. Implications and recommendations for successful VA-academic partnerships are described to help future projects minimize challenges and maximize rewards.

  3. Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology.

    PubMed

    Sie, Daoud; Snijders, Peter J F; Meijer, Gerrit A; Doeleman, Marije W; van Moorsel, Marinda I H; van Essen, Hendrik F; Eijk, Paul P; Grünberg, Katrien; van Grieken, Nicole C T; Thunnissen, Erik; Verheul, Henk M; Smit, Egbert F; Ylstra, Bauke; Heideman, Daniëlle A M

    2014-10-01

    Next generation DNA sequencing (NGS) holds promise for diagnostic applications, yet implementation in routine molecular pathology practice requires performance evaluation on DNA derived from routine formalin-fixed paraffin-embedded (FFPE) tissue specimens. The current study presents a comprehensive analysis of TruSeq Amplicon Cancer Panel-based NGS using a MiSeq Personal sequencer (TSACP-MiSeq-NGS) for somatic mutation profiling. TSACP-MiSeq-NGS (testing 212 hotspot mutation amplicons of 48 genes) and a data analysis pipeline were evaluated in a retrospective learning/test set approach (n = 58/n = 45 FFPE-tumor DNA samples) against 'gold standard' high-resolution-melting (HRM)-sequencing for the genes KRAS, EGFR, BRAF and PIK3CA. Next, the performance of the validated test algorithm was assessed in an independent, prospective cohort of FFPE-tumor DNA samples (n = 75). In the learning set, a number of minimum parameter settings was defined to decide whether a FFPE-DNA sample is qualified for TSACP-MiSeq-NGS and for calling mutations. The resulting test algorithm revealed 82% (37/45) compliance to the quality criteria and 95% (35/37) concordant assay findings for KRAS, EGFR, BRAF and PIK3CA with HRM-sequencing (kappa = 0.92; 95% CI = 0.81-1.03) in the test set. Subsequent application of the validated test algorithm to the prospective cohort yielded a success rate of 84% (63/75), and a high concordance with HRM-sequencing (95% (60/63); kappa = 0.92; 95% CI = 0.84-1.01). TSACP-MiSeq-NGS detected 77 mutations in 29 additional genes. TSACP-MiSeq-NGS is suitable for diagnostic gene mutation profiling in oncopathology.

  4. 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.

  5. Multiplex PCR, amplicon size and hybridization efficiency on the NanoChip electronic microarray.

    PubMed

    Børsting, Claus; Sanchez, Juan J; Morling, Niels

    2004-04-01

    We tested the SNP typing protocol developed for the NanoChip electronic microarray by analyzing the four Y chromosome loci SRY1532, SRY8299, TAT, and 92R7. Amplicons of different lengths containing the same locus were purified and addressed to the NanoChip array and fluorescently labelled reporter probes were hybridized to the amplicons. We demonstrated that as little as 10-30 fmol of 50 bp DNA amplicons was sufficient to obtain strong and reproducible results. The hybridization to 50 bp amplicons was up to 10 times more efficient than the hybridization to 200 bp amplicons containing the same SNP. Hybridization to individual amplicons in multiplexes was less efficient suggesting that intramolecular and intermolecular interactions may block access to the target sequence on the NanoChip array. We observed a high risk of contamination with amplicons shorter than 60 bp and therefore, we recommend the use of 60-200 bp amplicons for SNP typing analysis on the NanoChip platform. In a comparative study, we typed the 5 Y chromosome loci M173, 92R7, P25, SRY1532, and M9 in 400 males using the NanoChip SNP typing protocol and the SNaPshot kit. Concording results were obtained for all samples demonstrating the accuracy of the NanoChip SNP typing protocol.

  6. 78 FR 32126 - VA Dental Insurance Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-29

    ... AFFAIRS 38 CFR Part 17 RIN 2900-AN99 VA Dental Insurance Program AGENCY: Department of Veterans Affairs... rules and procedures for the VA Dental Insurance Program (VADIP), a pilot program that offers premium-based dental insurance to enrolled veterans and certain survivors and dependents of veterans. Under...

  7. 48 CFR 801.690 - VA's COCP.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false VA's COCP. 801.690 Section 801.690 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS GENERAL DEPARTMENT OF... Responsibilities 801.690 VA's COCP. ...

  8. 48 CFR 801.690 - VA's COCP.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false VA's COCP. 801.690 Section 801.690 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS GENERAL DEPARTMENT OF... Responsibilities 801.690 VA's COCP. ...

  9. Metagenomic 16S rDNA Illumina tags are a powerful alternative to amplicon sequencing to explore diversity and structure of microbial communities.

    PubMed

    Logares, Ramiro; Sunagawa, Shinichi; Salazar, Guillem; Cornejo-Castillo, Francisco M; Ferrera, Isabel; Sarmento, Hugo; Hingamp, Pascal; Ogata, Hiroyuki; de Vargas, Colomban; Lima-Mendez, Gipsi; Raes, Jeroen; Poulain, Julie; Jaillon, Olivier; Wincker, Patrick; Kandels-Lewis, Stefanie; Karsenti, Eric; Bork, Peer; Acinas, Silvia G

    2014-09-01

    Sequencing of 16S rDNA polymerase chain reaction (PCR) amplicons is the most common approach for investigating environmental prokaryotic diversity, despite the known biases introduced during PCR. Here we show that 16S rDNA fragments derived from Illumina-sequenced environmental metagenomes (mi tags) are a powerful alternative to 16S rDNA amplicons for investigating the taxonomic diversity and structure of prokaryotic communities. As part of the Tara Oceans global expedition, marine plankton was sampled in three locations, resulting in 29 subsamples for which metagenomes were produced by shotgun Illumina sequencing (ca. 700 Gb). For comparative analyses, a subset of samples was also selected for Roche-454 sequencing using both shotgun (m454 tags; 13 metagenomes, ca. 2.4 Gb) and 16S rDNA amplicon (454 tags; ca. 0.075 Gb) approaches. Our results indicate that by overcoming PCR biases related to amplification and primer mismatch, mi tags may provide more realistic estimates of community richness and evenness than amplicon 454 tags. In addition, mi tags can capture expected beta diversity patterns. Using mi tags is now economically feasible given the dramatic reduction in high-throughput sequencing costs, having the advantage of retrieving simultaneously both taxonomic (Bacteria, Archaea and Eukarya) and functional information from the same microbial community.

  10. Amplicon sequencing of bacterial microbiota in abortion material from cattle.

    PubMed

    Vidal, Sara; Kegler, Kristel; Posthaus, Horst; Perreten, Vincent; Rodriguez-Campos, Sabrina

    2017-10-10

    Abortions in cattle have a significant economic impact on animal husbandry and require prompt diagnosis for surveillance of epizootic infectious agents. Since most abortions are not epizootic but sporadic with often undetected etiologies, this study examined the bacterial community present in the placenta (PL, n = 32) and fetal abomasal content (AC, n = 49) in 64 cases of bovine abortion by next generation sequencing (NGS) of the 16S rRNA gene. The PL and AC from three fetuses of dams that died from non-infectious reasons were included as controls. All samples were analyzed by bacterial culture, and 17 were examined by histopathology. We observed 922 OTUs overall and 267 taxa at the genus level. No detectable bacterial DNA was present in the control samples. The microbial profiles of the PL and AC differed significantly, both in their composition (PERMANOVA), species richness and Chao-1 (Mann-Whitney test). In both organs, Pseudomonas was the most abundant genus. The combination of NGS and culture identified opportunistic pathogens of interest in placentas with lesions, such as Vibrio metschnikovii, Streptococcus uberis, Lactococcus lactis and Escherichia coli. In placentas with lesions where culturing was unsuccessful, Pseudomonas and unidentified Aeromonadaceae were identified by NGS displaying high number of reads. Three cases with multiple possible etiologies and placentas presenting lesions were detected by NGS. Amplicon sequencing has the potential to uncover unknown etiological agents. These new insights on cattle abortion extend our focus to previously understudied opportunistic abortive bacteria.

  11. Determination of VA health care costs.

    PubMed

    Barnett, Paul G

    2003-09-01

    In the absence of billing data, alternative methods are used to estimate the cost of hospital stays, outpatient visits, and treatment innovations in the U.S. Department of Veterans Affairs (VA). The choice of method represents a trade-off between accuracy and research cost. The direct measurement method gathers information on staff activities, supplies, equipment, space, and workload. Since it is expensive, direct measurement should be reserved for finding short-run costs, evaluating provider efficiency, or determining the cost of treatments that are innovative or unique to VA. The pseudo-bill method combines utilization data with a non-VA reimbursement schedule. The cost regression method estimates the cost of VA hospital stays by applying the relationship between cost and characteristics of non-VA hospitalizations. The Health Economics Resource Center uses pseudo-bill and cost regression methods to create an encounter-level database of VA costs. Researchers are also beginning to use the VA activity-based cost allocation system.

  12. Post-amplification Klenow fragment treatment alleviates PCR bias caused by partially single-stranded amplicons.

    PubMed

    Egert, Markus; Friedrich, Michael W

    2005-04-01

    Partially single-stranded amplicons, formed during PCR amplification of single and mixed templates, are a potential source of bias in genetic diversity studies. The analysis of 16S rRNA gene diversity in mixed template samples by the fingerprinting technique terminal restriction fragment length polymorphism (T-RFLP) analysis can be biased by the occurrence of pseudo-T-RFs, i.e., restriction fragments occurring in addition to the expected terminal restriction fragments of single amplicons. This bias originates from PCR products, which are single-stranded at their terminal restriction site. Here we show that treatment of PCR amplicons with Klenow fragment prior to restriction digest and T-RFLP analysis minimized effectively the occurrence of pseudo-T-RFs. Klenow fragment activity filled in bases into the partially single-stranded amplicons and thereby restored the affected amplicons to complete double strands. Our method allowed to improve the assessment of genetic diversity and gene ratios from T-RFLP analysis of an original environmental sample. Since partially single-stranded amplicons might influence many PCR-based techniques, post-amplification treatment with Klenow fragment may be useful for a wide range of applications, which assess the composition of amplicon pools, e.g., the analysis of marker gene diversity in mixed template samples by fingerprinting techniques or the analysis of sequence diversity by cloning.

  13. Construction of a cytomegalovirus-based amplicon: a vector with a unique transfer capacity.

    PubMed

    Borst, Eva Maria; Messerle, Martin

    2003-07-01

    Cytomegalovirus (CMV) has a number of interesting properties that qualifies it as a vector for gene transfer. Especially appealing is the ability of the CMV genome to persist in hematopoietic progenitor cells and the packaging capacity of the viral capsid that accommodates a DNA genome of 230 kbp. In order to exploit the packaging capacity of the CMV capsid we investigated whether the principles of an amplicon vector can be applied to CMV. Amplicons are herpesviral vectors, which contain only the cis-active sequences required for replication and packaging of the vector genome. For construction of a CMV amplicon the sequences comprising the lytic origin of replication (orilyt) and the cleavage packaging recognition sites (pac) of human CMV were cloned onto a plasmid. A gene encoding the green fluorescent protein was used as a model transgene. The amplicon plasmid replicated in the presence of a CMV helper virus and was packaged into CMV particles, with replication and packaging being dependent on the presence of the orilyt and pac sequences. The packaged amplicon could be transferred to recipient cells and reisolated from the transduced cells. Analysis of the DNA isolated from CMV capsids revealed that the CMV amplicon was packaged as a concatemer with a size of approximately 210 kbp. The CMV amplicon vector has the potential to transfer therapeutic genes with a size of more than 200 kbp and thus provides a unique transfer capacity among viral vectors.

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

    PubMed

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

    2015-10-20

    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.

  15. 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. PMID:27600235

  16. DAMe: a toolkit for the initial processing of datasets with PCR replicates of double-tagged amplicons for DNA metabarcoding analyses.

    PubMed

    Zepeda-Mendoza, Marie Lisandra; Bohmann, Kristine; Carmona Baez, Aldo; Gilbert, M Thomas P

    2016-05-03

    DNA metabarcoding is an approach for identifying multiple taxa in an environmental sample using specific genetic loci and taxa-specific primers. When combined with high-throughput sequencing it enables the taxonomic characterization of large numbers of samples in a relatively time- and cost-efficient manner. One recent laboratory development is the addition of 5'-nucleotide tags to both primers producing double-tagged amplicons and the use of multiple PCR replicates to filter erroneous sequences. However, there is currently no available toolkit for the straightforward analysis of datasets produced in this way. We present DAMe, a toolkit for the processing of datasets generated by double-tagged amplicons from multiple PCR replicates derived from an unlimited number of samples. Specifically, DAMe can be used to (i) sort amplicons by tag combination, (ii) evaluate PCR replicates dissimilarity, and (iii) filter sequences derived from sequencing/PCR errors, chimeras, and contamination. This is attained by calculating the following parameters: (i) sequence content similarity between the PCR replicates from each sample, (ii) reproducibility of each unique sequence across the PCR replicates, and (iii) copy number of the unique sequences in each PCR replicate. We showcase the insights that can be obtained using DAMe prior to taxonomic assignment, by applying it to two real datasets that vary in their complexity regarding number of samples, sequencing libraries, PCR replicates, and used tag combinations. Finally, we use a third mock dataset to demonstrate the impact and importance of filtering the sequences with DAMe. DAMe allows the user-friendly manipulation of amplicons derived from multiple samples with PCR replicates built in a single or multiple sequencing libraries. It allows the user to: (i) collapse amplicons into unique sequences and sort them by tag combination while retaining the sample identifier and copy number information, (ii) identify sequences carrying

  17. "COV'COP" allows to detect CNVs responsible for inherited diseases among amplicons sequencing data.

    PubMed

    Derouault, P; Parfait, B; Moulinas, R; Barrot, C-C; Sturtz, F; Merillou, S; Lia, A-S

    2017-01-30

    In order to help molecular geneticists to rapidly identify CNVs responsible for inherited diseases among amplicons sequencing data generated by NGS, we designed a user-friendly tool "Cov'Cop". Using the run's coverage file provided by the sequencer, Cov'Cop simultaneously analyzes all the patients of the run using a two-stage algorithm containing correction and normalization levels and provides an easily understandable output, showing with various colors, potentially deleted and duplicated amplicons.

  18. 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

  19. Gene transfer into hepatocytes mediated by herpes simplex virus-Epstein-Barr virus hybrid amplicons.

    PubMed

    Müller, Lars; Saydam, Okay; Saeki, Yoshinaga; Heid, Irma; Fraefel, Cornel

    2005-01-01

    Gene transfer into hepatocytes is highly desirable for the long-term goal of replacing deficient proteins and correcting metabolic disorders. Vectors based on herpes simplex virus type-1 (HSV-1) have been demonstrated to mediate efficient gene transfer into hepatocytes both in vitro and in vivo. Large transgene capacity and extrachromosomal persistence make HSV-1/EBV hybrid amplicon vectors an attractive candidate for hepatic gene replacement therapy. To assess liver-directed gene transfer, we constructed (i) a conventional HSV-1 amplicon vector encoding a secreted reporter protein (secreted alkaline phosphatase, SEAP) under the control of the HSV-1 immediate-early 4/5 promoter; (ii) a HSV-1 amplicon encoding SEAP under the control of the artificial CAG promoter (the chicken beta-actin promoter and cytomegalovirus (CMV) immediate-early enhancer); and (iii) a HSV-1/EBV hybrid amplicon, also encoding SEAP under the control of the CAG promoter. While all three vector constructs yielded high SEAP concentrations in vitro and in vivo, use of HSV-1/EBV hybrid amplicon vectors significantly prolonged the duration of gene expression. Using conventional amplicon vectors in cultured hepatocytes, SEAP was detected for two weeks, whereas SEAP was detected for at least six weeks when HSV-1/EBV amplicons were used. Intraparenchymal injection into the liver of SICD mice yielded high (up to 77 ng of SEAP per milliliter serum) and sustained (greater than three weeks) expression of SEAP. Serum transaminases (ALT/AST) were measured at different time points to monitor for hepatocellular damage. While initially elevated four times above baseline, the transaminase levels returned to normal after three to seven days. These results demonstrate the usefulness of HSV-1-based amplicons and SEAP for the evaluation of gene replacement strategies in the liver.

  20. 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). (c) 2016 APA, all rights reserved).

  1. Imaging herpes simplex virus type 1 amplicon vector-mediated gene expression in human glioma spheroids.

    PubMed

    Kaestle, Christine; Winkeler, Alexandra; Richter, Raphaela; Sauer, Heinrich; Hescheler, Jürgen; Fraefel, Cornel; Wartenberg, Maria; Jacobs, Andreas H

    2011-06-01

    Vectors derived from herpes simplex virus type 1 (HSV-1) have great potential for transducing therapeutic genes into the central nervous system; however, inefficient distribution of vector particles in vivo may limit their therapeutic potential in patients with gliomas. This study was performed to investigate the extent of HSV-1 amplicon vector-mediated gene expression in a three-dimensional glioma model of multicellular spheroids by imaging highly infectious HSV-1 virions expressing green fluorescent protein (HSV-GFP). After infection or microscopy-guided vector injection of glioma spheroids at various spheroid sizes, injection pressures and injection times, the extent of HSV-1 vector-mediated gene expression was investigated via laser scanning microscopy. Infection of spheroids with HSV-GFP demonstrated a maximal depth of vector-mediated GFP expression at 70 to 80 μm. A > 80% transduction efficiency was reached only in small spheroids with a diameter of < 150 μm. Guided vector injection into the spheroids showed transduction efficiencies ranging between < 10 and > 90%. The results demonstrated that vector-mediated gene expression in glioma spheroids was strongly dependent on the mode of vector application-injection pressure and injection time being the most important parameters. The assessment of these vector application parameters in tissue models will contribute to the development of safe and efficient gene therapy protocols for clinical application.

  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. 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…

  4. 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

  5. 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/. © The Author(s) 2015. Published by Oxford University Press.

  6. Generic Amplicon Deep Sequencing to Determine Ilarvirus Species Diversity in Australian Prunus

    PubMed Central

    Kinoti, Wycliff M.; Constable, Fiona E.; Nancarrow, Narelle; Plummer, Kim M.; Rodoni, Brendan

    2017-01-01

    The distribution of Ilarvirus species populations amongst 61 Australian Prunus trees was determined by next generation sequencing (NGS) of amplicons generated using a genus-based generic RT-PCR targeting a conserved region of the Ilarvirus RNA2 component that encodes the RNA dependent RNA polymerase (RdRp) gene. Presence of Ilarvirus sequences in each positive sample was further validated by Sanger sequencing of cloned amplicons of regions of each of RNA1, RNA2 and/or RNA3 that were generated by species specific PCRs and by metagenomic NGS. Prunus necrotic ringspot virus (PNRSV) was the most frequently detected Ilarvirus, occurring in 48 of the 61 Ilarvirus-positive trees and Prune dwarf virus (PDV) and Apple mosaic virus (ApMV) were detected in three trees and one tree, respectively. American plum line pattern virus (APLPV) was detected in three trees and represents the first report of APLPV detection in Australia. Two novel and distinct groups of Ilarvirus-like RNA2 amplicon sequences were also identified in several trees by the generic amplicon NGS approach. The high read depth from the amplicon NGS of the generic PCR products allowed the detection of distinct RNA2 RdRp sequence variant populations of PNRSV, PDV, ApMV, APLPV and the two novel Ilarvirus-like sequences. Mixed infections of ilarviruses were also detected in seven Prunus trees. Sanger sequencing of specific RNA1, RNA2, and/or RNA3 genome segments of each virus and total nucleic acid metagenomics NGS confirmed the presence of PNRSV, PDV, ApMV and APLPV detected by RNA2 generic amplicon NGS. However, the two novel groups of Ilarvirus-like RNA2 amplicon sequences detected by the generic amplicon NGS could not be associated to the presence of sequence from RNA1 or RNA3 genome segments or full Ilarvirus genomes, and their origin is unclear. This work highlights the sensitivity of genus-specific amplicon NGS in detection of virus sequences and their distinct populations in multiple samples, and the need

  7. Electroanalytical study of proflavine intercalation in 5-methyl or inosine-containing amplicons.

    PubMed

    Alexiadou, Despina K; Ioannou, Andrea K; Kouidou-Andreou, Sofia A; Voulgaropoulos, Anastasios N; Girousi, Stella Th

    2008-10-01

    Amplicons corresponding to the GC-rich p53 exon 5 and its analogues, synthesized by substituting 60% of cytosine by 5-methyl-cytosine, or 60% of guanosine by inosine and GC-poor p53 exon 6 were synthesized and investigated electrochemically, in the presence and absence of proflavine, by differential pulse voltammetry (DPV). Incorporation of base analogues and the thermal stability of the resulting amplicons were tested in the presence of a fluorescent probe (Sybr-Green). Peak current at 1.0 V was lower for methylated than for unmethylated PCR amplicons and was similarly affected by proflavine intercalation. In contrast, considerable peak current differences were observed in the presence of proflavine for unmodified exon 5 v.s. exon 6 or inosine-containing amplicons. Thermal analysis verified the expected shifts in melting temperature (T (m)) due to the base analogue incorporation and GC-content variations. In conclusion, methylated and unmethylated PCR amplicons could be distinguished in model DNA systems using differential pulse voltammetry (DPV) and use of proflavine could serve as an electrochemical probe for identifying different DNA conformations.

  8. 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.

  9. 48 CFR 801.690 - VA's COCP.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false VA's COCP. 801.690 Section 801.690 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS GENERAL DEPARTMENT OF VETERANS AFFAIRS ACQUISITION REGULATION SYSTEM Career Development, Contracting Authority,...

  10. Genetics Home Reference: carbonic anhydrase VA deficiency

    MedlinePlus

    ... Lonlay P, Burlina A, van Karnebeek CD, Häberle J. Defective hepatic bicarbonate production due to carbonic anhydrase ... on PubMed GeneReview: Carbonic Anhydrase VA Deficiency Häberle J. Clinical and biochemical aspects of primary and secondary ...

  11. Sex determination in beef by melting curve analysis of PCR amplicons from the amelogenin locus.

    PubMed

    Ballin, Nicolai Z; Madsen, Knud G

    2007-11-01

    Sex determination of beef is important to meet the rules of the Commission Regulation (EC) 765/2002 that qualify for export refunds. A SYBR Green sex identification assay based on melting curve analysis of PCR amplicons from the amelogenin locus (AMELX and AMELY) was developed. The PCR amplicons of 130/130 and 130/67 base pairs produced from female and male beef, respectively, are easily distinguished by both melting curve analysis and gel electrophoresis. Results from the melting curve analysis of amplicons are ready in less than three minutes, and requires no additional work in addition to the PCR setup. Applicability of the sex determination assay was studied by analysis of 12 unknown beef samples and the results were compared to an accredited method based on gel electrophoresis. In addition, six different cattle breeds were examined. All test results were correct in respect to sex.

  12. Glioma-specific and cell cycle-regulated herpes simplex virus type 1 amplicon viral vector.

    PubMed

    Ho, Ivy A W; Hui, Kam M; Lam, Paula Y P

    2004-05-01

    We have engineered a novel herpes simplex virus type 1 (HSV-1)-based amplicon viral vector, whereby gene expression is controlled by cell cycle events. In nondividing cells, trans-activation of the cyclin A promoter via interaction of the Gal4/NF-YA fusion protein with the Gal4-binding sites is prevented by the presence of a repressor protein, cell cycle-dependent factor 1 (CDF-1). CDF-1 is specifically expressed during the G(0)/G(1) phase of the cell cycle and its binding site is located within the cyclin A promoter. In actively proliferating cells, trans-activation could take place because of the absence of CDF-1. Our results showed that when all these cell cycle-specific regulatory elements are incorporated in cis into a single HSV-1 amplicon plasmid vector backbone (pC8-36), reporter luciferase activity is greatly enhanced. Transgene expression mediated by this series of HSV-1 amplicon plasmid vectors and amplicon viral vectors could be regulated in a cell cycle-dependent manner in a variety of cell lines. In a further attempt to target transgene expression to a selected group of actively proliferating cells such as glial cells, we have replaced the cytomegalovirus promoter of the pC8-36 amplicon plasmid with the glial cell-specific GFAP enhancer element. With this latter viral construct, cell type-specific and cell cycle-dependent transgene expression could subsequently be demonstrated specifically in glioma-bearing animals. Taken together, our results suggest that this series of cell cycle-regulatable HSV-1 amplicon viral vectors could potentially be adapted as useful tools for the treatment of human cancers.

  13. Improved packaging system for generation of high-level noncytotoxic HSV-1 amplicon vectors using Cre-loxP site-specific recombination to delete the packaging signals of defective helper genomes.

    PubMed

    Zaupa, Cécile; Revol-Guyot, Valerie; Epstein, Alberto L

    2003-07-20

    Amplicons are promising helper-dependent HSV-1-derived vectors that allow the transfer and expression of very large foreigner DNA into dividing and quiescent cells. We had already described an approach to prepare large amounts of high-titer amplicon vectors, using Cre-loxP site-specific recombination system to delete the packaging ("a") signals of an HSV-1 recombinant helper virus (HSV-1 LaL). Amplicon vectors prepared using such a system showed a level of contamination with helper particles lower than 1%. The residual helper particles generated by this system are, however, replication-competent, thus precluding their use in gene therapy. To avoid such potential spread of residual particles, we present here the development of a defective Cre-loxP-based helper virus (HSV-1 LaL Delta J), deleted of the genes encoding ICP4 and ICP34.5 proteins from the helper genome, in addition to the native "a" signals. HSV-1 LaL Delta J carries a single floxed "a" signal in gC locus. To produce HSV-1 LaL Delta J and to prepare the amplicon vectors, we have constructed two novel cell lines expressing the essential ICP4 protein, either alone or in combination with Cre recombinase. These cell lines were conceived to complement ICP4 while minimizing the probability of generating replication-competent particles. In this paper we present results demonstrating that the novel helper system allows ready production of large amounts of high-titer amplicon vectors. Residual helper particles generated still do not exceed 0.5% of the viral population and can grow only in cells expressing ICP4. Amplicon vectors produced with this method showed no cytotoxicty for infected cells.

  14. Use of AmpliWax to optimize amplicon sterilization by isopsoralen.

    PubMed Central

    De la Viuda, M; Fille, M; Ruiz, J; Aslanzadeh, J

    1996-01-01

    The photochemical inactivation of amplicons by isopsoralen (IP-10) has been suggested as a possible means to prevent PCR carryover contamination. To evaluate the technique, serial dilutions of amplicons (10(11) to 10(3)) from the Borrelia burgdorferi OSP A gene were amplified in the presence of 0, 25, 50, and 100 micrograms of IP-10 per ml for 45 cycles. The PCR products were exposed to UV light for 15 min to activate IP-10 and sterilize the amplicons. One microliter of each sterilized sample was reamplified for an additional 45 cycles. The PCR products were then resolved in an agarose gel, blotted onto a nylon membrane, and probed with an alkaline phosphatase-conjugated chemiluminescent probe. Although IP-10 at concentrations of 50 and 100 micrograms/ml effectively sterilized up to 10(11) amplicons, the compound was inhibitory to PCR. IP-10 at a concentration of 25 micrograms/ml had slight inhibitory effect on PCR and did not completely sterilized all of the amplicons. Therefore, in subsequent experiments AmpliWax was substituted for mineral oil, and PCR was performed on 10(9) to 10(3) amplicons as described above. Following the amplification, the PCR tubes were cooled to solidify the AmpliWax and inoculated with various concentrations of IP-10. With this technique, PCR products produced from as many as 10(9) target amplicons were effectively sterilized with 200 micrograms of IP-10 per ml. Similarly, the addition of IP-10 (50 micrograms/ml) before and after PCR was evaluated for the detection of B. burgdorferi in 62 ticks from a region of Southern Connecticut where the organism is highly endemic. PCR performed in the presence of 50 micrograms of IP-10 per ml detected B. burgdorferi-specific DNA in 17 of 62 ticks (27%) following gel electrophoresis and in 34 of 62 ticks (55%) following Southern blot hybridization of the PCR products. In contrast, post-PCR addition of IP-10 detected borrelia-specific DNA in 31 of 62 ticks (50%) following gel electrophoresis and in

  15. Accessing VA Healthcare During Large-Scale Natural Disasters.

    PubMed

    Der-Martirosian, Claudia; Pinnock, Laura; Dobalian, Aram

    2017-01-01

    Natural disasters can lead to the closure of medical facilities including the Veterans Affairs (VA), thus impacting access to healthcare for U.S. military veteran VA users. We examined the characteristics of VA patients who reported having difficulty accessing care if their usual source of VA care was closed because of natural disasters. A total of 2,264 veteran VA users living in the U.S. northeast region participated in a 2015 cross-sectional representative survey. The study used VA administrative data in a complex stratified survey design with a multimode approach. A total of 36% of veteran VA users reported having difficulty accessing care elsewhere, negatively impacting the functionally impaired and lower income VA patients.

  16. PANGEA: pipeline for analysis of next generation amplicons.

    PubMed

    Giongo, Adriana; Crabb, David B; Davis-Richardson, Austin G; Chauliac, Diane; Mobberley, Jennifer M; Gano, Kelsey A; Mukherjee, Nabanita; Casella, George; Roesch, Luiz F W; Walts, Brandon; Riva, Alberto; King, Gary; Triplett, Eric W

    2010-07-01

    High-throughput DNA sequencing can identify organisms and describe population structures in many environmental and clinical samples. Current technologies generate millions of reads in a single run, requiring extensive computational strategies to organize, analyze and interpret those sequences. A series of bioinformatics tools for high-throughput sequencing analysis, including pre-processing, clustering, database matching and classification, have been compiled into a pipeline called PANGEA. The PANGEA pipeline was written in Perl and can be run on Mac OSX, Windows or Linux. With PANGEA, sequences obtained directly from the sequencer can be processed quickly to provide the files needed for sequence identification by BLAST and for comparison of microbial communities. Two different sets of bacterial 16S rRNA sequences were used to show the efficiency of this workflow. The first set of 16S rRNA sequences is derived from various soils from Hawaii Volcanoes National Park. The second set is derived from stool samples collected from diabetes-resistant and diabetes-prone rats. The workflow described here allows the investigator to quickly assess libraries of sequences on personal computers with customized databases. PANGEA is provided for users as individual scripts for each step in the process or as a single script where all processes, except the chi(2) step, are joined into one program called the 'backbone'.

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

    PubMed

    Salentijn, Elma Mj; Esselink, Danny G; Goryunova, Svetlana V; van der Meer, Ingrid M; Gilissen, Luud J W J; Smulders, Marinus J M

    2013-12-19

    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. 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. 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 be useful as a reference in

  18. 78 FR 48609 - Safety Zone; James River; Newport News, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-09

    ... SECURITY Coast Guard 33 CFR Part 165 RIN 1625-AA00 Safety Zone; James River; Newport News, VA AGENCY: Coast... as follows: Sec. 165.T05-0670 Safety Zone, James River, Newport News, VA. (a) Definitions. For the...'' N longitude 076 38'40'' W, located near Fort Eustis in Newport News, VA. (c) Regulations. (1)...

  19. 38 CFR 1.920 - Referral of VA debts.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ..., the date on which payment became due, and the date VA's right to collect the debt first accrued. (c) This certification will also state that VA provided the debtor with written notice of: (1) The nature... opportunity to inspect and copy VA records pertaining to the debt; (4) The right to contest both the existence...

  20. 48 CFR 819.7109 - VA review of application.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false VA review of application... SOCIOECONOMIC PROGRAMS SMALL BUSINESS PROGRAMS VA Mentor-Protégé Program 819.7109 VA review of application. (a... that the information that is in VAAR 819.7108 is included. If the application relates to a specific...

  1. 38 CFR 74.27 - How will VA store information?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2012-07-01 2012-07-01 false How will VA store information? 74.27 Section 74.27 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) VETERANS SMALL BUSINESS REGULATIONS Records Management § 74.27 How will VA store information? VA...

  2. 38 CFR 3.1700 - Types of VA burial benefits.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2014-07-01 2014-07-01 false Types of VA burial... ADJUDICATION Burial Benefits Burial Benefits: General § 3.1700 Types of VA burial benefits. Pt. 3, Subpt. B, Nt. (a) Burial benefits. VA provides the following types of burial benefits, which are discussed in §§...

  3. Electronic microarray analysis of 16S rDNA amplicons for bacterial detection.

    PubMed

    Barlaan, Edward A; Sugimori, Miho; Furukawa, Seiji; Takeuchi, Kazuhisa

    2005-01-12

    Electronic microarray technology is a potential alternative in bacterial detection and identification. However, conditions for bacterial detection by electronic microarray need optimization. Using the NanoChip electronic microarray, we investigated eight marine bacterial species. Based on the 16S rDNA sequences of these species, we constructed primers, reporter probes, and species-specific capture probes. We carried out two separate analyses for longer (533 bp) and shorter (350 and 200 bp) amplified products (amplicons). To detect simultaneously the hybridization signals for the 350- and 200-bp amplicons, we designed a common reporter probe from an overlapping sequence within both fragments. We developed methods to optimize detection of hybridization signals for processing the DNA chips. A matrix analysis was performed for different bacterial species and complementary capture probes on electronic microarrays. Results showed that, when using the longer amplicon, not all bacterial targets hybridized with the complementary capture probes, which was characterized by the presence of false-positive signals. However, with the shorter amplicons, all bacterial species were correctly and completely detected using the constructed complementary capture probes.

  4. High-throughput amplicon sequencing reveals distinct communities within a corroding concrete sewer system.

    PubMed

    Cayford, Barry I; Dennis, Paul G; Keller, Jurg; Tyson, Gene W; Bond, Philip L

    2012-10-01

    Microbially induced concrete corrosion (MICC) is an important problem in sewers. Here, small-subunit (SSU) rRNA gene amplicon pyrosequencing was used to characterize MICC communities. Microbial community composition differed between wall- and ceiling-associated MICC layers. Acidithiobacillus spp. were present at low abundances, and the communities were dominated by other sulfur-oxidizing-associated lineages.

  5. 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

  6. High-Throughput Amplicon Sequencing Reveals Distinct Communities within a Corroding Concrete Sewer System

    PubMed Central

    Dennis, Paul G.; Keller, Jurg; Tyson, Gene W.

    2012-01-01

    Microbially induced concrete corrosion (MICC) is an important problem in sewers. Here, small-subunit (SSU) rRNA gene amplicon pyrosequencing was used to characterize MICC communities. Microbial community composition differed between wall- and ceiling-associated MICC layers. Acidithiobacillus spp. were present at low abundances, and the communities were dominated by other sulfur-oxidizing-associated lineages. PMID:22843532

  7. VaST: Variability Search Toolkit

    NASA Astrophysics Data System (ADS)

    Sokolovsky, Kirill V.; Lebedev, Alexandr A.

    2017-04-01

    VaST (Variability Search Toolkit) finds variable objects on a series of astronomical images in FITS format. The software performs object detection and aperture photometry using SExtractor (ascl:1010.064) on each image, cross-matches lists of detected stars, performs magnitude calibration with respect to the first (reference) image and constructs a lightcurve for each object. The sigma-magnitude, Stetson's L variability index, Robust Median Statistic (RoMS) and other plots may be used to visually identify variable star candidates. The two distinguishing features of VaST are its ability to perform accurate aperture photometry of images obtained with non-linear detectors and to handle complex image distortions. VaST can be used in cases of unstable PSF (e.g., bad guiding or with digitized wide-field photographic images), and has been successfully applied to images obtained with telescopes ranging from 0.08 to 2.5m in diameter equipped with a variety of detectors including CCD, CMOS, MIC and photographic plates.

  8. Protocols for metagenomic DNA extraction and Illumina amplicon library preparation for faecal and swab samples.

    PubMed

    Vo, A-T E; Jedlicka, J A

    2014-11-01

    Next-generation sequencing (NGS) technology has extraordinarily enhanced the scope of research in the life sciences. To broaden the application of NGS to systems that were previously difficult to study, we present protocols for processing faecal and swab samples into amplicon libraries amenable to Illumina sequencing. We developed and tested a novel metagenomic DNA extraction approach using solid phase reversible immobilization (SPRI) beads on Western Bluebird (Sialia mexicana) samples stored in RNAlater. Compared with the MO BIO PowerSoil Kit, the current standard for the Human and Earth Microbiome Projects, the SPRI-based method produced comparable 16S rRNA gene PCR amplification from faecal extractions but significantly greater DNA quality, quantity and PCR success for both cloacal and oral swab samples. We furthermore modified published protocols for preparing highly multiplexed Illumina libraries with minimal sample loss and without post-adapter ligation amplification. Our library preparation protocol was successfully validated on three sets of heterogeneous amplicons (16S rRNA gene amplicons from SPRI and PowerSoil extractions as well as control arthropod COI gene amplicons) that were sequenced across three independent, 250-bp, paired-end runs on Illumina's MiSeq platform. Sequence analyses revealed largely equivalent results from the SPRI and PowerSoil extractions. Our comprehensive strategies focus on maximizing efficiency and minimizing costs. In addition to increasing the feasibility of using minimally invasive sampling and NGS capabilities in avian research, our methods are notably not avian-specific and thus applicable to many research programmes that involve DNA extraction and amplicon sequencing. © 2014 John Wiley & Sons Ltd.

  9. 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

  10. 76 FR 61150 - Enhanced-Use Lease (EUL) of Department of Veterans Affairs (VA) Real Property at the VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-03

    ..., manage, maintain and operate the EUL development. As consideration for the lease, the lessee will be... AFFAIRS Enhanced-Use Lease (EUL) of Department of Veterans Affairs (VA) Real Property at the VA... Intent to Enter into an Enhanced-Use Lease. SUMMARY: The Secretary of VA intends to enter into an EUL...

  11. Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line.

    PubMed

    Ma, C; Looney, J E; Leu, T H; Hamlin, J L

    1988-06-01

    We have recently isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase (dhfr) amplicon types from the methotrexate-resistant Chinese hamster ovary (CHO) cell line CHOC 400. In the work described in this report, we used pulse-field gradient gel electrophoresis to analyze large SfiI restriction fragments arising from the amplified dhfr domains. The junction between the 260-kilobase type I amplicons (which are arranged in head-to-tail configurations in the genome) has been localized, allowing the construction of a linear map of the parental dhfr locus. We also show that the 220-kilobase type II amplicons are arranged as inverted repeat structures in the CHOC 400 genome and arose from the type I sequence relatively early in the amplification process. Our data indicate that there are a number of minor amplicon types in the CHOC 400 cell line that were not detected in previous studies; however, the type II amplicons represent ca. 75% of all the amplicons in the CHOC 400 genome. Both the type I and type II amplicons are shown to be composed entirely of sequences that were present in the parental dhfr locus. Studies of less resistant cell lines show that initial amplicons can be larger than those observed in CHOC 400. Once established, a given amplicon type appears to be relatively stable throughout subsequent amplification steps. We also present a modification of an in-gel renaturation method that gives a relatively complete picture of the size and variability of amplicons in the genome.

  12. Decontamination of 16S rRNA gene amplicon sequence datasets based on bacterial load assessment by qPCR.

    PubMed

    Lazarevic, Vladimir; Gaïa, Nadia; Girard, Myriam; Schrenzel, Jacques

    2016-04-23

    Identification of unexpected taxa in 16S rRNA surveys of low-density microbiota, diluted mock communities and cultures demonstrated that a variable fraction of sequence reads originated from exogenous DNA. The sources of these contaminants are reagents used in DNA extraction, PCR, and next-generation sequencing library preparation, and human (skin, oral and respiratory) microbiota from the investigators. For in silico removal of reagent contaminants, a pipeline was used which combines the relative abundance of operational taxonomic units (OTUs) in V3-4 16S rRNA gene amplicon datasets with bacterial DNA quantification based on qPCR targeting of the V3 segment of the 16S rRNA gene. Serially diluted cultures of Escherichia coli and Staphylococcus aureus were used for 16S rDNA profiling, and DNA from each of these species was used as a qPCR standard. OTUs assigned to Escherichia or Staphylococcus were virtually unaffected by the decontamination procedure, whereas OTUs from Pseudomonas, which is a major reagent contaminant, were completely or nearly completely removed. The decontamination procedure also attenuated the trend of increase in OTU richness in serially diluted cultures. Removal of contaminant sequences derived from reagents based on use of qPCR data may improve taxonomic representation in samples with low DNA concentration. Using the described pipeline, OTUs derived from cross-contamination of negative extraction controls were not recognized as contaminants and not removed from the sample dataset.

  13. 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

  14. 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 for...

  15. 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 for...

  16. 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 for...

  17. 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 for...

  18. 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 for...

  19. Potential of pmoA amplicon pyrosequencing for methanotroph diversity studies.

    PubMed

    Lüke, Claudia; Frenzel, Peter

    2011-09-01

    We analyzed the potential of pmoA amplicon pyrosequencing compared to that of Sanger sequencing with paddy soils as a model environment. We defined operational taxonomic unit (OTU) cutoff values of 7% and 18%, reflecting methanotrophic species and major phylogenetic pmoA lineages, respectively. Major lineages were already well covered by clone libraries; nevertheless, pyrosequencing provided a higher level of diversity at the species level.

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

    PubMed

    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.

  1. 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.

  2. Characterization of Novel Genes Within 8P11-12 Amplicon in Breast Cancer

    DTIC Science & Technology

    2007-06-01

    C-myc amplification in breast cancer: a meta - analysis of its occurrence and prognostic relevance. Br J Cancer, 83: 1688-1695, 2000. 2. Hui, R...Nass SJ, Dickson RB, Trock BJ. C-myc amplification in breast cancer: a meta - analysis of its occurrence and prognostic relevance. Br J Cancer 2000;83...a detailed genomic and expression analysis of the 8p11-p12 amplicon in breast cancer cell lines and identified several novel candidate genes

  3. 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.

  4. 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:…

  5. Automated amplicon design suitable for analysis of DNA variants by melting techniques.

    PubMed

    Ekstrøm, Per Olaf; Nakken, Sigve; Johansen, Morten; Hovig, Eivind

    2015-11-11

    The technological development of DNA analysis has had tremendous development in recent years, and the present deep sequencing techniques present unprecedented opportunities for detailed and high-throughput DNA variant detection. Although DNA sequencing has had an exponential decrease in cost per base pair analyzed, focused and target-specific methods are however still much in use for analysis of DNA variants. With increasing capacity in the analytical procedures, an equal demand in automated amplicon and primer design has emerged. We have constructed a web-based tool that is able to batch design DNA variant assay suitable for analysis by denaturing gel/capillary electrophoresis and high resolution melting. The tool is developed as a computational workflow that implements one of the most widely used primer design tools, followed by validation of primer specificity, as well as calculation and visualization of the melting properties of the resulting amplicon, with or without an artificial high melting domain attached. The tool will be useful for scientists applying DNA melting techniques in analysis of DNA variations. The tool is freely available at http://meltprimer.ous-research.no/ . Herein, we demonstrate a novel tool with respect to covering the whole amplicon design workflow necessary for groups that use melting equilibrium techniques to separate DNA variants.

  6. 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

  7. Microbes in deep marine sediments viewed through amplicon sequencing and metagenomics

    NASA Astrophysics Data System (ADS)

    Biddle, J.; Leon, Z. R.; Russell, J. A., III; Martino, A. J.

    2016-12-01

    Nearly twenty percent of microbial biomass on Earth can be found in the marine subsurface. The majority of this is concentrated on continental margins, which have been investigated by scientific drilling. On the Costa Rica Margin, Iberian Margin and Peru Margins, sediment samples have been investigated through DNA extraction followed by amplicon and metagenomic sequencing. Overall samples show a high degree of microbial diversity, including many lineages of newly defined groups. In this talk, metagenome assembled genomes of unusual lineages will be presented, including their relationships to shallower relatives. From Costa Rica, in particular, we have retrieved deep relatives of Lokiarchaeota and Thorarchaeota, as well as other deeply branching archaeal relatives. We discuss their genome similarities to both other archaea and eukaryotes. From the Iberian Margin, relatives of Atribacteria and Aerophobetes will be discussed. Finally, we will detail the knowledge lost or gained depending on whether samples are studied via amplicon sequencing or total metagenomics, as studies in other environments have shown that up to 15% of microbial diversity is ignored when samples are studied via amplicon sequencing alone.

  8. Extending the transposable payload limit of Sleeping Beauty (SB) using the Herpes Simplex Virus (HSV)/SB amplicon vector platform

    PubMed Central

    de Silva, Suresh; Mastrangelo, Michael A.; Lotta, Louis T.; Burris, Clark A.; Federoff, Howard J.; Bowers, William J.

    2009-01-01

    The ability of a viral vector to safely deliver and stably integrate large transgene units (transgenons), which not only include one or several therapeutic genes but also requisite native transcriptional regulatory elements, would be of significant benefit for diseases presently refractory to available technologies. The herpes simplex virus type-1 (HSV-1) amplicon vector has the largest known payload capacity of approximately 130 kb, but its episomal maintenance within the transduced cell nucleus and induction of host cell silencing mechanisms limits the duration of the delivered therapeutic gene(s). Our laboratory developed an integration-competent version of the HSV-1 amplicon by adaptation of the Sleeping Beauty (SB) transposon system, which significantly extends transgene expression in vivo. The maximum size limit of the amplicon-vectored transposable element remains unknown, but previously published plasmid-centric studies have established that DNA segments larger than 6-kb are inefficiently transposed. Herein, we compared the transposition efficiency of SB transposase in the context of both the HSV amplicon vector as well as the HSV amplicon plasmid harboring 7-kb and 12-kb transposable reporter transgene units. Our results indicate that the transposition efficiency of the 12-kb transposable unit via SB transposase was significantly reduced compared to the 7-kb transposable unit when the plasmid version of the HSV amplicon was used. However, the packaged HSV amplicon vector form provided a more amenable platform from which the 12-kb transposable unit was mobilized at a similar efficiency to that of the 7-kb transposable unit via the SB transposase. Overall, our results indicate that SB is competent in stably integrating transgenon units of at least 12 kb in size within the human genome upon delivery of the platform via HSV amplicons. PMID:19865178

  9. 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

  10. Mining environmental high-throughput sequence data sets to identify divergent amplicon clusters for phylogenetic reconstruction and morphotype visualization.

    PubMed

    Gimmler, Anna; Stoeck, Thorsten

    2015-08-01

    Environmental high-throughput sequencing (envHTS) is a very powerful tool, which in protistan ecology is predominantly used for the exploration of diversity and its geographic and local patterns. We here used a pyrosequenced V4-SSU rDNA data set from a solar saltern pond as test case to exploit such massive protistan amplicon data sets beyond this descriptive purpose. Therefore, we combined a Swarm-based blastn network including 11 579 ciliate V4 amplicons to identify divergent amplicon clusters with targeted polymerase chain reaction (PCR) primer design for full-length small subunit of the ribosomal DNA retrieval and probe design for fluorescence in situ hybridization (FISH). This powerful strategy allows to benefit from envHTS data sets to (i) reveal the phylogenetic position of the taxon behind divergent amplicons; (ii) improve phylogenetic resolution and evolutionary history of specific taxon groups; (iii) solidly assess an amplicons (species') degree of similarity to its closest described relative; (iv) visualize the morphotype behind a divergent amplicons cluster; (v) rapidly FISH screen many environmental samples for geographic/habitat distribution and abundances of the respective organism and (vi) to monitor the success of enrichment strategies in live samples for cultivation and isolation of the respective organisms.

  11. 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

  12. Proposed comprehensive ototoxicity monitoring program for VA healthcare (COMP-VA)

    PubMed Central

    Konrad-Martin, Dawn; Reavis, Kelly M.; McMillan, Garnett; Helt, Wendy J.; Dille, Marilyn

    2015-01-01

    Prevention and rehabilitation of hearing loss and tinnitus, the two most commonly awarded service-connected disabilities, are high priority initiatives in the Department of Veterans Affairs (VA). At least 4,000 Veterans, most with significant hearing loss, will receive cisplatin this year, with more than half sustaining permanent hearing shift and nearly 40% developing new tinnitus. With improved survivability following cancer treatment, Veterans treated with cisplatin are approached with the dual goals of effective treatment and preserved quality of life. This article describes COMP-VA, a comprehensive ototoxicity monitoring program developed for VA patients receiving cisplatin. The program includes an individualized pretreatment prediction model that identifies the likelihood of hearing shift given cisplatin dose and patient factors. It supports both manual and automated hearing testing with a newly developed portable audiometer capable of performing the recommended procedures on the chemotherapy unit during treatment. It also includes objective methods for identifying outer hair cell changes and predicting audiogram changes using distortion-product otoacoustic emissions. We describe this program of evidence-based ototoxicity monitoring protocols using a case example to give the reader an understanding of how this program would be applied, along with a plan for future work to accomplish the final stages of program development. PMID:24805896

  13. 78 FR 71041 - VA Compensation and Pension Regulation Rewrite Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-27

    ...The Department of Veterans Affairs (VA) proposes to reorganize and rewrite its compensation and pension regulations in a logical, claimant-focused, and user-friendly format. The intended effect of the proposed revisions is to assist claimants, beneficiaries, veterans' representatives, and VA personnel in locating and understanding these...

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

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-22

    ... Federal Aviation Administration 14 CFR Part 71 Establishment of Class E Airspace; Forest, VA AGENCY... Airspace at Forest, VA, to accommodate the new Area Navigation (RNAV) Global Positioning System (GPS... published in the Federal Register a notice of proposed rulemaking to establish Class E airspace at Forest...

  15. 76 FR 34576 - Amendment of Class E Airspace; Waynesboro, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-14

    ... Approach Procedures developed for Eagle's Nest Airport. This action enhances the safety and management of...) to amend Class E airspace at Eagle's Nest Airport, Waynesboro, VA (75 FR 14820) Docket No. FAA-2010... procedures developed at Eagle's Nest Airport, Waynesboro, VA. This action is necessary for the safety and...

  16. VA Dental Insurance Program--federalism. Direct final rule.

    PubMed

    2013-10-22

    The Department of Veterans Affairs (VA) is taking direct final action to amend 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 veterans. Specifically, this rule will add language to clarify the limited preemptive effect of certain criteria in the VADIP regulations.

  17. 38 CFR 17.71 - Revocation of VA approval.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2010-07-01 2010-07-01 false Revocation of VA approval... Community Residential Care § 17.71 Revocation of VA approval. (a) If a hearing official determines under... residential care facility and notify the community residential care facility of this revocation. (b)...

  18. 33 CFR 80.510 - Chesapeake Bay Entrance, VA.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 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. A...

  19. 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. A...

  20. 33 CFR 80.510 - Chesapeake Bay Entrance, VA.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 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. A...

  1. 33 CFR 80.510 - Chesapeake Bay Entrance, VA.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 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. A...

  2. 33 CFR 80.510 - Chesapeake Bay Entrance, VA.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 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. A...

  3. 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...

  4. 78 FR 62441 - VA Dental Insurance Program-Federalism

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-22

    ... AFFAIRS 38 CFR Part 17 RIN 2900-AO85 VA Dental Insurance Program--Federalism AGENCY: Department of... direct final action to amend 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...

  5. 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…

  6. 38 CFR 74.27 - How will VA store information?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false How will VA store information? 74.27 Section 74.27 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) VETERANS SMALL BUSINESS REGULATIONS Records Management § 74.27 How will VA store information?...

  7. Regional effects on chimera formation in 454 pyrosequenced amplicons from a mock community.

    PubMed

    Shin, Sunguk; Lee, Tae Kwon; Han, Jung Min; Park, Joonhong

    2014-07-01

    Chimeras are a frequent artifact in polymerase chain reaction and could be the underlying causes of erroneous taxonomic identifications, overestimated microbial diversity, and spurious sequences. However, little is known about the regional effects on chimera formation. Therefore, we investigated the chimera formation rates in different regions of phylogenetically important biomarker genes to test the regional effects on chimera formation. An empirical study of chimera formation rates was performed using the Roche GSFLX™ system with sequences of the V1/V2/V3 and V4/V5 regions of the 16S rRNA gene and sequences of the nifH gene from a mock microbial community. The chimera formation rates for the 16S V1/V2/V3 region, V4/V5 region, and nifH gene were 22.1-38.5%, 3.68-3.88%, and 0.31-0.98%, respectively. Some amplicons from the V1/V2/V3 regions were shorter than the typical length (∼7-31%), reflecting incomplete extension. In the V1/V2/V3 and V4/V5 regions, conserved and hypervariable regions were identified. Chimeric hot spots were located in parts of conserved regions near the ends of the amplicons. The 16S V1/V2/V3 region had the highest chimera formation rate, likely because of long template lengths and incomplete extension. The amplicons of the nifH gene had the lowest frequency of chimera formation most likely because of variations in their wobble positions in triplet codons. Our results suggest that the main reasons for chimera formation are sequence similarity and premature termination of DNA extension near primer regions. Other housekeeping genes can be a good substitute for 16S rRNA genes in molecular microbial studies to reduce the effects of chimera formation.

  8. Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths.

    PubMed

    Ditommaso, Savina; Giacomuzzi, Monica; Ricciardi, Elisa; Zotti, Carla M

    2015-08-01

    Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31).

  9. 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

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

    PubMed

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

    2015-09-01

    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.

  11. Functional characterization of the 19q12 amplicon in grade III breast cancers

    PubMed Central

    2012-01-01

    Introduction The 19q12 locus is amplified in a subgroup of oestrogen receptor (ER)-negative grade III breast cancers. This amplicon comprises nine genes, including cyclin E1 (CCNE1), which has been proposed as its 'driver'. The aim of this study was to identify the genes within the 19q12 amplicon whose expression is required for the survival of cancer cells harbouring their amplification. Methods We investigated the presence of 19q12 amplification in a series of 313 frozen primary breast cancers and 56 breast cancer cell lines using microarray comparative genomic hybridisation (aCGH). The nine genes mapping to the smallest region of amplification on 19q12 were silenced using RNA interference in phenotypically matched breast cancer cell lines with (MDA-MB-157 and HCC1569) and without (Hs578T, MCF7, MDA-MB-231, ZR75.1, JIMT1 and BT474) amplification of this locus. Genes whose silencing was selectively lethal in amplified cells were taken forward for further validation. The effects of cyclin-dependent kinase 2 (CDK2) silencing and chemical inhibition were tested in cancer cells with and without CCNE1 amplification. Results 19q12 amplification was identified in 7.8% of ER-negative grade III breast cancer. Of the nine genes mapping to this amplicon, UQCRFS1, POP4, PLEKHF1, C19ORF12, CCNE1 and C19ORF2 were significantly over-expressed when amplified in primary breast cancers and/or breast cancer cell lines. Silencing of POP4, PLEKHF1, CCNE1 and TSZH3 selectively reduced cell viability in cancer cells harbouring their amplification. Cancer cells with CCNE1 amplification were shown to be dependent on CDK2 expression and kinase activity for their survival. Conclusions The 19q12 amplicon may harbour more than a single 'driver', given that expression of POP4, PLEKHF1, CCNE1 and TSZH3 is required for the survival of cancer cells displaying their amplification. The observation that cancer cells harbouring CCNE1 gene amplification are sensitive to CDK2 inhibitors provides a

  12. 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

  13. 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 with...

  14. 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 with...

  15. 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 with...

  16. 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 with...

  17. 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 with...

  18. 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…

  19. 78 FR 76064 - Authorization for Non-VA Medical Services; Withdrawal

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-16

    ... AFFAIRS 38 CFR Part 17 RIN 2900-AO47 Authorization for Non-VA Medical Services; Withdrawal AGENCY... amended its regulations regarding payment by VA for medical services under VA's statutory authority to provide non-VA medical care. VA sought to remove an outdated regulatory limitation on veterans...

  20. 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.

  1. VA Health Care: Further Action Needed to Address Weaknesses in Management and Oversight of Non-VA Medical Care

    DTIC Science & Technology

    2014-06-18

    medical care when a VA facility is unable to provide certain specialty care services, such as cardiology or orthopedics, or when a veteran would have...needing treatment in several specialties—including audiology, cardiology , and ophthalmology—were referred to non-VA providers for this reason

  2. Veteran Use of Health Care Systems in Rural States: Comparing VA and Non-VA Health Care Use Among Privately Insured Veterans Under Age 65.

    PubMed

    Charlton, Mary E; Mengeling, Michelle A; Schlichting, Jennifer A; Jiang, Lan; Turvey, Carolyn; Trivedi, Amal N; Kizer, Kenneth W; West, Alan N

    2016-09-01

    To quantify use of VA and non-VA care among working-age veterans with private insurance by linking VA data to private health insurance plan (PHIP) data. Demographics and utilization were compared between dual users of VA and non-VA systems versus single-system users for veterans < 65 living in 2 rural Midwestern states concurrently enrolled in VA health care and a PHIP for ≥ 1 complete federal fiscal year from 2000 to 2010. Chi-square and t-tests were used for univariate analyses. VA reliance was computed as the percentage of visits, admissions and prescriptions in VA. Multinomial logistic regression was used to compare characteristics by dual use versus non-VA only or VA only use. Of 16,330 eligible veterans, 54% used both VA and non-VA services, 39% used non-VA only, and 5% used VA only. Compared with single-system use, dual use was associated with older age, priority levels 1-4, service-connected conditions, rural residence, greater years of study eligibility, and enrollment in the PHIP before VA. VA reliance was 33% for outpatient care, 14% for inpatient, and 40% for pharmacy. PHIP data substantially underestimated VA use compared to VA data; 26% who used VA health care had no VA claims in the PHIP dataset. Over half of working-age veterans enrolled in VA and private insurance used services in both systems. Care coordination efforts across systems should include veterans of all ages, particularly rural veterans more likely to be dual users, and better methods are needed to identify veterans with private insurance and their private providers. © 2016 National Rural Health Association.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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. © 2015 John Wiley & Sons Ltd.

  8. Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis.

    PubMed

    Lonowski, Lindsey A; Narimatsu, Yoshiki; Riaz, Anjum; Delay, Catherine E; Yang, Zhang; Niola, Francesco; Duda, Katarzyna; Ober, Elke A; Clausen, Henrik; Wandall, Hans H; Hansen, Steen H; Bennett, Eric P; Frödin, Morten

    2017-03-01

    This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.

  9. ThermoAlign: a genome-aware primer design tool for tiled amplicon resequencing

    PubMed Central

    Francis, Felix; Dumas, Michael D.; Wisser, Randall J.

    2017-01-01

    Isolating and sequencing specific regions in a genome is a cornerstone of molecular biology. This has been facilitated by computationally encoding the thermodynamics of DNA hybridization for automated design of hybridization and priming oligonucleotides. However, the repetitive composition of genomes challenges the identification of target-specific oligonucleotides, which limits genetics and genomics research on many species. Here, a tool called ThermoAlign was developed that ensures the design of target-specific primer pairs for DNA amplification. This is achieved by evaluating the thermodynamics of hybridization for full-length oligonucleotide-template alignments — thermoalignments — across the genome to identify primers predicted to bind specifically to the target site. For amplification-based resequencing of regions that cannot be amplified by a single primer pair, a directed graph analysis method is used to identify minimum amplicon tiling paths. Laboratory validation by standard and long-range polymerase chain reaction and amplicon resequencing with maize, one of the most repetitive genomes sequenced to date (≈85% repeat content), demonstrated the specificity-by-design functionality of ThermoAlign. ThermoAlign is released under an open source license and bundled in a dependency-free container for wide distribution. It is anticipated that this tool will facilitate multiple applications in genetics and genomics and be useful in the workflow of high-throughput targeted resequencing studies. PMID:28300202

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

    PubMed Central

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

    2016-01-01

    The human microbiome has emerged as a major player in regulating human health and disease. Translation 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 shotgun whole genome sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1×106 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 shotgun whole genome 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

  11. Unscheduled DNA replication origin activation at inserted HPV 18 sequences in a HPV-18/MYC amplicon.

    PubMed

    Conti, Chiara; Herrick, John; Bensimon, Aaron

    2007-08-01

    Oncogene amplification is a critical step leading to tumorigenesis, but the underlying mechanisms are still poorly understood. Despite data suggesting that DNA replication is a major source of genomic instability, little is known about replication origin usage and replication fork progression in rearranged regions. Using a single DNA molecule approach, we provide here the first study of replication kinetics on a previously characterized MYC/papillomavirus (HPV18) amplicon in a cervical cancer. Using this amplicon as a model, we investigated the role DNA replication control plays in generating amplifications in human cancers. The data reveal severely perturbed DNA replication kinetics in the amplified region when compared with other regions of the same genome. It was found that DNA replication is initiated from both genomic and viral sequences, resulting in a higher median frequency of origin firings. In addition, it was found that the higher initiation frequency was associated with an equivalent increase in the number of stalled replication forks. These observations raise the intriguing possibility that unscheduled replication origin activation at inserted HPV-18 viral DNA sequences triggers DNA amplification in this cancer cell line and the subsequent overexpression of the MYC oncogene.

  12. 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

  13. Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform

    PubMed Central

    Schirmer, Melanie; Ijaz, Umer Z.; D'Amore, Rosalinda; Hall, Neil; Sloan, William T.; Quince, Christopher

    2015-01-01

    With read lengths of currently up to 2 × 300 bp, high throughput and low sequencing costs Illumina's MiSeq is becoming one of the most utilized sequencing platforms worldwide. The platform is manageable and affordable even for smaller labs. This enables quick turnaround on a broad range of applications such as targeted gene sequencing, metagenomics, small genome sequencing and clinical molecular diagnostics. However, Illumina error profiles are still poorly understood and programs are therefore not designed for the idiosyncrasies of Illumina data. A better knowledge of the error patterns is essential for sequence analysis and vital if we are to draw valid conclusions. Studying true genetic variation in a population sample is fundamental for understanding diseases, evolution and origin. We conducted a large study on the error patterns for the MiSeq based on 16S rRNA amplicon sequencing data. We tested state-of-the-art library preparation methods for amplicon sequencing and showed that the library preparation method and the choice of primers are the most significant sources of bias and cause distinct error patterns. Furthermore we tested the efficiency of various error correction strategies and identified quality trimming (Sickle) combined with error correction (BayesHammer) followed by read overlapping (PANDAseq) as the most successful approach, reducing substitution error rates on average by 93%. PMID:25586220

  14. Amplicon pyrosequencing reveals spatial and temporal consistency in diazotroph assemblages of the Acropora millepora microbiome.

    PubMed

    Lema, Kimberley A; Willis, Bette L; Bourne, David G

    2014-10-01

    Diazotrophic bacteria potentially play an important functional role in supplying fixed nitrogen to the coral holobiont, but the value of such a partnership depends on the stability of the association. Here we evaluate the composition of diazotroph assemblages associated with the coral Acropora millepora throughout four seasons and at two reefs, an inshore and an offshore (mid-shelf) reef on the Great Barrier Reef, Australia. Amplicon pyrosequencing of the nifH gene revealed that diazotrophs are ubiquitous members of the bacterial community associated with A. millepora. Rhizobia (65% of the overall nifH sequences retrieved) and particularly Bradyrhizobia sp.-affiliated sequences (> 50% of rhizobia sequences) dominated diazotrophic assemblages across all coral samples from the two sites throughout the year. In contrast to this consistency in the spatial and temporal patterns of occurrence of diazotroph assemblages, the overall coral-associated bacterial community, assessed through amplicon sequencing of the general bacterial 16S ribosomal RNA gene, differed between inshore and mid-shelf reef locations. Sequences associated with the Oceanospirillales family, particularly with Endozoicomonas sp., dominated bacterial communities associated with inshore corals. Although rhizobia represented a variable and generally small fraction of the overall bacterial community associated with A. millepora, consistency in the structure of these diazotrophic assemblages suggests that they have a functional role in the coral holobiont. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  15. 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

  16. 75 FR 73016 - Proposed Establishment of Class E Airspace; Kenbridge, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-29

    ... examined during normal business hours at the office of the Eastern Service Center, Federal Aviation... the Earth. * * * * * AEA VA E5 Kenbridge, VA Lunenburg County Airport, VA (Lat. 36 57'37'' N.,...

  17. 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.

  18. Haemophilia utilization group study - Part Va (HUGS Va): design, methods and baseline data.

    PubMed

    Zhou, Z-Y; Wu, J; Baker, J; Curtis, R; Forsberg, A; Huszti, H; Koerper, M; Lou, M; Miller, R; Parish, K; Riske, B; Shapiro, A; Ullman, M; Johnson, K

    2011-09-01

    To describe the study design, procedures and baseline characteristics of the Haemophilia Utilization Group Study - Part Va (HUGS Va), a US multi-center observational study evaluating the cost of care and burden of illness in persons with factor VIII deficiency. Patients with factor VIII level ≤ 30%, age 2-64 years, receiving treatment at one of six federally supported haemophilia treatment centres (HTCs) were enrolled in the study. Participants completed an initial interview including questions on socio-demographical characteristics, health insurance status, co-morbidities, access to care, haemophilia treatment regimen, factor utilization, self-reported joint pain and motion limitation and health-related quality of life. A periodic follow-up survey collected data regarding time lost from usual activities, disability days, health care utilization and outcomes of care. HTC clinicians documented participants' baseline clinical characteristics and pharmacy dispensing records for 2 years. Between July 2005 and July 2007, 329 participants were enrolled. Average age was 9.7 years for children and 33.5 years for adults; two-thirds had severe haemophilia. The distributions of age, marital status, education level and barriers to haemophilia care were relatively consistent across haemophilic severity categories. Differences were found in participants' employment status, insurance status and income. Overall, children with haemophilia had quality of life scores comparable to healthy counterparts. Adults had significantly lower physical functioning than the general US population. As one of the largest economic studies of haemophilia care, HUGS Va will provide detailed information regarding the burden of illness and health care utilization in the US haemophilia A population.

  19. Effect of promoter strength on protein expression and immunogenicity of an HSV-1 amplicon vector encoding HIV-1 Gag

    PubMed Central

    Santos, Kathlyn; Duke, Cindy M.P.; Rodriguez-Colon, Sol M.; Dakwar, Anthony; Fan, Shongshan; Keefer, Michael C.; Federoff, Howard J.; Frelinger, John G.; Bowers, William J.; Dewhurst., Stephen

    2007-01-01

    Helper-free herpes simplex virus type-1 (HSV-1) amplicon vectors elicit robust immune responses to encoded proteins, including human immunodeficiency virus type-1 (HIV-1) antigens. To improve this vaccine delivery system, seven amplicon vectors were constructed, each encoding HIV-1 Gag under the control of a different promoter. Gag expression levels were analyzed in murine and human cell lines, as well as in biopsied tissue samples from injected mice; these data were then compared with Gag-specific T cell responses in BALB/c mice. The magnitude of the amplicon-induced immune response was found to correlate strongly with the level of Gag production both in vitro and in vivo. Interestingly, the best correlation of the strength of the amplicon-induced immune response was with antigen expression in cultured DC rather than expression at the tissue site of injection or in cultured cell lines. These findings may have implications for the generation of improved HSV-1 amplicon vectors for HIV-1 vaccine delivery. PMID:17145123

  20. PCR-based detection of chlamydial infection in swine and subsequent PCR-coupled genotyping of chlamydial omp1-gene amplicons by DNA-hybridization, RFLP-analysis, and nucleotide sequence analysis.

    PubMed Central

    Hoelzle, L. E.; Steinhausen, G.; Wittenbrink, M. M.

    2000-01-01

    Lung and intestine of 49 pigs with respiratory diseases and endocervical swabs from 205 sows with reproductive disorders were investigated for chlamydial infection by polymerase chain reaction. PCR primers targeted DNA sequences on the chlamydial omp1 or omp2 genes. PCR amplicons were generated from 49.0% of pigs with respiratory disease, from 60.0% of sows with reproductive disorders, from 24.5% of respiratory healthy controls, but from no endocervical swabs from fertile sows. By DNA hybridization, a high prevalence of mixed infections with Chlamydophila abortus and Chlamydia suis in the porcine lung and intestine was found and confirmed by RFLP and nucleotide analysis. Of the omp1-PCR amplicons from endocervical swabs 81.3% were identified as Chlamydophila abortus, indicating an association of this chlamydial species with reproductive disorders in sows. Nucleotide sequence analysis of omp1-amplicons identified as deriving from Chlamydia suis shared a maximum of 82.7% homology with the reference strain S45. PMID:11117968

  1. 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.

  2. 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

  3. An academic-VA partnership: Student interprofessional teams integrated with VA PACT teams.

    PubMed

    Swenty, Constance L; Schaar, Gina L; Butler, Ryan M

    2016-12-01

    Veterans are challenged with multiple unique healthcare issues related to their military service environment. Likewise, health care providers must understand the special concerns associated with military conflict and recognize how the veteran's care can be optimized by interprofessional care delivery. Little is taught didactically or clinically that supports nursing students in addressing the unique issues of the veteran or the student's need to work collaboratively with allied health team members to enhance the veteran's care. Because of limited exposure to the veteran's special conditions, nursing students who may seek a career with the veteran population often face challenges in rendering appropriate care. The VA offers an invaluable opportunity for health profession students to collaborate with VA interprofessional Patient Aligned Care Teams (PACT) ultimately optimizing veteran health outcomes. This academic partnership, that implements an interprofessional model, will prepare students to better embrace the veteran population. This article describes the immersion of health profession students in interprofessional collaborative practice (IPCP) using PACT team principles which ultimately promotes the students' ability to link theory content to patient care delivery. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Ultra-Deep Pyrosequencing (UDPS) Data Treatment to Study Amplicon HCV Minor Variants

    PubMed Central

    Gregori, Josep; Esteban, Juan I.; Cubero, María; Garcia-Cehic, Damir; Perales, Celia; Casillas, Rosario; Alvarez-Tejado, Miguel; Rodríguez-Frías, Francisco; Guardia, Jaume; Domingo, Esteban; Quer, Josep

    2013-01-01

    We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS) methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior) after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GS-FLX+. The observed CVs for the normalized Shannon entropy (Sn), the mutation frequency (Mf), and the nucleotidic diversity (Pi) were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each), in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%. Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity. PMID:24391758

  5. Evaluation of the reproducibility of amplicon sequencing with Illumina MiSeq platform

    PubMed Central

    Van Nostrand, Joy D.; Ning, Daliang; Sun, Bo; Xue, Kai; Liu, Feifei; Deng, Ye; Liang, Yuting; Zhou, Jizhong

    2017-01-01

    Illumina’s MiSeq has become the dominant platform for gene amplicon sequencing in microbial ecology studies; however, various technical concerns, such as reproducibility, still exist. To assess reproducibility, 16S rRNA gene amplicons from 18 soil samples of a reciprocal transplantation experiment were sequenced on an Illumina MiSeq. The V4 region of 16S rRNA gene from each sample was sequenced in triplicate with each replicate having a unique barcode. The average OTU overlap, without considering sequence abundance, at a rarefaction level of 10,323 sequences was 33.4±2.1% and 20.2±1.7% between two and among three technical replicates, respectively. When OTU sequence abundance was considered, the average sequence abundance weighted OTU overlap was 85.6±1.6% and 81.2±2.1% for two and three replicates, respectively. Removing singletons significantly increased the overlap for both (~1–3%, p<0.001). Increasing the sequencing depth to 160,000 reads by deep sequencing increased OTU overlap both when sequence abundance was considered (95%) and when not (44%). However, if singletons were not removed the overlap between two technical replicates (not considering sequence abundance) plateaus at 39% with 30,000 sequences. Diversity measures were not affected by the low overlap as α-diversities were similar among technical replicates while β-diversities (Bray-Curtis) were much smaller among technical replicates than among treatment replicates (e.g., 0.269 vs. 0.374). Higher diversity coverage, but lower OTU overlap, was observed when replicates were sequenced in separate runs. Detrended correspondence analysis indicated that while there was considerable variation among technical replicates, the reproducibility was sufficient for detecting treatment effects for the samples examined. These results suggest that although there is variation among technical replicates, amplicon sequencing on MiSeq is useful for analyzing microbial community structure if used appropriately

  6. 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

  7. Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

    PubMed Central

    Blomquist, Thomas M.; Crawford, Erin L.; Lovett, Jennie L.; Yeo, Jiyoun; Stanoszek, Lauren M.; Levin, Albert; Li, Jia; Lu, Mei; Shi, Leming; Muldrew, Kenneth; Willey, James C.

    2013-01-01

    Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1

  8. RadNet Air Data From Virginia Beach, VA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Virginia Beach, VA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  9. 75 FR 35511 - Virginia Disaster Number VA-00028

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-22

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster Number VA-00028 AGENCY: Small Business Administration. ACTION: Amendment 3..., is hereby amended to include the following areas as adversely affected by the disaster....

  10. 76 FR 43575 - Amendment of Class E Airspace; Staunton, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-21

    ... 30320; telephone (404) 305-6364. SUPPLEMENTARY INFORMATION: History On March 18, 2011, the FAA published... areas extending upward from 700 feet or more above the surface of the earth. * * * * * AEA VA...

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

    NASA Image and Video Library

    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...

  12. 77 FR 67063 - VA Directive 0005 on Scientific Integrity

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-08

    ... technological information from political or commercial influence; Prohibit suppression or alteration of... ``inappropriate influence.'' The definition of ``inappropriate influence'' should be more explicit. VA Response... analyses will be protected from political and commercial influence. The term ``inappropriate...

  13. Eastern Colorado Health Care System (VA Hospital) NPDES Permit

    EPA Pesticide Factsheets

    Under NPDES permit CO-0034991, the U.S. Department of Veterans Affairs (VA) is authorized to discharge from its wastewater treatment facility in Adams County, Colorado, to a storm sewer to Toll Gate Creek, a tributary of Sand Creek.

  14. VA INFORMATION TECHNOLOGY: Important Initiatives Begun, Yet Serious Vulnerabilities Persist

    DTIC Science & Technology

    2007-11-02

    Technology Management Issues United States General Accounting Office GAO Testimony Before the Subcommittee on Oversight and Investigations, Committee on...VA INFORMATION TECHNOLOGY Important Initiatives Begun, Yet Serious Vulnerabilities Persist Statement of David L. McClure Director, Information

  15. ENTRANCE TO CEMETERY FROM VA MEDICAL CENTER CAMPUS, WITH ADMINISTRATION ...

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

    ENTRANCE TO CEMETERY FROM VA MEDICAL CENTER CAMPUS, WITH ADMINISTRATION BUILDING IN BACKGROUND. VIEW TO NORTH. - Bath National Cemetery, Department of Veterans Affairs Medical Center, San Juan Avenue, Bath, Steuben County, NY

  16. VA Is Here for the People Who Support Our Veterans

    MedlinePlus

    ... on track. Calls can be referred to local Suicide Prevention Coordinators and other VA providers who specialize in issues such as: Post-traumatic stress (PTS/PTSD) Traumatic brain injury (TBI) Military sexual ...

  17. RadNet Air Data From Richmond, VA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Richmond, VA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  18. RadNet Air Data From Harrisonburg, VA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Harrisonburg, VA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  19. Single-Step Conversion of Cells to Retrovirus Vector Producers with Herpes Simplex Virus–Epstein-Barr Virus Hybrid Amplicons

    PubMed Central

    Sena-Esteves, Miguel; Saeki, Yoshinaga; Camp, Sara M.; Chiocca, E. Antonio; Breakefield, Xandra O.

    1999-01-01

    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 106 and 107 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

  20. Quantitative characterization of cell transduction by HSV-1 amplicons using flow cytometry and real-time PCR.

    PubMed

    El-Sherbini, Yasser M; Stevenson, Mark M; Seymour, Leonard W; Wade-Martins, Richard

    2009-08-01

    Herpes simplex virus type 1 (HSV-1) amplicon preparations are usually quantified as transducing units/ml (TU/ml), with little information on genomic copy/TU ratios. In the present study, two HSV-1 amplicons expressing enhanced green fluorescent protein (EGFP) were analysed by quantitative PCR (qPCR) and transducing activity to obtain genomic copy/TU ratios. One vector (pHSV-GL) contains the HSV-1 packaging signal (pac) and origin of replication (oriS) and the other (pHSV/EBV-GL) includes Epstein-Barr virus (EBV) episomal maintenance elements. The pHSV-GL and pHSV/EBV-GL amplicons were prepared at titres of 7.55x10(7) and 7.24x10(7)TU/ml, containing 2.56x10(9) and 1.33x10(9) genomic copies/ml respectively. This produced preliminary estimates of genomic copy/TU ratios of 34:1 and 18:1. However standard transduction conditions did not deplete fully the supernatant of transducing particles since the same supernatant was subsequently able to achieve 25% the initial transduction efficiency, although centrifugation of amplicon particles onto cells improved infectivity by 1.8-fold. Finally, qPCR analysis of FACS-purified EGFP-expressing cells showed the presence of approximately 3 amplicon genomes/transduced cell, independent of the infection dose. Accordingly, the initial estimated genomic copy/TU ratio for pHSV-GL was revised to 6.3:1. Measuring the genomic copy/TU ratios is an important parameter for comparing the quality of amplicon preparations and standardizing experimental conditions.

  1. Infectivity of Herpes Simplex Virus Type-1 (HSV-1) amplicon vectors in dendritic cells is determined by the helper virus strain used for packaging

    PubMed Central

    Santos, Kathlyn; Sanfilippo, Christine M.; Narrow, Wade C.; Casey, Ann E.; Rodriguez-Colon, Sol M.; McDermott, Michael P.; Federoff, Howard J.; Bowers, William J.; Dewhurst, Stephen

    2007-01-01

    Herpes Simplex Virus Type-1 (HSV-1) amplicon vectors are being explored for a wide range of potential applications, including vaccine delivery and immunotherapy of cancer. While extensive effort has been directed towards the improvement of the amplicon “payload” in these vectors, relatively little attention has been paid to the effect of the packaging HSV-1 strains on the biological properties of co-packaged amplicon vectors. We therefore compared the biological properties of amplicon stocks prepared using a panel of primary HSV-1 isolates, a molecularly cloned strain used to package helper-free amplicons (designated here as F5), and two laboratory isolates (KOS and strain 17, which is the parent of the F5 clone). This analysis revealed considerable inter-strain variability in the ability of amplicon stocks packaged by different primary HSV-1 isolates to efficiently transduce established cell lines and primary human dendritic cells (DC). Amplicons packaged by both the F5 molecularly cloned virus and its lab-adapted parent (strain 17) were very inefficient at transducing DC, when compared to amplicons packaged by KOS or by several of the primary virus isolates. These finding have important implications for the future development of improved amplicon-based vaccine delivery systems and suggest that DC tropism may be an instrinsic property of some HSV-1 strains, independent of passage history or molecular cloning. PMID:17606303

  2. Preparation of Amplicon Libraries for Metabarcoding of Marine Eukaryotes Using Illumina MiSeq: The Dual-PCR Method.

    PubMed

    Bourlat, Sarah J; Haenel, Quiterie; Finnman, Jennie; Leray, Matthieu

    2016-01-01

    This protocol details the preparation of multiplexed amplicon libraries for metabarcoding (amplicon-based) studies of microscopic marine eukaryotes. Metabarcoding studies, based on the amplification of a taxonomically informative marker from a collection of organisms or an environmental sample, can be performed to analyze biodiversity patterns or predator-prey interactions. For Metazoa, we use the mitochondrial cytochrome oxidase 1 (CO1) or the small ribosomal subunit (SSU) markers. Here, we describe a strategy for the preparation of multiplexed Illumina MiSeq libraries using a dual-PCR approach for the addition of index and adaptor sequences.

  3. Using Amplicon Deep Sequencing to Detect Genetic Signatures of Plasmodium vivax Relapse

    PubMed Central

    Lin, Jessica T.; Hathaway, Nicholas J.; Saunders, David L.; Lon, Chanthap; Balasubramanian, Sujata; Kharabora, Oksana; Gosi, Panita; Sriwichai, Sabaithip; Kartchner, Laurel; Chuor, Char Meng; Satharath, Prom; Lanteri, Charlotte; Bailey, Jeffrey A.; Juliano, Jonathan J.

    2015-01-01

    Plasmodium vivax infections often recur due to relapse of hypnozoites from the liver. In malaria-endemic areas, tools to distinguish relapse from reinfection are needed. We applied amplicon deep sequencing to P. vivax isolates from 78 Cambodian volunteers, nearly one-third of whom suffered recurrence at a median of 68 days. Deep sequencing at a highly variable region of the P. vivax merozoite surface protein 1 gene revealed impressive diversity—generating 67 unique haplotypes and detecting on average 3.6 cocirculating parasite clones within individuals, compared to 2.1 clones detected by a combination of 3 microsatellite markers. This diversity enabled a scheme to classify over half of recurrences as probable relapses based on the low probability of reinfection by multiple recurring variants. In areas of high P. vivax diversity, targeted deep sequencing can help detect genetic signatures of relapse, key to evaluating antivivax interventions and achieving a better understanding of relapse-reinfection epidemiology. PMID:25748326

  4. High‑throughput sequencing of amplicons for monitoring yeast biodiversity in must and during alcoholic fermentation.

    PubMed

    David, Vanessa; Terrat, Sébastien; Herzine, Khaled; Claisse, Olivier; Rousseaux, Sandrine; Tourdot-Maréchal, Raphaëlle; Masneuf-Pomarede, Isabelle; Ranjard, Lionel; Alexandre, Hervé

    2014-05-01

    We compared pyrosequencing technology with the PCR-ITS-RFLP analysis of yeast isolates and denaturing gradient gel electrophoresis (DGGE). These methods gave divergent findings for the yeast population. DGGE was unsuitable for the quantification of biodiversity and its use for species detection was limited by the initial abundance of each species. The isolates identified by PCR-ITSRFLP were not fully representative of the true population. For population dynamics, high-throughput sequencing technology yielded results differing in some respects from those obtained with other approaches. This study demonstrates that 454 pyrosequencing of amplicons is more relevant than other methods for studying the yeast community on grapes and during alcoholic fermentation. Indeed, this high-throughput sequencing method detected larger numbers of species on grapes and identified species present during alcoholic fermentation that were undetectable with the other techniques.

  5. Amplicon Competition Enables End-Point Quantitation of Nucleic Acids Following Isothermal Amplification.

    PubMed

    Jiang, Yu Sherry; Stacy, Apollo; Whiteley, Marvin; Ellington, Andrew D; Bhadra, Sanchita

    2017-09-05

    It is inherently difficult to quantitate nucleic acid analytes with most isothermal amplification assays. We developed loop-mediated isothermal amplification (LAMP) reactions in which competition between defined numbers of "false" and "true" amplicons leads to order of magnitude quantitation by a single endpoint determination. These thresholded LAMP reactions were successfully used to directly and quantitatively estimate the numbers of nucleic acids in complex biospecimens, including directly from cells and in sewage, with the values obtained closely correlating with qPCR quantitations. Thresholded LAMP reactions are amenable to endpoint readout by cell phone, unlike other methods that require continuous monitoring, and should therefore prove extremely useful in developing one-pot reactions for point-of-care diagnostics without needing sophisticated material or informatics infrastructure. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. 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.

  7. Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology.

    PubMed

    Moralli, Daniela; Monaco, Zoia L

    2015-02-01

    De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.

  8. Nitrogenase gene amplicons from global marine surface waters are dominated by genes of non-cyanobacteria.

    PubMed

    Farnelid, Hanna; Andersson, Anders F; Bertilsson, Stefan; Al-Soud, Waleed Abu; Hansen, Lars H; Sørensen, Søren; Steward, Grieg F; Hagström, Åke; Riemann, Lasse

    2011-04-29

    Cyanobacteria are thought to be the main N(2)-fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III) were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N(2) fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant.

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

    PubMed Central

    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

  10. Similar Diversity of Alphaproteobacteria and Nitrogenase Gene Amplicons on Two Related Sphagnum Mosses

    PubMed Central

    Bragina, Anastasia; Maier, Stefanie; Berg, Christian; Müller, Henry; Chobot, Vladimir; Hadacek, Franz; Berg, Gabriele

    2012-01-01

    Sphagnum mosses represent a main vegetation component in ombrotrophic wetlands. They harbor a specific and diverse microbial community with essential functions for the host. To understand the extend of host specificity and impact of environment, Sphagnum fallax and Sphagnum angustifolium, two phylogenetically closely related species, which show distinct habitat preference with respect to the nutrient level, were analyzed by a multifaceted approach. Microbial fingerprints obtained by PCR-single-strand conformation polymorphism of 16S rRNA and nitrogenase-encoding (nifH) genes were highly similar for both Sphagnum species. Similarity was confirmed for colonization patterns obtained by fluorescence in situ hybridization (FISH) coupled with confocal laser scanning microscopy (CLSM): Alphaproteobacteria were the main colonizers inside the hyaline cells of Sphagnum leaves. A deeper survey of Alphaproteobacteria by 16S rRNA gene amplicon sequencing reveals a high diversity with Acidocella, Acidisphaera, Rhodopila, and Phenylobacterium as major genera for both mosses. Nitrogen fixation is an important function of Sphagnum-associated bacteria, which is fulfilled by microbial communities of Sphagna in a similar way. NifH libraries of Sphagnum-associated microbial communities were characterized by high diversity and abundance of Alphaproteobacteria but contained also diverse amplicons of other taxa, e.g., Cyanobacteria and Deltaproteobacteria. Statistically significant differences between the microbial communities of both Sphagnum species could not be discovered in any of the experimental approach. Our results show that the same close relationship, which exists between the physical, morphological, and chemical characteristics of Sphagnum mosses and the ecology and function of bog ecosystems, also connects moss plantlets with their associated bacterial communities. PMID:22294982

  11. Pitfalls of converting practice guidelines into quality measures: lessons learned from a VA performance measure.

    PubMed

    Walter, Louise C; Davidowitz, Natalie P; Heineken, Paul A; Covinsky, Kenneth E

    2004-05-26

    The Department of Veterans Affairs (VA) manages the largest health care system in the United States, and the Institute of Medicine has recommended that many practices of VA quality measurement be applied to the US health care system as a whole. The VA measures quality of care at all of its sites by assessing adherence rates to performance measures, which generally are derived from evidence-based practice guidelines. Higher adherence rates are used as evidence of better quality of care. However, there are problems with converting practice guidelines, intended to offer guidance to clinicians, into performance measures that are meant to identify poor-quality care. We suggest a more balanced perspective on the use of performance measures to define quality by delineating conceptual problems with the conversion of practice guidelines into quality measures. Focusing on colorectal cancer screening, we use a case study at 1 VA facility to illustrate pitfalls that can cause adherence rates to guideline-based performance measures to be poor indicators of the quality of cancer screening. Pitfalls identified included (1) not properly considering illness severity of the sample population audited for adherence to screening, (2) not distinguishing screening from diagnostic procedures when setting achievable target screening rates, and (3) not accounting for patient preferences or clinician judgment when scoring performance measures. For many patients with severe comorbid illnesses or strong preferences against screening, the risks of colorectal cancer screening outweigh the benefits, and the decision to not screen may reflect good quality of care. Performance measures require more thoughtful specification and interpretation to avoid defining high testing rates as good quality of care regardless of who received the test, why it was performed, or whether the patient wanted it.

  12. A ``Visual Binary'' Orbit of the Low-Luminosity Hyad VA351

    NASA Astrophysics Data System (ADS)

    Franz, O. G.; Wasserman, L. H.; Bradley, A. J.; Benedict, G. F.; Duncombe, R. L.; Hemenway, P. D.; Jefferys, W. H.; McArthur, B.; Nelan, E.; Shelus, P. J.; Story, D.; Whipple, A. L.; Fredrick, L. W.; van Altena, Wm. F.

    1996-05-01

    VA351=G7-203 \\{V=13.2, (B-V)=1.5; RA=04:25:13.4, Dec=17:16:06 (2000)\\} was first resolved as a 0.051-arcsec pair on 1994 d45 in the course of our search for binaries among faint members of the Hyades cluster conducted with HST Fine Guidance Sensor #3 in the Transfer Function Scan mode (Franz et al. 1994, BAAS 26, 929). With four additional measures obtained during 1994 d296 through 1996 d78, our data now cover a position-angle interval of 156 deg. From these five HST-FGS measures we have computed a first ``visual'' orbit of VA351 with P=3.36 +/- 0.12\\ y and a=0.069 +/- 0.001 arcsec. Using a cluster parallax p=0.021 arcsec we derive a total mass M=1.8 +/- 0.3 Msun, a value clearly too large for a pair of near-equal M-dwarfs. It may, however, be in accord with the conclusion by Latham (1955, private comm.), based upon radial velocities, that VA351 is a triple system. Spectra we have obtained at McDonald Observatory and at Lowell Observatory appear to provide independent evidence for the presence of additional components. Under a proposal approved for HST Cycle 6 we expect to obtain six additional FGS observations which will combine transfer function mode with position mode relative to local reference stars. These observations, together with the data now in hand, will yield true barycentric orbits and thus masses of the resolved components of VA351 with an expected accuracy of five percent or better. Moreover, the combination of HST-FGS astrometry and radial velocity measures should permit the conclusive analysis of this low-luminosity Hyad system. This work is supported by NASA under grant NAG5-1603 to UTexas.

  13. VA Health Care. Additional Efforts to Better Assess Joint Ventures Needed

    DTIC Science & Technology

    2008-03-01

    Kans. Okla. Minn. Iowa Mo. Ark. La. Ill. Miss. Ind. Ky. Tenn. Ala. Ga. S.C. N.C. Va. Ohio N.H. Mass. Mich . Calif. Wash. Wis. N.Y. Maine Vt. W.Va...train VA personnel in a variety of areas, including basic life support and advanced cardiac life support. Finally, VA officials and academic

  14. 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... information technology. Title: VA Police Officer Pre-Employment Screening Checklist, VA Form 0120. OMB Control... complete VA Form 0120 to document pre- employment history and conduct background checks on...

  15. Sequence-specific validation of LAMP amplicons in real-time optomagnetic detection of Dengue serotype 2 synthetic DNA.

    PubMed

    Minero, Gabriel Antonio S; Nogueira, Catarina; Rizzi, Giovanni; Tian, Bo; Fock, Jeppe; Donolato, Marco; Strömberg, Mattias; Hansen, Mikkel F

    2017-09-08

    We report on an optomagnetic technique optimised for real-time molecular detection of Dengue fever virus under ideal as well as non-ideal laboratory conditions using two different detection approaches. The first approach is based on the detection of the hydrodynamic volume of streptavidin coated magnetic nanoparticles attached to biotinylated LAMP amplicons. We demonstrate detection of sub-femtomolar Dengue DNA target concentrations in the ideal contamination-free lab environment within 20 min. The second detection approach is based on sequence-specific binding of functionalised magnetic nanoparticles to loops of LAMP amplicons. Melting studies reveal that true positive and spurious amplicons have different melting points and this allows us to discriminate between them. This is found to be in a good agreement with subsequent studies on real-time sequence-specific discrimination of LAMP amplicons. The specific binding causes clustering of magnetic nanoparticles via binding to multiple sites (loops) emerging in the elongation phase of LAMP. Formation of nanoclusters is monitored via the depletion of the optomagnetic signal due to free nanoparticles. After sequence-specific validation, we claim detection of down to 100 fM of Dengue target after 20 min of LAMP with a contamination background.

  16. Isolation of cultivation-resistant oomycetes, first detected as amplicon sequences, from roots of herbicide-terminated winter rye

    USDA-ARS?s Scientific Manuscript database

    The dynamics of microbial communities associated with dying cover crops are of interest because of potential impacts on disease in a subsequent crop, and because of the importance of microbial activity on plant residue to soil organic matter dynamics and nutrient cycling. High throughput amplicon se...

  17. The HER2 amplicon includes several genes required for the growth and survival of HER2 positive breast cancer cells.

    PubMed

    Sahlberg, Kristine Kleivi; Hongisto, Vesa; Edgren, Henrik; Mäkelä, Rami; Hellström, Kirsi; Due, Eldri U; Moen Vollan, Hans Kristian; Sahlberg, Niko; Wolf, Maija; Børresen-Dale, Anne-Lise; Perälä, Merja; Kallioniemi, Olli

    2013-06-01

    About 20% of breast cancers are characterized by amplification and overexpression of the HER2 oncogene. Although significant progress has been achieved for treating such patients with HER2 inhibitor trastuzumab, more than half of the patients respond poorly or become resistant to the treatment. Since 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 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. Silencing of HER2 caused a greater growth arrest and apoptosis in the responding compared to the non-responding cell lines, indicating that the resistant cells are inherently less dependent on the HER2 pathway. Several other genes in the amplicon also showed a more pronounced effect when silenced; indicating that expression of HER2 co-amplified genes may be needed to sustain the growth of breast cancer cells. Importantly, co-silencing of STARD3, GRB7, PSMD3 and PERLD1 together with HER2 led to an additive inhibition of cell viability as well as induced apoptosis. These studies indicate that breast cancer cells may become addicted to the amplification of several genes that reside in the HER2 amplicon. The simultaneous targeting of these genes may increase the efficacy of the anti-HER2 therapies and possibly also counteract trastuzumab resistance. The observed additive effects seem to culminate to both apoptosis and cell proliferation pathways indicating that these pathways may be interesting targets for combinatorial treatment of HER2+ breast cancers.

  18. High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Array™ System for simplified amplicon library preparation.

    PubMed

    Moonsamy, P V; Williams, T; Bonella, P; Holcomb, C L; Höglund, B N; Hillman, G; Goodridge, D; Turenchalk, G S; Blake, L A; Daigle, D A; Simen, B B; Hamilton, A; May, A P; Erlich, H A

    2013-03-01

    The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome; distinguishing the thousands of HLA alleles is challenging. Next generation sequencing of exonic amplicons with the 454 genome sequence (GS) FLX System and Conexio Assign ATF 454 software provides high resolution, high throughput HLA genotyping for eight class I and class II loci. HLA typing of potential donors for unrelated bone marrow donor registries typically uses a subset of these loci at high sample throughput and low cost per sample. The Fluidigm Access Array System enables the incorporation of 48 different multiplex identifiers (MIDs) corresponding to 48 genomic DNA samples with up to 48 different primer pairs in a microfluidic device generating 2304 parallel polymerase chain reactions (PCRs). Minimal volumes of reagents are used. During genomic PCR, in this 4-primer system, the outer set of primers containing the MID and the 454 adaptor sequences are incorporated into an amplicon generated by the inner HLA target-specific primers each containing a common sequence tag at the 5' end of the forward and reverse primers. Pools of the resulting amplicons are used for emulsion PCR and clonal sequencing on the 454 Life Sciences GS FLX System, followed by genotyping with Conexio software. We have genotyped 192 samples with 100% concordance to known genotypes using 8 primer pairs (covering exons 2 and 3 of HLA-A, B and C, and exon 2 of DRB1, 3/4/5 and DQB1) and 96 MIDs in a single GS FLX run. An average of 166 reads per amplicon was obtained. We have also genotyped 96 samples at high resolution (14 primer pairs covering exons 2, 3, and 4 of the class I loci and exons 2 of DRB1, 3/4/5, DQA1, DQB1, DPB1, and exon 3 of DQB1), recovering an average of 173 sequence reads per amplicon. © 2013 John Wiley & Sons A/S.

  19. Roles of factor Va heavy and light chains in protein and lipid rearrangements associated with the formation of a bovine factor Va-membrane complex.

    PubMed Central

    Koppaka, V; Talbot, W F; Zhai, X; Lentz, B R

    1997-01-01

    Factor Va is an essential protein cofactor of the enzyme factor Xa, which activates prothrombin to thrombin during blood coagulation. Peptides with an apparent Mr of approximately 94,000 (heavy chain; HC) and approximately 74,000 or 72,000 (light chain; LC) interact in the presence of Ca2+ to form active Va. The two forms of Va-LC differ in their carboxyl-terminal C2 domain. Using Va reconstituted with either LC form, we examined the effects of the two LC species on membrane binding and on the activity of membrane-bound Va. We found that 1) Va composed of the 72,000 LC bound only slightly more tightly to membranes composed of a mixture of neutral and acidic lipids, the Kd being reduced by a factor of approximately 3 at 5 mM and by a factor of 6 at 2 mM Ca2+. 2) The two forms of Va seemed to undergo different conformational changes when bound to a membrane. 3) The activity of bovine Va varied somewhat with LC species, the difference being greatest at limiting Xa concentration. We have also addressed the role of the two Va peptides in membrane lipid rearrangements and binding: 1) Va binding increased lateral packing density in mixed neutral/acidic lipid membranes. In the solid phase, Va-HC had no effect, whereas Va-LC and whole Va had similar but small effects. In the fluid phase, Va-HC and whole Va both altered membrane packing, with Va-HC having the largest effect. 2) Va-HC bound reversibly and in a Ca2+-independent fashion to membranes composed of neutral phospholipid (Kd, approximately 0.3 microM; stoichiometry approximately 91). High ionic strength had little effect on binding. 3) The substantial effect of Va on packing within neutral phospholipid membranes was mimicked by Va-HC. 4) Based on measurements of membrane phase behavior, binding of Va or its peptide components did not induce thermodynamically discernible lateral membrane domains. These results suggest that the membrane association of factor Va is a complex process involving both chains of Va, changes

  20. Poststroke Rehabilitation and Restorative Care Utilization: A Comparison Between VA Community Living Centers and VA-contracted Community Nursing Homes.

    PubMed

    Jia, Huanguang; Pei, Qinglin; Sullivan, Charles T; Cowper Ripley, Diane C; Wu, Samuel S; Bates, Barbara E; Vogel, W Bruce; Bidelspach, Douglas E; Wang, Xinping; Hoffman, Nannette

    2016-03-01

    Effective poststroke rehabilitation care can speed patient recovery and minimize patient functional disabilities. Veterans affairs (VA) community living centers (CLCs) and VA-contracted community nursing homes (CNHs) are the 2 major sources of institutional long-term care for Veterans with stroke receiving care under VA auspices. This study compares rehabilitation therapy and restorative nursing care among Veterans residing in VA CLCs versus those Veterans in VA-contracted CNHs. Retrospective observational. All Veterans diagnosed with stroke, newly admitted to the CLCs or CNHs during the study period who completed at least 2 Minimum Data Set assessments postadmission. The outcomes were numbers of days for rehabilitation therapy and restorative nursing care received by the Veterans during their stays in CLCs or CNHs as documented in the Minimum Data Set databases. For rehabilitation therapy, the CLC Veterans had lower user rates (75.2% vs. 76.4%, P=0.078) and fewer observed therapy days (4.9 vs. 6.4, P<0.001) than CNH Veterans. However, the CLC Veterans had higher adjusted odds for therapy (odds ratio=1.16, P=0.033), although they had fewer average therapy days (coefficient=-1.53±0.11, P<0.001). For restorative nursing care, CLC Veterans had higher user rates (33.5% vs. 30.6%, P<0.001), more observed average care days (9.4 vs. 5.9, P<0.001), higher adjusted odds (odds ratio=2.28, P<0.001), and more adjusted days for restorative nursing care (coefficient=5.48±0.37, P<0.001). Compared with their counterparts at VA-contracted CNHs, Veterans at VA CLCs had fewer average rehabilitation therapy days (both unadjusted and adjusted), but they were significantly more likely to receive restorative nursing care both before and after risk adjustment.

  1. 16S rRNA Amplicon Sequencing for Epidemiological Surveys of Bacteria in Wildlife

    PubMed Central

    Razzauti, Maria; Bard, Emilie; Bernard, Maria; Brouat, Carine; Charbonnel, Nathalie; Dehne-Garcia, Alexandre; Loiseau, Anne; Tatard, Caroline; Tamisier, Lucie; Vayssier-Taussat, Muriel; Vignes, Helene

    2016-01-01

    ABSTRACT The human impact on natural habitats is increasing the complexity of human-wildlife interactions and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of the large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents, including 208 Mus musculus domesticus, 189 Rattus rattus, 93 Mastomys natalensis, and 221 Mastomys erythroleucus, collected from 24 villages in Senegal. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia, Bartonella, Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia. Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species, and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of bacterial

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

    PubMed

    2012-03-05

    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.

  3. VA's National PTSD Brain Bank: a National Resource for Research.

    PubMed

    Friedman, Matthew J; Huber, Bertrand R; Brady, Christopher B; Ursano, Robert J; Benedek, David M; Kowall, Neil W; McKee, Ann C

    2017-08-25

    The National PTSD Brain Bank (NPBB) is a brain tissue biorepository established to support research on the causes, progression, and treatment of PTSD. It is a six-part consortium led by VA's National Center for PTSD with participating sites at VA medical centers in Boston, MA; Durham, NC; Miami, FL; West Haven, CT; and White River Junction, VT along with the Uniformed Services University of Health Sciences. It is also well integrated with VA's Boston-based brain banks that focus on Alzheimer's disease, ALS, chronic traumatic encephalopathy, and other neurological disorders. This article describes the organization and operations of NPBB with specific attention to: tissue acquisition, tissue processing, diagnostic assessment, maintenance of a confidential data biorepository, adherence to ethical standards, governance, accomplishments to date, and future challenges. Established in 2014, NPBB has already acquired and distributed brain tissue to support research on how PTSD affects brain structure and function.

  4. Technology Changes and VA Mental Health Computer Applications

    PubMed Central

    Gottfredson, Douglas; Finkelstein, Allan; Christensen, Phillip; Weaver, Richard; Sells, Jeffery; Miller, David; Anderson, Ronald

    1993-01-01

    Since 1972, the Department of Veterans Affairs has had mental health computer applications for clinicians, managers, and researchers, operating on main frame and mini computers. The advent of personal computers has provided the opportunity to further enhance mental health automation. With Congressional support, VA's Mental Health and Behavioral Sciences Service placed micro computers in 168 VA Medical Centers and developed additional mental health applications. Using wide area networking procedures, a National Mental Health Database System (NMHDS) was established. In addition, a Computer-assisted Assessment, Psychotherapy, Education, and Research system (CAPER), a Treatment Planner, a Suicide and Assaultive Behavior Monitoring system, and a national registry of VA mental health treatment resources were developed. Each of these computer applications is demonstrated and discussed.

  5. VA Telemedicine: An Analysis of Cost and Time Savings.

    PubMed

    Russo, Jack E; McCool, Ryan R; Davies, Louise

    2016-03-01

    The Veterans Affairs (VA) healthcare system provides beneficiary travel reimbursement ("travel pay") to qualifying patients for traveling to appointments. Travel pay is a large expense for the VA and hence the U.S. Government, projected to cost nearly $1 billion in 2015. Telemedicine in the VA system has the potential to save money by reducing patient travel and thus the amount of travel pay disbursed. In this study, we quantify this savings and also report trends in VA telemedicine volumes over time. All telemedicine visits based at the VA Hospital in White River Junction, VT between 2005 and 2013 were reviewed (5,695 visits). Travel distance and time saved as a result of telemedicine were calculated. Clinical volume in the mental health department, which has had the longest participation in telemedicine, was analyzed. Telemedicine resulted in an average travel savings of 145 miles and 142 min per visit. This led to an average travel payment savings of $18,555 per year. Telemedicine volume grew significantly over the study period such that by the final year the travel pay savings had increased to $63,804, or about 3.5% of the total travel pay disbursement for that year. The number of mental health telemedicine visits rose over the study period but remained small relative to the number of face-to-face visits. A higher proportion of telemedicine visits involved new patients. Telemedicine at the VA saves travel distance and time, although the reduction in travel payments remains modest at current telemedicine volumes.

  6. 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

  7. What the VA can teach us about geriatric care.

    PubMed

    Ratner, Edward R; West, Melissa; Hartwig, Kristopher N; Meyer, Bruce C

    2013-01-01

    The innovation now being demanded by Medicare is creating new opportunities for health care organizations to redesign how they deliver care for elderly people. For many years, the VA Health System has experimented with ways to deliver care more effectively and efficiently. Hospital-based postacute and palliative care and home-based primary care are two examples of successful approaches that non-VA providers should be looking at as they move away from fee-for-service reimbursement and invent new care-delivery models.

  8. Effects of VA-045 on learning and memory deficits in traumatic brain injury (TBI)-induced retrograde and anterograde amnesic mice

    PubMed Central

    Tang, Ya-Ping; Noda, Yukihiro; Hasegawa, Takaaki; Nabeshima, Toshitaka

    1997-01-01

    No specific regimen has been developed to treat post-traumatic amnesia in man. In the present study, we examined the effects of (+)-eburnamenine-14-carboxylic acid (2-nitroxyethyl) ester (VA-045), a novel derivative of apovincaminic acid, on learning and memory deficits associated with a mild traumatic brain injury (TBI) in mice. Two kinds of amnesia, TBI-induced retrograde amnesia (TRA) and anterograde amnesia (TAA), were produced by means of post- and pre-acquisition head injury, respectively, by a simple weight-drop device. A novel procedure of water-finding task was used to assess learning and memory functions. Both TRA and TAA mice were dramatically impaired in the task performance, with prolonged latencies for finding and drinking in either retention test or retest, indicating that retention was impaired in TRA mice while learning and retention were impaired in TAA mice. VA-045 administered 30 min post-trauma in TRA mice dramatically shortened the prolonged latencies for finding and drinking in both retention test and retest, indicating that VA-045 significantly improved the retention deficit observed in TRA mice. VA-045 administered 30 min post-trauma in TAA mice dramatically attenuated the prolonged latencies for finding and drinking in both retention test and retest, indicating that VA-045 significantly improved the learning and retention deficits observed in TAA mice. Administration of VA-045 30 min pre-trauma in normal mice markedly attenuated the delay of latencies for finding and drinking after trauma in both retention test and retest, which shows that VA-045 significantly prevented learning and retention deficits after TBI. Motor activities were not significantly affected by either the TBI or the chemical treatment at the time of task examination in either experimental model. It is concluded that VA-045 may have potential effects on learning and memory deficits observed in either TBI-induced retrograde or anterograde amnesia. PMID:9313933

  9. 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.

  10. 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

  11. Gene-specific FACS sorting method for target selection in high-throughput amplicon sequencing

    PubMed Central

    2010-01-01

    Background In addition to shotgun sequencing, next generation sequencing has been shown to be suitable for deep sequencing of many specific PCR-amplified target genes in parallel. However, unspecific product formation is a common problem in amplicon sequencing since these fragments are difficult to fully remove by gel purification, and their presence inevitably reduces the number of mappable sequence reads that can be obtained in each sequencing run. Results We have used a novel flow cytometric sorting approach to specifically enrich Roche/454 DNA Capture beads carrying target DNA sequences on their surface, and reject beads carrying unspecific sequences. This procedure gives a nearly three-fold increase in the fraction of informative sequences obtained. Presented results also show that there are no significant differences in the distribution or presence of different genotypes between a FACS-enriched sample and a standard-enriched control sample. Conclusions Target-specific FACS enrichment prior to Roche/454 sequencing provides a quick, inexpensive way of increasing the amount of high quality data obtained in a single sequencing run, without introducing any sequence bias. PMID:20184782

  12. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. PARTIE: a partition engine to separate metagenomic and amplicon projects in the Sequence Read Archive.

    PubMed

    Torres, Pedro J; Edwards, Robert A; McNair, Katelyn A

    2017-08-01

    The Sequence Read Archive (SRA) contains raw data from many different types of sequence projects. As of 2017, the SRA contained approximately ten petabases of DNA sequence (10 16 bp). Annotations of the data are provided by the submitter, and mining the data in the SRA is complicated by both the amount of data and the detail within those annotations. Here, we introduce PARTIE, a partition engine optimized to differentiate sequence read data into metagenomic (random) and amplicon (targeted) sequence data sets. PARTIE subsamples reads from the sequencing file and calculates four different statistics: k -mer frequency, 16S abundance, prokaryotic- and viral-read abundance. These metrics are used to create a RandomForest decision tree to classify the sequencing data, and PARTIE provides mechanisms for both supervised and unsupervised classification. We demonstrate the accuracy of PARTIE for classifying SRA data, discuss the probable error rates in the SRA annotations and introduce a resource assessing SRA data. PARTIE and reclassified metagenome SRA entries are available from https://github.com/linsalrob/partie. redwards@mail.sdsu.edu. Supplementary data are available at Bioinformatics online.

  14. 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.

  15. Accuracy of the high-throughput amplicon sequencing to identify species within the genus Aspergillus.

    PubMed

    Lee, Seungeun; Yamamoto, Naomichi

    2015-12-01

    This study characterized the accuracy of high-throughput amplicon sequencing to identify species within the genus Aspergillus. To this end, we sequenced the internal transcribed spacer 1 (ITS1), β-tubulin (BenA), and calmodulin (CaM) gene encoding sequences as DNA markers from eight reference Aspergillus strains with known identities using 300-bp sequencing on the Illumina MiSeq platform, and compared them with the BLASTn outputs. The identifications with the sequences longer than 250 bp were accurate at the section rank, with some ambiguities observed at the species rank due to mostly cross detection of sibling species. Additionally, in silico analysis was performed to predict the identification accuracy for all species in the genus Aspergillus, where 107, 210, and 187 species were predicted to be identifiable down to the species rank based on ITS1, BenA, and CaM, respectively. Finally, air filter samples were analysed to quantify the relative abundances of Aspergillus species in outdoor air. The results were reproducible across biological duplicates both at the species and section ranks, but not strongly correlated between ITS1 and BenA, suggesting the Aspergillus detection can be taxonomically biased depending on the selection of the DNA markers and/or primers.

  16. 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

  17. 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.

  18. 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.

  19. Shedding light on the microbial community of the macropod foregut using 454-amplicon pyrosequencing.

    PubMed

    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.

  20. 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

  1. Targeted Amplicon Sequencing (TAS): A Scalable Next-Gen Approach to Multilocus, Multitaxa Phylogenetics

    PubMed Central

    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

  2. Polymicrobial nature of chronic diabetic foot ulcer biofilm infections determined using bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP).

    PubMed

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

    2008-10-03

    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. 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. 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 definitely warranted and needed in order to prove

  3. Inhibition of adenovirus replication by the E1A antisense transcript initiated from hsp70 and VA-1 promoters.

    PubMed

    Miroshnichenko, O I; Borisenko, A S; Ponomareva, T I; Tikhonenko, T I

    1990-03-01

    The E1A region of the adenoviral genome, important for initiation of virus infection and activation of other viral genes, was chosen as a target for engineering antisense RNA (asRNA) to inhibit adenovirus 5 (Ad5) replication in COS-1 cell culture in vitro. The hsp70 promoter, taken from the appropriate heat-shock-protein gene of Drosophila melanogaster, and the VA-1 RNA promoter, derived from the Ad5 gene coding for low-molecular-mass VA-1 RNA and recognized by RNA polymerase III were used as regulatory elements of transcription. The two types of recombinant constructs contained E1A fragments of 710 bp (hsp70 constructs) or 380 or 740 bp (VA-1 RNA constructs) in reverse orientation relative to the promoter position, as well as a transcription termination signal, the SV40 ori, and the gene controlling Geneticin (antibiotic G418) resistance (G418R). After selection of transfected COS-1 cells in the presence of G418, a number of stable G418R cell lines were raised which expressed engineered asRNAs. Plating of Ad5 suspensions of known titre on monolayers of transfected COS-1 cells clearly showed strong inhibition of adenovirus replication by asRNAs: 75% with the hsp70 promoter and 90% with the VA-1 RNA promoter.

  4. 48 CFR 801.695 - VA's Appointment of HCAs Program.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false VA's Appointment of HCAs Program. 801.695 Section 801.695 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS GENERAL DEPARTMENT OF VETERANS AFFAIRS ACQUISITION REGULATION SYSTEM Career Development,...

  5. 12 CFR 217.205 - VaR-based measure.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... portfolio of correlation trading positions that is modeled under § 217.209. A Board-regulated institution... differences in volatility and less than perfect correlation of rates along the yield curve. (2) The VaR-based measure may incorporate empirical correlations within and across risk categories, provided the Board...

  6. 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... less than six—to capture differences in volatility and less than perfect correlation of rates along the yield curve. (2) The VaR-based measure may incorporate empirical correlations within and across risk...

  7. 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... differences in volatility and less than perfect correlation of rates along the yield curve. (2) The VaR-based measure may incorporate empirical correlations within and across risk categories, provided the FDIC...

  8. VA contracts give unfair advantage to Picker--study.

    PubMed

    Wagner, M

    1991-08-19

    Service contracts for computed tomography scanners in Dept. of Veterans Affairs hospitals have given an unfair advantage to Picker International, according to a study by the VA's inspector general's office. The report is the latest round in a controversy that has pitted Picker against at least two independent service organizations and enmeshed it in a $1.65 billion lawsuit.

  9. 76 FR 72838 - Amendment of Class E Airspace; Luray, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-28

    ... management of Instrument Flight Rules (IFR) operations within the National Airspace System. This action also..., 2011, and effective September 15, 2011, which is incorporated by reference in 14 CFR 71.1. The Class E... Federal Aviation Administration 14 CFR Part 71 Amendment of Class E Airspace; Luray, VA AGENCY:...

  10. Military and VA general dentistry training: a national resource.

    PubMed

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

    2002-06-01

    In 1999, HRSA contracted with the UCLA School of Dentistry to evaluate the postgraduate general dentistry (PDG) training programs. The purpose of this article is to compare the program characteristics of the PGD training programs sponsored by the Armed Services (military) and VA. Surveys mailed to sixty-six VA and forty-two military program directors in fall 2000 sought information regarding the infrastructure of the program, the program emphasis, resident preparation prior to entering the program, and a description of patients served and types of services provided. Of the eighty-one returned surveys (75 percent response rate), thirty were received from military program directors and fifty-one were received from VA program directors. AEGDs reported treating a higher proportion of children patients and GPRs more medically intensive, disadvantaged and HIV/AIDS patients. Over half of the directors reported increases in curriculum emphasis in implantology. The program directors reported a high level of inadequate preparation among incoming dental residents. Having a higher ratio of residents to total number of faculty predicted inadequate preparation (p=.022) although the model was weak. Although HRSA doesn't financially support federally sponsored programs, their goal of improved dental training to care for medically compromised individuals is facilitated through these programs, thus making military and VA general dentistry programs a national resource.

  11. 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…

  12. 78 FR 2708 - Virginia Disaster Number VA-00052

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-14

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster Number VA-00052 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... completed loan applications to: U.S. Small Business Administration, Processing and Disbursement...

  13. 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…

  14. 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…

  15. 48 CFR 801.695 - VA's Appointment of HCAs Program.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false VA's Appointment of HCAs Program. 801.695 Section 801.695 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS GENERAL DEPARTMENT OF VETERANS AFFAIRS ACQUISITION REGULATION SYSTEM Career Development, Contracting...

  16. 48 CFR 801.695 - VA's Appointment of HCAs Program.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false VA's Appointment of HCAs Program. 801.695 Section 801.695 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS GENERAL DEPARTMENT OF VETERANS AFFAIRS ACQUISITION REGULATION SYSTEM Career Development, Contracting...

  17. 48 CFR 801.695 - VA's Appointment of HCAs Program.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false VA's Appointment of HCAs Program. 801.695 Section 801.695 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS GENERAL DEPARTMENT OF VETERANS AFFAIRS ACQUISITION REGULATION SYSTEM Career Development, Contracting...

  18. 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…

  19. 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... for Lake Anna Airport. This action is necessary for the safety and management of IFR operations at the... within the scope of that authority as it establishes controlled airspace at Lake Anna Airport, Bumpass...

  20. 75 FR 2895 - Virginia Disaster Number VA-00027

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-19

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster Number VA-00027 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... the original declaration remains unchanged. (Catalog of Federal Domestic Assistance Numbers 59002 and...

  1. 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…

  2. 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.... ADDRESSES: Submit completed loan applications to: U.S. Small Business Administration, Processing and..., Office of Disaster Assistance, U.S. Small Business Administration, 409 3rd Street, SW., Suite...

  3. An Overview of Patient Safety Climate in the VA

    PubMed Central

    Hartmann, Christine W; Rosen, Amy K; Meterko, Mark; Shokeen, Priti; Zhao, Shibei; Singer, Sara; Falwell, Alyson; Gaba, David M

    2008-01-01

    Objective To assess variation in safety climate across VA hospitals nationally. Study Setting Data were collected from employees at 30 VA hospitals over a 6-month period using the Patient Safety Climate in Healthcare Organizations survey. Study Design We sampled 100 percent of senior managers and physicians and a random 10 percent of other employees. At 10 randomly selected hospitals, we sampled an additional 100 percent of employees working in units with intrinsically higher hazards (high-hazard units [HHUs]). Data Collection Data were collected using an anonymous survey design. Principal Findings We received 4,547 responses (49 percent response rate). The percent problematic response—lower percent reflecting higher levels of patient safety climate—ranged from 12.0–23.7 percent across hospitals (mean=17.5 percent). Differences in safety climate emerged by management level, clinician status, and workgroup. Supervisors and front-line staff reported lower levels of safety climate than senior managers; clinician responses reflected lower levels of safety climate than those of nonclinicians; and responses of employees in HHUs reflected lower levels of safety climate than those of workers in other areas. Conclusions This is the first systematic study of patient safety climate in VA hospitals. Findings indicate an overall positive safety climate across the VA, but there is room for improvement. PMID:18355257

  4. 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.

  5. Efficacy of Multiagent Herpes Simplex Virus Amplicon-Mediated Immunotherapy as Adjuvant Treatment for Experimental Hepatic Cancer

    PubMed Central

    Delman, Keith A.; Zager, Jonathan S.; Bennett, Joseph J.; Malhotra, Sandeep; Ebright, Michael I.; McAuliffe, Priscilla F.; Halterman, Marc W.; Federoff, Howard J.; Fong, Yuman

    2002-01-01

    Objective To evaluate the use of herpes simplex viral (HSV) amplicon vectors for production of tumor vaccines and to determine if such vaccines expressing combinations of immunostimulatory agents may be effective in the treatment of experimental liver cancer. Methods A hepatic metastatic tumor model using CT-26 colorectal cancer in syngeneic Balb/C mice was utilized. Tumor vaccines were produced by brief (20 minutes) exposure of irradiated tumor cells to herpes amplicon vectors carrying the transgene for RANTES, B7.1, or GM-CSF. The antitumor efficacy of vaccination using tumor cells secreting GM-CSF (single agent) or a combination of RANTES/B7.1/GM-CSF (multiagent) was tested. The effect of 60% hepatectomy or T-cell depletion was also tested in this model. Results In vitro assays confirmed high-level cytokine or costimulatory molecule production by cells transduced with amplicons. Antitumor efficacy was observed with single-agent or multiagent treatment. Without hepatectomy, immunization with single-agent or multiagent vaccine therapy appears equivalent. When administered in the setting of hepatectomy, multiagent regimens produced a higher cure rate than single-agent therapy (50% vs. 12.5%, P = .03). Animals treated with GM-CSF alone had an average nodule count of 40 ± 19 (P < .006 vs. Hep control 232 ± 30), while animals treated with multiagent therapy had an average nodule count of 11 ± 7 (P < .0004 vs. control). CD4+ and CD8+ lymphocyte blockade abrogated observed efficacy, confirming a lymphocyte-mediated response. Conclusions Tumor vaccines produced using HSV amplicon-mediated gene transfer may be useful in the treatment of liver malignancies. In the setting of hepatectomy, multiagent vaccine therapy offers an advantage over single-agent therapy. These data encourage consideration of such HSV-based neoadjuvant immunotherapy for treatment of liver malignancies. PMID:12192320

  6. Prostate-Specific and Tumor-Specific Targeting of an Oncolytic HSV-1 Amplicon/Helper Virus for Prostate Cancer Treatment

    DTIC Science & Technology

    2009-11-01

    Targeting of an Oncolytic HSV - 1 Amplicon/Helper Virus for Prostate Cancer Treatment PRINCIPAL INVESTIGATOR: Cleo Lee CONTRACTING...5a. CONTRACT NUMBER Prostate-Specific and Tumor-Specific Targeting of an Oncolytic HSV - 1 Amplicon/Helper Virus for Prostate Cancer Treatment...untranslated region (3’UTR) of a herpes simplex virus- 1 ( HSV - 1 ) essential viral gene, ICP4, to create CMV-ICP4-143T and CMV-ICP4-145T amplicon viruses. Our

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

    PubMed

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

    2016-01-01

    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.

  8. 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

  9. 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

    ...The Veterans Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public comment on the proposed collection of certain information by the agency. Under the Paperwork Reduction Act (PRA) of 1995, Federal agencies are required to publish notice in the Federal Register concerning each proposed collection of information, including each proposed......

  10. 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

    ...The Veterans Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public comment on the proposed collection of certain information by the agency. Under the Paperwork Reduction Act (PRA) of 1995, Federal agencies are required to publish notice in the Federal Register concerning each proposed collection of information, including each proposed......

  11. EPA, W. Va. DEP File Settlement with Justice Companies to Restore and Protect Monroe Co., W. Va. Waterways

    EPA Pesticide Factsheets

    CHARLESTON, W.Va. (Dec. 10, 2015) Today, the U.S. Environmental Protection Agency and the West Virginia Department of Environmental Protection filed a settlement with James C. Justice II, the James C. Justice Companies, Inc. and High Mountain Living

  12. 77 FR 76451 - Designation for the West Sacramento, CA; Frankfort, IN; and Richmond, VA Areas.

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-28

    ... Administration Designation for the West Sacramento, CA; Frankfort, IN; and Richmond, VA Areas. AGENCY: Grain... Frankfort, IN(765) 258-3624........ 1/1/2013 12/31/2015 Virginia Richmond, VA(757) 494-2464............

  13. 66. ISTHMUS BRIDGE (HAER No. VA48P) TO JAMESTOWN ISLAND, GENERAL ...

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

    66. ISTHMUS BRIDGE (HAER No. VA-48-P) TO JAMESTOWN ISLAND, GENERAL VIEW FROM SOUTHEAST ON ROAD. NOTE FERRY PIER IN BACKGROUND. - Colonial Parkway, Yorktown to Jamestown Island, Yorktown, York County, VA

  14. 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

  15. 77 FR 20889 - Proposed Information Collection (Request One-VA Identification Verification Card) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-06

    ... AFFAIRS Proposed Information Collection (Request One-VA Identification Verification Card) Activity... Identity Verification (PIV) identification card. DATES: Written comments and recommendations on the... use of other forms of information technology. Title: Request for One-VA Identification Card. OMB...

  16. 75 FR 33898 - Agency Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-15

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF VETERANS AFFAIRS Agency Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity.... 2900-0021.'' SUPPLEMENTARY INFORMATION: Title: VA Loan Electronic Reporting Interface (VALERI)...

  17. 78 FR 36642 - Proposed Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-18

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF VETERANS AFFAIRS Proposed Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity... techniques or the use of other forms of information technology. Title: VA Loan Electronic Reporting...

  18. SiNG-PCRseq: Accurate inter-sequence quantification achieved by spiking-in a neighbor genome for competitive PCR amplicon sequencing.

    PubMed

    Oh, Soo A; Yang, Inchul; Hahn, Yoonsoo; Kang, Yong-Kook; Chung, Sun-Ku; Jeong, Sangkyun

    2015-07-06

    Despite the recent technological advances in DNA quantitation by sequencing, accurate delineation of the quantitative relationship among different DNA sequences is yet to be elaborated due to difficulties in correcting the sequence-specific quantitation biases. We here developed a novel DNA quantitation method via spiking-in a neighbor genome for competitive PCR amplicon sequencing (SiNG-PCRseq). This method utilizes genome-wide chemically equivalent but easily discriminable homologous sequences with a known copy arrangement in the neighbor genome. By comparing the amounts of selected human DNA sequences simultaneously to those of matched sequences in the orangutan genome, we could accurately draw the quantitative relationships for those sequences in the human genome (root-mean-square deviations <0.05). Technical replications of cDNA quantitation performed using different reagents at different time points also resulted in excellent correlations (R(2) > 0.95). The cDNA quantitation using SiNG-PCRseq was highly concordant with the RNA-seq-derived version in inter-sample comparisons (R(2) = 0.88), but relatively discordant in inter-sequence quantitation (R(2) < 0.44), indicating considerable level of sequence-dependent quantitative biases in RNA-seq. Considering the measurement structure explicitly relating the amount of different sequences within a sample, SiNG-PCRseq will facilitate sharing and comparing the quantitation data generated under different spatio-temporal settings.

  19. 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...

  20. 78 FR 56271 - FY 2014-2020 Draft VA Strategic Plan

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-12

    ... AFFAIRS FY 2014-2020 Draft VA Strategic Plan AGENCY: Department of Veterans Affairs. ACTION: Notice of... availability of the FY 2014-2020 Draft VA Strategic Plan (Strategic Plan) for public review and comment, as.... Comments should indicate that they are submitted in response to the ``FY 2014-2020 Draft VA Strategic Plan...

  1. 48 CFR 803.7000 - Display of the VA Hotline poster.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false Display of the VA Hotline... 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...

  2. 48 CFR 803.7000 - Display of the VA Hotline poster.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false Display of the VA Hotline... 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...

  3. 48 CFR 803.7000 - Display of the VA Hotline poster.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false Display of the VA Hotline... 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...

  4. 48 CFR 803.7000 - Display of the VA Hotline poster.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false Display of the VA Hotline... 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...

  5. 38 CFR 63.10 - Selection of non-VA community-based providers.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... community-based providers. 63.10 Section 63.10 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF... community-based providers. (a) Who can apply. VA may award per diem contracts to non-VA community-based... non-VA community-based provider; however, the negotiated rate must be based on local community needs...

  6. 38 CFR 63.10 - Selection of non-VA community-based providers.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... community-based providers. 63.10 Section 63.10 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF... community-based providers. (a) Who can apply. VA may award per diem contracts to non-VA community-based... non-VA community-based provider; however, the negotiated rate must be based on local community needs...

  7. 38 CFR 3.1706 - Burial allowance for a veteran who died while hospitalized by VA.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... veteran who died while hospitalized by VA. 3.1706 Section 3.1706 Pensions, Bonuses, and Veterans' Relief... A08se3. § 3.1706 Burial allowance for a veteran who died while hospitalized by VA. Pt. 3, Subpt. B, Nt...-connected and who died while hospitalized by VA. For purposes of this allowance, a veteran was hospitalized...

  8. 78 FR 59773 - Proposed Information Collection (VA Request for Determination of Reasonable Value) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-27

    ... AFFAIRS Proposed Information Collection (VA Request for Determination of Reasonable Value) Activity... solicits comments for information needed to determine the reasonable value of properties for guaranteed or... information technology. Title: VA Request for Determination of Reasonable Value, VA Form 26-1805 and 26-1805-1...

  9. 76 FR 14820 - Proposed Amendment of Class E Airspace; Waynesboro, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-18

    ... Instrument Approach Procedures (SIAPs) developed for Eagle's Nest Airport. This action would enhance the... Waynesboro, VA to accommodate new standard instrument approach procedures developed for Eagle's Nest Airport... Feet or More Above the Surface of the Earth. * * * * * AEA VA E5 Waynesboro, VA Eagle's Nest Airport...

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

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2013-07-01 2013-07-01 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 21.1032 - VA has a duty to assist claimants in obtaining evidence.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2014-07-01 2014-07-01 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...

  12. 77 FR 2348 - Agency Information Collection (VA Enrollment Certification): Activity Under OMB Review

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-17

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF VETERANS AFFAIRS Agency Information Collection (VA Enrollment Certification): Activity Under OMB Review AGENCY... INFORMATION: Title: VA Enrollment Certification, VA Form 22-1999. OMB Control Number: 2900-0073. Type of...

  13. 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…

  14. 48 CFR 803.7000 - Display of the VA Hotline poster.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Display of the VA Hotline... 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...

  15. 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 on...

  16. 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…

  17. 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... collection. Abstract: VA personnel use the form to document pre-employment history and conduct...

  18. 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.

  19. VA/DoD Collaboration Guidebook for Healthcare Research

    DTIC Science & Technology

    2011-01-24

    VA Medical Center Providence, RI COL Rachel K Evans, PT, PhD Research Director Center for the Intrepid Brooke Army Medical Center Fort Sam...through the process of setting up the study by the CFI’s Research Director, COL Rachel Evans. At the initial planning meeting, COL Evans arranged...WA MAMC and WBAMC have IRBs Evans Army Community Hospital, Fort Carson , CO Bassett Army Community Hospital, Fort Wainwright, AK Weed Army

  20. Inpatient Mental Health Recaptre using Dod/VA Sharing

    DTIC Science & Technology

    2009-10-03

    Their Mission and Vision state and following: DoD/VA Sharing 15 Mission: Provide high quality, customer focused accessible and comprehensive...being accountable to our nation. Vision : We are the face of Army Medicine: Quality health care for our Army starts here! Access to world-class care...Army’s Regional Medical Commands (RMCs) who report to the MEDCOM. CTVHCS current mission and vision are: Mission: Honor America’s veterans by providing

  1. '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.

  2. 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}.

  3. 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.

  4. Unraveling Core Functional Microbiota in Traditional Solid-State Fermentation by High-Throughput Amplicons and Metatranscriptomics Sequencing.

    PubMed

    Song, Zhewei; Du, Hai; Zhang, Yan; Xu, Yan

    2017-01-01

    Fermentation microbiota is specific microorganisms that generate different types of metabolites in many productions. In traditional solid-state fermentation, the structural composition and functional capacity of the core microbiota determine the quality and quantity of products. As a typical example of food fermentation, Chinese Maotai-flavor liquor production involves a complex of various microorganisms and a wide variety of metabolites. However, the microbial succession and functional shift of the core microbiota in this traditional food fermentation remain unclear. Here, high-throughput amplicons (16S rRNA gene amplicon sequencing and internal transcribed space amplicon sequencing) and metatranscriptomics sequencing technologies were combined to reveal the structure and function of the core microbiota in Chinese soy sauce aroma type liquor production. In addition, ultra-performance liquid chromatography and headspace-solid phase microextraction-gas chromatography-mass spectrometry were employed to provide qualitative and quantitative analysis of the major flavor metabolites. A total of 10 fungal and 11 bacterial genera were identified as the core microbiota. In addition, metatranscriptomic analysis revealed pyruvate metabolism in yeasts (genera Pichia, Schizosaccharomyces, Saccharomyces, and Zygosaccharomyces) and lactic acid bacteria (genus Lactobacillus) classified into two stages in the production of flavor components. Stage I involved high-level alcohol (ethanol) production, with the genus Schizosaccharomyces serving as the core functional microorganism. Stage II involved high-level acid (lactic acid and acetic acid) production, with the genus Lactobacillus serving as the core functional microorganism. The functional shift from the genus Schizosaccharomyces to the genus Lactobacillus drives flavor component conversion from alcohol (ethanol) to acid (lactic acid and acetic acid) in Chinese Maotai-flavor liquor production. Our findings provide insight into the

  5. Unraveling Core Functional Microbiota in Traditional Solid-State Fermentation by High-Throughput Amplicons and Metatranscriptomics Sequencing

    PubMed Central

    Song, Zhewei; Du, Hai; Zhang, Yan; Xu, Yan

    2017-01-01

    Fermentation microbiota is specific microorganisms that generate different types of metabolites in many productions. In traditional solid-state fermentation, the structural composition and functional capacity of the core microbiota determine the quality and quantity of products. As a typical example of food fermentation, Chinese Maotai-flavor liquor production involves a complex of various microorganisms and a wide variety of metabolites. However, the microbial succession and functional shift of the core microbiota in this traditional food fermentation remain unclear. Here, high-throughput amplicons (16S rRNA gene amplicon sequencing and internal transcribed space amplicon sequencing) and metatranscriptomics sequencing technologies were combined to reveal the structure and function of the core microbiota in Chinese soy sauce aroma type liquor production. In addition, ultra-performance liquid chromatography and headspace-solid phase microextraction-gas chromatography-mass spectrometry were employed to provide qualitative and quantitative analysis of the major flavor metabolites. A total of 10 fungal and 11 bacterial genera were identified as the core microbiota. In addition, metatranscriptomic analysis revealed pyruvate metabolism in yeasts (genera Pichia, Schizosaccharomyces, Saccharomyces, and Zygosaccharomyces) and lactic acid bacteria (genus Lactobacillus) classified into two stages in the production of flavor components. Stage I involved high-level alcohol (ethanol) production, with the genus Schizosaccharomyces serving as the core functional microorganism. Stage II involved high-level acid (lactic acid and acetic acid) production, with the genus Lactobacillus serving as the core functional microorganism. The functional shift from the genus Schizosaccharomyces to the genus Lactobacillus drives flavor component conversion from alcohol (ethanol) to acid (lactic acid and acetic acid) in Chinese Maotai-flavor liquor production. Our findings provide insight into the

  6. Impact of the REACH II and REACH VA Dementia Caregiver Interventions on Healthcare Costs.

    PubMed

    Nichols, Linda O; Martindale-Adams, Jennifer; Zhu, Carolyn W; Kaplan, Erin K; Zuber, Jeffrey K; Waters, Teresa M

    2017-05-01

    Examine caregiver and care recipient healthcare costs associated with caregivers' participation in Resources for Enhancing Alzheimer's Caregivers Health (REACH II or REACH VA) behavioral interventions to improve coping skills and care recipient management. RCT (REACH II); propensity-score matched, retrospective cohort study (REACH VA). Five community sites (REACH II); 24 VA facilities (REACH VA). Care recipients with Alzheimer's disease and related dementias (ADRD) and their caregivers who participated in REACH II study (analysis sample of 110 caregivers and 197 care recipients); care recipients whose caregivers participated in REACH VA and a propensity matched control group (analysis sample of 491). Previously collected data plus Medicare expenditures (REACH II) and VA costs plus Medicare expenditures (REACH VA). There was no increase in VA or Medicare expenditures for care recipients or their caregivers who participated in either REACH intervention. For VA care recipients, REACH was associated with significantly lower total VA costs of care (33.6%). VA caregiver cost data was not available. In previous research, both REACH II and REACH VA have been shown to provide benefit for dementia caregivers at a cost of less than $5/day; however, concerns about additional healthcare costs may have hindered REACH's widespread adoption. Neither REACH intervention was associated with additional healthcare costs for caregivers or patients; in fact, for VA patients, there were significantly lower healthcare costs. The VA costs savings may be related to the addition of a structured format for addressing the caregiver's role in managing complex ADRD care to an existing, integrated care system. These findings suggest that behavioral interventions are a viable mechanism to support burdened dementia caregivers without additional healthcare costs. © 2017, Copyright the Authors Journal compilation © 2017, The American Geriatrics Society.

  7. A comprehensive method for amplicon-based and metagenomic characterization of viruses, bacteria, and eukaryotes in freshwater samples.

    PubMed

    Uyaguari-Diaz, Miguel I; Chan, Michael; Chaban, Bonnie L; Croxen, Matthew A; Finke, Jan F; Hill, Janet E; Peabody, Michael A; Van Rossum, Thea; Suttle, Curtis A; Brinkman, Fiona S L; Isaac-Renton, Judith; Prystajecky, Natalie A; Tang, Patrick

    2016-07-19

    Studies of environmental microbiota typically target only specific groups of microorganisms, with most focusing on bacteria through taxonomic classification of 16S rRNA gene sequences. For a more holistic understanding of a microbiome, a strategy to characterize the viral, bacterial, and eukaryotic components is necessary. We developed a method for metagenomic and amplicon-based analysis of freshwater samples involving the concentration and size-based separation of eukaryotic, bacterial, and viral fractions. Next-generation sequencing and culture-independent approaches were used to describe and quantify microbial communities in watersheds with different land use in British Columbia. Deep amplicon sequencing was used to investigate the distribution of certain viruses (g23 and RdRp), bacteria (16S rRNA and cpn60), and eukaryotes (18S rRNA and ITS). Metagenomic sequencing was used to further characterize the gene content of the bacterial and viral fractions at both taxonomic and functional levels. This study provides a systematic approach to separate and characterize eukaryotic-, bacterial-, and viral-sized particles. Methodologies described in this research have been applied in temporal and spatial studies to study the impact of land use on watershed microbiomes in British Columbia.

  8. 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

  9. 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

  10. Parallel tagged amplicon sequencing reveals major lineages and phylogenetic structure in the North American tiger salamander (Ambystoma tigrinum) species complex.

    PubMed

    O'Neill, Eric M; Schwartz, Rachel; Bullock, C Thomas; Williams, Joshua S; Shaffer, H Bradley; Aguilar-Miguel, X; Parra-Olea, Gabriela; Weisrock, David W

    2013-01-01

    Modern analytical methods for population genetics and phylogenetics are expected to provide more accurate results when data from multiple genome-wide loci are analysed. We present the results of an initial application of parallel tagged sequencing (PTS) on a next-generation platform to sequence thousands of barcoded PCR amplicons generated from 95 nuclear loci and 93 individuals sampled across the range of the tiger salamander (Ambystoma tigrinum) species complex. To manage the bioinformatic processing of this large data set (344 330 reads), we developed a pipeline that sorts PTS data by barcode and locus, identifies high-quality variable nucleotides and yields phased haplotype sequences for each individual at each locus. Our sequencing and bioinformatic strategy resulted in a genome-wide data set with relatively low levels of missing data and a wide range of nucleotide variation. structure analyses of these data in a genotypic format resulted in strongly supported assignments for the majority of individuals into nine geographically defined genetic clusters. Species tree analyses of the most variable loci using a multi-species coalescent model resulted in strong support for most branches in the species tree; however, analyses including more than 50 loci produced parameter sampling trends that indicated a lack of convergence on the posterior distribution. Overall, these results demonstrate the potential for amplicon-based PTS to rapidly generate large-scale data for population genetic and phylogenetic-based research.

  11. 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

  12. Improved Efficiency and Reliability of NGS Amplicon Sequencing Data Analysis for Genetic Diagnostic Procedures Using AGSA Software.

    PubMed

    Poulet, Axel; Privat, Maud; Ponelle, Flora; Viala, Sandrine; Decousus, Stephanie; Perin, Axel; Lafarge, Laurence; Ollier, Marie; El Saghir, Nagi S; Uhrhammer, Nancy; Bignon, Yves-Jean; Bidet, Yannick

    Screening for BRCA mutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of the BRCA genes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm) of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts.

  13. Improved Efficiency and Reliability of NGS Amplicon Sequencing Data Analysis for Genetic Diagnostic Procedures Using AGSA Software

    PubMed Central

    Poulet, Axel; Privat, Maud; Viala, Sandrine; Decousus, Stephanie; Perin, Axel; Lafarge, Laurence; Ollier, Marie; El Saghir, Nagi S.

    2016-01-01

    Screening for BRCA mutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of the BRCA genes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm) of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts. PMID:27656653

  14. Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi.

    PubMed

    Lindner, Daniel L; Carlsen, Tor; Henrik Nilsson, R; Davey, Marie; Schumacher, Trond; Kauserud, Håvard

    2013-06-01

    The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular investigations of environmental samples. In this study 454 amplicon pyrosequencing of the ITS1 region was applied to 99 phylogenetically diverse axenic single-spore cultures of fungi (Dikarya: Ascomycota and Basidiomycota) to investigate levels of intragenomic variation. Three species (one Basidiomycota and two Ascomycota), in addition to a positive control species known to contain ITS paralogs, displayed levels of molecular variation indicative of intragenomic variation; taxon inflation due to presumed intragenomic variation was ≈9%. Intragenomic variability in the ITS region appears to be widespread but relatively rare in fungi (≈3-5% of species investigated in this study), suggesting this problem may have minor impacts on species richness estimates relative to PCR and/or pyrosequencing errors. Our results indicate that 454 amplicon pyrosequencing represents a powerful tool for investigating levels of ITS intragenomic variability across taxa, which may be valuable for better understanding the fundamental mechanisms underlying concerted evolution of repetitive DNA regions.

  15. 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

  16. Deep sequencing of amplicons reveals widespread intraspecific hybridization and multiple origins of polyploidy in big sagebrush (Artemisia tridentata; Asteraceae).

    PubMed

    Richardson, Bryce A; Page, Justin T; Bajgain, Prabin; Sanderson, Stewart C; Udall, Joshua A

    2012-12-01

    Hybridization has played an important role in the evolution and ecological adaptation of diploid and polyploid plants. Artemisia tridentata (Asteraceae) tetraploids are extremely widespread and of great ecological importance. These tetraploids are often taxonomically identified as A. tridentata subsp. wyomingensis or as autotetraploids of diploid subspecies tridentata and vaseyana. Few details are available as to how these tetraploids are formed or how they are related to diploid subspecies. • We used amplicon sequencing to assess phylogenetic relationships among three recognized subspecies: tridentata, vaseyana, and wyomingensis. DNA sequence data from putative genes were pyrosequenced and assembled from 329 samples. Nucleotide diversity and putative haplotypes were estimated from the high-read coverage. Phylogenies were constructed from Bayesian coalescence and neighbor-net network analyses. • Analyses support distinct diploid subspecies of tridentata and vaseyana in spite of known hybridization in ecotones. Nucleotide diversity estimates of populations compared to the total diversity indicate the relationships are predominately driven by a small proportion of the amplicons. Tetraploids, including subspecies wyomingensis, are polyphyletic occurring within and between diploid subspecies groups. • Artemisia tridentata is a species comprising phylogenetically distinct diploid progenitors and a tetraploid complex with varying degrees of phylogenetic and morphological affinities to the diploid subspecies. These analyses suggest tetraploids are formed locally or regionally from diploid tridentata and vaseyana populations via autotetraploidy, followed by introgression between tetraploid groups. Understanding the phylogenetic vs. ecological relationships of A. tridentata subspecies will have bearing on how to restore these desert ecosystems.

  17. Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi

    PubMed Central

    Lindner, Daniel L; Carlsen, Tor; Henrik Nilsson, R; Davey, Marie; Schumacher, Trond; Kauserud, Håvard

    2013-01-01

    The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular investigations of environmental samples. In this study 454 amplicon pyrosequencing of the ITS1 region was applied to 99 phylogenetically diverse axenic single-spore cultures of fungi (Dikarya: Ascomycota and Basidiomycota) to investigate levels of intragenomic variation. Three species (one Basidiomycota and two Ascomycota), in addition to a positive control species known to contain ITS paralogs, displayed levels of molecular variation indicative of intragenomic variation; taxon inflation due to presumed intragenomic variation was ≈9%. Intragenomic variability in the ITS region appears to be widespread but relatively rare in fungi (≈3–5% of species investigated in this study), suggesting this problem may have minor impacts on species richness estimates relative to PCR and/or pyrosequencing errors. Our results indicate that 454 amplicon pyrosequencing represents a powerful tool for investigating levels of ITS intragenomic variability across taxa, which may be valuable for better understanding the fundamental mechanisms underlying concerted evolution of repetitive DNA regions. PMID:23789083

  18. Noncoherent detection of DQPSK in OFDM systems using predictive VA

    NASA Astrophysics Data System (ADS)

    Veludandi, Vineel K.; Vasudevan, K.

    2017-01-01

    Noncoherent detection of differential quaternary phase shift keying (DQPSK) signals in OFDM systems is efficiently implemented using a predictive Viterbi algorithm (VA) operating on a trellis with just S T = MP‑1 states instead of M P states, where M denotes an M-ary PSK constellation and P denotes the order of the prediction filter. The prediction filter coefficients are generated based on the channel DFT alone making a high SNR approximation, since the estimation of the noise-variance using training symbols results in loss of throughput.

  19. The Association Between Military Sexual Trauma and Use of VA and non-VA Healthcare Services among Female Veterans with Military Service in Iraq or Afghanistan

    PubMed Central

    Calhoun, Patrick S.; Schry, Amie R.; Dennis, Paul A.; Wagner, H. Ryan; Kimbrel, Nathan A.; Bastian, Lori A.; Beckham, Jean C.; Kudler, Harold; Straits-Tröster, Kristy

    2016-01-01

    Military sexual trauma (MST) has been linked with increased rates of mental health disorders among veterans. Few studies have addressed how military sexual trauma is related to use of VA and non-VA health care. The purpose of the current study was to (1) examine the association between MST, combat experiences and mental health outcomes (i.e., PTSD and depression) and (2) examine the association of MST and use of VA and non-VA health care services among female veterans who served in Iraq and Afghanistan. Female respondents to a survey assessing OEF/OIF veterans’ needs and health (N=185) completed measures of demographic variables, military history, combat exposure, MST, PTSD and depression symptoms, and use of VA and non-VA healthcare. Overall, 70% of the sample experienced one or more combat-related experiences and 15.7% endorsed MST during deployment to Iraq or Afghanistan. MST and combat exposure were both positively associated with PTSD and depression symptoms even after controlling for the effects of demographic and military history variables; the interaction between MST and combat exposure was not a significant predictor of PTSD or depression symptoms. MST was associated with increased use of VA mental health services in bivariate results but was not independently related to VA service utilization after accounting for PTSD and depression symptoms. MST and combat exposure are significantly related to PTSD and depression symptoms among female OEF/OIF veterans, but there was not a synergistic effect of these two types of trauma in the current study. Approximately half of women who report MST have not used VA healthcare. Continued outreach and education initiatives may be needed to ensure veterans understand the resources available to address MST related mental and physical health problems through the VA. PMID:26802046

  20. A random effects multinomial logit analysis of using Medicare and VA healthcare among veterans with dementia

    PubMed Central

    Zhu, Carolyn W.; Livote, Elayne E.; Ross, Joseph S.; Penrod, Joan D.

    2011-01-01

    Aims To examine longitudinal patterns of VA-only use, dual VA and Medicare use, or Medicare-only use among veterans with dementia. Methods Data on VA and Medicare use (1998–2001) were obtained from and VA administrative datasets and Medicare claims for 2,137 male veterans with a formal diagnosis of Alzheimer’s disease or vascular dementia enrolled in the National Longitudinal Caregiver Study. A random effects multinomial logit model accounting for unobserved individual heterogeneity was used to estimate the effects of patient and caregiver characteristics on use group over time. Results Compared to VA-only use, dual VA and Medicare use was associated with being white, married, higher education, having private insurance, Medicaid, low VA priority level, more functional limitations, and having lived in a nursing home or died in that year. Medicare-only use was associated with older age, being married, higher education, having private insurance, low VA priority level, living further from a VA Medical Center, having more comorbidities, functional limitations, and having lived in a nursing home or died. Veterans whose caregivers reported better health were more likely to be dual users, but those whose caregivers reported more comorbidities were more likely to use Medicare only. Discussion Different aspects of veterans’ needs and caregiver characteristics have differential effect on where veterans seek care. Efforts to coordinate care between VA and Medicare providers are necessary to ensure patients receive high quality care. PMID:20635273

  1. HSV-1 amplicon vectors elicit polyfunctional T cell responses to HIV-1 Env, and strongly boost responses to an adenovirus prime

    PubMed Central

    Duke, Cindy M.P.; Maguire, Casey A.; Keefer, Michael C.; Federoff, Howard J.; Bowers, William J.; Dewhurst, Stephen

    2007-01-01

    HSV-1 amplicon vectors elicit strong T-cell responses to encoded antigens but the qualitative nature of these responses is poorly understood. Antigen-specific CD4+ and CD8+ T-cell responses to amplicon and adenovirus (rAd5) vectors encoding HIV-1 gp120 were assessed following immunization of mice, by performing intracellular cytokine staining for IFNγ, IL-2 and TNFα, following stimulation of splenocytes with a HIV-1 Env peptide pool. The quality of the primary T-cell response to amplicon and rAd5 vectors was strikingly similar, but there were qualitative differences in responses to amplicon vectors that incorporated different promoters upstream of gp120 - suggesting that promoters can significantly influence immune response quality. When prime-boost combinations were studied, a rAd5 prime and amplicon boost elicited the highest T-cell response. Furthermore, protocols that incorporated a rAd5 prime consistently elicited a greater proportion of polyfunctional CD4+ T-cells - regardless of boost. This suggests that initial priming can shape immune response quality after a boost. Overall, these findings provide insight into effective vector combinations for HIV-1 vaccine development. PMID:17868958

  2. 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.

  3. Use of VA and Medicare Services By Dually Eligible Veterans with Psychiatric Problems

    PubMed Central

    Carey, Kathleen; Montez-Rath, Maria E; Rosen, Amy K; Christiansen, Cindy L; Loveland, Susan; Ettner, Susan L

    2008-01-01

    Objective To examine how service accessibility measured by geographic distance affects service sector choices for veterans who are dually eligible for veterans affairs (VA) and Medicare services and who are diagnosed with mental health and/or substance abuse (MH/SA) disorders. Data Sources Primary VA data sources were the Patient Treatment (acute care), Extended Care (long-term care), and Outpatient Clinic files. VA cost data were obtained from (1) inpatient and outpatient cost files developed by the VA Health Economics and Resource Center and (2) outpatient VA Decision Support System files. Medicare data sources were the denominator, Medicare Provider Analysis Review (MEDPAR), Provider-of-Service, Outpatient Standard Analytic and Physician/Supplier Standard Analytic files. Additional sources included the Area Resource File and Census Bureau data. Study Design We identified dually eligible veterans who had either an inpatient or outpatient MH/SA diagnosis in the VA system during fiscal year (FY)'99. We then estimated one- and two-part regression models to explain the effects of geographic distance on both VA and Medicare total and MH/SA costs. Principal Findings Results provide evidence for substitution between the VA and Medicare, demonstrating that poorer geographic access to VA inpatient and outpatient clinics decreased VA expenditures but increased Medicare expenditures, while poorer access to Medicare-certified general and psychiatric hospitals decreased Medicare expenditures but increased VA expenditures. Conclusions As geographic distance to VA medical facility increases, Medicare plays an increasingly important role in providing mental health services to veterans. PMID:18355256

  4. Intravenously Administered Alphavirus Vector VA7 Eradicates Orthotopic Human Glioma Xenografts in Nude Mice

    PubMed Central

    Ruotsalainen, Janne J.; Martikainen, Miika W.; Stanford, Marianne M.; McCart, J. Andrea; Bell, John C.; Hinkkanen, Ari E.

    2010-01-01

    Background VA7 is a neurotropic alphavirus vector based on an attenuated strain of Semliki Forest virus. We have previously shown that VA7 exhibits oncolytic activity against human melanoma xenografts in immunodeficient mice. The purpose of this study was to determine if intravenously administered VA7 would be effective against human glioma. Methodology/Principal Findings In vitro, U87, U251, and A172 human glioma cells were infected and killed by VA7-EGFP. In vivo, antiglioma activity of VA7 was tested in Balb/c nude mice using U87 cells stably expressing firefly luciferase in subcutaneous and orthotopic tumor models. Intravenously administered VA7-EGFP completely eradicated 100% of small and 50% of large subcutaneous U87Fluc tumors. A single intravenous injection of either VA7-EGFP or VA7 expressing Renilla luciferase (VA7-Rluc) into mice bearing orthotopic U87Fluc tumors caused a complete quenching of intracranial firefly bioluminescence and long-term survival in total 16 of 17 animals. In tumor-bearing mice injected with VA7-Rluc, transient intracranial and peripheral Renilla bioluminescence was observed. Virus was well tolerated and no damage to heart, liver, spleen, or brain was observed upon pathological assessment at three and ninety days post injection, despite detectable virus titers in these organs during the earlier time point. Conclusion VA7 vector is apathogenic and can enter and destroy brain tumors in nude mice when administered systemically. This study warrants further elucidation of the mechanism of tumor destruction and attenuation of the VA7 virus. PMID:20066051

  5. Factors determining medical students' and residents' satisfaction during VA-based training: findings from the VA Learners' Perceptions Survey.

    PubMed

    Cannon, Grant W; Keitz, Sheri A; Holland, Gloria J; Chang, Barbara K; Byrne, John M; Tomolo, Anne; Aron, David C; Wicker, Annie B; Kashner, T Michael

    2008-06-01

    To compare medical students' and physician residents' satisfaction with Veterans Affairs (VA) training to determine the factors that were most strongly associated with trainee satisfaction ratings. Each year from 2001 to 2006, all medical students and residents in VA teaching facilities were invited to complete the Learners' Perceptions Survey. Participants rated their overall training satisfaction on a 100-point scale and ranked specific satisfaction in four separate educational domains (learning environment, clinical faculty, working environment, and physical environment) on a five-point Likert scale. Each domain was composed of unique items. A total of 6,527 medical students and 16,583 physician residents responded to the survey. The overall training satisfaction scores for medical students and physician residents were 84 and 79, respectively (P < .001), with significant differences in satisfaction reported across the training continuum. For both medical students and residents, the rating of each of the four educational domains was statistically significantly associated with the overall training satisfaction score (P < .001). The learning environment domain had the strongest association with overall training satisfaction score, followed by the clinical preceptor, working environment, and physical environment domains; no significant differences were found between medical students and physician residents in the rank order. Satisfaction with quality of care and faculty teaching contributed significantly to training satisfaction. Factors that influence training satisfaction were similar for residents and medical students. The domain with the highest association was the learning environment; quality of care was a key item within this domain.

  6. 75 FR 70351 - Termination of Environmental Review Process Cities of Chesapeake and Virginia Beach, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-17

    ... Virginia Beach, VA AGENCY: Federal Highway Administration (FHWA), DOT. ACTION: Termination of environmental... the Cities of Chesapeake and Virginia Beach, Virginia, is terminated. FOR FURTHER INFORMATION CONTACT...

  7. Penn/VA center for studies of addiction.

    PubMed

    O'Brien, Charles P; McLellan, A Thomas; Childress, Anna Rose; Woody, George E

    2009-01-01

    The Penn/VA Center was founded in 1971 because of great concern over the number of Vietnam veterans returning home addicted to heroin. At that time little was known about the science of addiction, so our program from the very beginning was designed to gather data about the nature of addiction and measure the effects of available treatments. In other words, the goals were always a combination of treatment and research. This combination has continued to the present day. A human laboratory for the study of addiction phenomena such as conditioned responses was also founded in 1971. The key clinician investigators in this group have remained in the Center since the 1970s with most of the research staff continuing to work together. Important new investigators have been added over the years. Treatment was empirically based with randomized, controlled clinical trials as the gold standard for determining evidence-based treatment. The patients coming to treatment do not distinguish between abuse of alcohol and other drugs, so the treatment and research programs have always focused on all drugs including ethyl alcohol and the combination of ethyl alcohol with other drugs such as cocaine and opioids. Most of the patients coming for treatment also suffered from additional psychiatric disorders such as depression, anxiety, bipolar disorder or schizophrenia. Thus, the addiction treatment program in 1980 absorbed the rest of the VA Psychiatry Service into the Substance Abuse Program forming a new Behavioral Health Service with responsibility for over 9000 patients. The integration of substance abuse treatment with overall mental health care was the most efficient way to handle patients with complicated combinations of disorders. While this continues to be the best way to treat patients, it has proven difficult in practice. The main reason for this difficulty is that most mental health therapists whether they are psychiatrists, psychologists or social workers feel very inadequate

  8. HPV Genotyping of Modified General Primer-Amplicons Is More Analytically Sensitive and Specific by Sequencing than by Hybridization

    PubMed Central

    Meisal, Roger; Rounge, Trine Ballestad; Christiansen, Irene Kraus; Eieland, Alexander Kirkeby; Worren, Merete Molton; Molden, Tor Faksvaag; Kommedal, Øyvind; Hovig, Eivind; Leegaard, Truls Michael

    2017-01-01

    Sensitive and specific genotyping of human papillomaviruses (HPVs) is important for population-based surveillance of carcinogenic HPV types and for monitoring vaccine effectiveness. Here we compare HPV genotyping by Next Generation Sequencing (NGS) to an established DNA hybridization method. In DNA isolated from urine, the overall analytical sensitivity of NGS was found to be 22% higher than that of hybridization. NGS was also found to be the most specific method and expanded the detection repertoire beyond the 37 types of the DNA hybridization assay. Furthermore, NGS provided an increased resolution by identifying genetic variants of individual HPV types. The same Modified General Primers (MGP)-amplicon was used in both methods. The NGS method is described in detail to facilitate implementation in the clinical microbiology laboratory and includes suggestions for new standards for detection and calling of types and variants with improved resolution. PMID:28045981

  9. Comparative analyses of the major royal jelly protein gene cluster in three Apis species with long amplicon sequencing.

    PubMed

    Helbing, Sophie; Lattorff, H Michael G; Moritz, Robin F A; Buttstedt, Anja

    2017-06-01

    The western honeybee, Apis mellifera is a prominent model organism in the field of sociogenomics and a recent upgrade substantially improved annotations of the reference genome. Nevertheless, genome assemblies based on short-sequencing reads suffer from problems in regions comprising e.g. multi-copy genes. We used single-molecule nanopore-based sequencing with extensive read-lengths to reconstruct the organization of the major royal jelly protein (mrjp) region in three species of the genus Apis. Long-amplicon sequencing provides evidence for lineage-specific evolutionary fates of Apis mrjps. Whereas the most basal species, A. florea, seems to encode ten mrjps, different patterns of gene loss and retention were observed for A. mellifera and A. dorsata. Furthermore, we show that a previously reported pseudogene in A. mellifera, mrjp2-like, is an assembly artefact arising from short read sequencing. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  10. Comparative analyses of the major royal jelly protein gene cluster in three Apis species with long amplicon sequencing

    PubMed Central

    Lattorff, H. Michael G.; Moritz, Robin F.A.; Buttstedt, Anja

    2017-01-01

    Abstract The western honeybee, Apis mellifera is a prominent model organism in the field of sociogenomics and a recent upgrade substantially improved annotations of the reference genome. Nevertheless, genome assemblies based on short-sequencing reads suffer from problems in regions comprising e.g. multi-copy genes. We used single-molecule nanopore-based sequencing with extensive read-lengths to reconstruct the organization of the major royal jelly protein (mrjp) region in three species of the genus Apis. Long-amplicon sequencing provides evidence for lineage-specific evolutionary fates of Apis mrjps. Whereas the most basal species, A. florea, seems to encode ten mrjps, different patterns of gene loss and retention were observed for A. mellifera and A. dorsata. Furthermore, we show that a previously reported pseudogene in A. mellifera, mrjp2-like, is an assembly artefact arising from short read sequencing. PMID:28170034

  11. From reads to operational taxonomic units: an ensemble processing pipeline for MiSeq amplicon sequencing data

    PubMed Central

    Mysara, Mohamed; Njima, Mercy; Leys, Natalie; Raes, Jeroen

    2017-01-01

    Abstract The development of high-throughput sequencing technologies has provided microbial ecologists with an efficient approach to assess bacterial diversity at an unseen depth, particularly with the recent advances in the Illumina MiSeq sequencing platform. However, analyzing such high-throughput data is posing important computational challenges, requiring specialized bioinformatics solutions at different stages during the processing pipeline, such as assembly of paired-end reads, chimera removal, correction of sequencing errors, and clustering of those sequences into Operational Taxonomic Units (OTUs). Individual algorithms grappling with each of those challenges have been combined into various bioinformatics pipelines, such as mothur, QIIME, LotuS, and USEARCH. Using a set of well-described bacterial mock communities, state-of-the-art pipelines for Illumina MiSeq amplicon sequencing data are benchmarked at the level of the amount of sequences retained, computational cost, error rate, and quality of the OTUs. In addition, a new pipeline called OCToPUS is introduced, which is making an optimal combination of different algorithms. Huge variability is observed between the different pipelines in respect to the monitored performance parameters, where in general the amount of retained reads is found to be inversely proportional to the quality of the reads. By contrast, OCToPUS achieves the lowest error rate, minimum number of spurious OTUs, and the closest correspondence to the existing community, while retaining the uppermost amount of reads when compared to other pipelines. The newly introduced pipeline translates Illumina MiSeq amplicon sequencing data into high-quality and reliable OTUs, with improved performance and accuracy compared to the currently existing pipelines. PMID:28369460

  12. From reads to operational taxonomic units: an ensemble processing pipeline for MiSeq amplicon sequencing data.

    PubMed

    Mysara, Mohamed; Njima, Mercy; Leys, Natalie; Raes, Jeroen; Monsieurs, Pieter

    2017-02-01

    The development of high-throughput sequencing technologies has provided microbial ecologists with an efficient approach to assess bacterial diversity at an unseen depth, particularly with the recent advances in the Illumina MiSeq sequencing platform. However, analyzing such high-throughput data is posing important computational challenges, requiring specialized bioinformatics solutions at different stages during the processing pipeline, such as assembly of paired-end reads, chimera removal, correction of sequencing errors, and clustering of those sequences into Operational Taxonomic Units (OTUs). Individual algorithms grappling with each of those challenges have been combined into various bioinformatics pipelines, such as mothur, QIIME, LotuS, and USEARCH. Using a set of well-described bacterial mock communities, state-of-the-art pipelines for Illumina MiSeq amplicon sequencing data are benchmarked at the level of the amount of sequences retained, computational cost, error rate, and quality of the OTUs. In addition, a new pipeline called OCToPUS is introduced, which is making an optimal combination of different algorithms. Huge variability is observed between the different pipelines in respect to the monitored performance parameters, where in general the amount of retained reads is found to be inversely proportional to the quality of the reads. By contrast, OCToPUS achieves the lowest error rate, minimum number of spurious OTUs, and the closest correspondence to the existing community, while retaining the uppermost amount of reads when compared to other pipelines. The newly introduced pipeline translates Illumina MiSeq amplicon sequencing data into high-quality and reliable OTUs, with improved performance and accuracy compared to the currently existing pipelines. © The Author 2017. Published by Oxford University Press.

  13. Construction and packaging of herpes simplex virus/adeno-associated virus (HSV/AAV) Hybrid amplicon vectors.

    PubMed

    Saydam, Okay; Glauser, Daniel L; Fraefel, Cornel

    2012-03-01

    Herpes simplex virus type 1 (HSV-1)-based amplicon vectors conserve most properties of the parental virus: broad host range, the ability to transduce dividing and nondiving cells, and a large transgene capacity. This permits incorporation of genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell-specific expression, multiple transgene cassettes, or genetic elements from other viruses. Hybrid vectors use elements from HSV-1 that allow replication and packaging of large-vector DNA into highly infectious particles, and elements from other viruses that confer genetic stability to vector DNA in the transduced cell. For example, adeno-associated virus (AAV) has the unique ability to integrate its genome into a specific site on human chromosome 19. The viral rep gene and the inverted terminal repeats (ITRs) that flank the AAV genome are sufficient for this process. However, AAV-based vectors have a very small transgene capacity and do not conventionally contain the rep gene to support site-specific genomic integration. HSV/AAV hybrid vectors contain both HSV-1 replication and packaging functions and the AAV rep gene and a transgene cassette flanked by the AAV ITRs. This combines the large transgene capacity of HSV-1 with the capability of site-specific genomic transgene integration and long-term transgene expression of AAV. This protocol describes the preparation of HSV/AAV hybrid vectors using a replication-competent/conditional, packaging-defective HSV-1 genome cloned as a bacterial artificial chromosome (BAC) to provide helper functions for vector replication and packaging. The advantages and limitations of such vectors compared to standard HSV-1 amplicon vectors are also discussed.

  14. Short-read assembly of full-length 16S amplicons reveals bacterial diversity in subsurface sediments.

    PubMed

    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.

  15. 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.

  16. Fireworks algorithm for mean-VaR/CVaR models

    NASA Astrophysics Data System (ADS)

    Zhang, Tingting; Liu, Zhifeng

    2017-10-01

    Intelligent algorithms have been widely applied to portfolio optimization problems. In this paper, we introduce a novel intelligent algorithm, named fireworks algorithm, to solve the mean-VaR/CVaR model for the first time. The results show that, compared with the classical genetic algorithm, fireworks algorithm not only improves the optimization accuracy and the optimization speed, but also makes the optimal solution more stable. We repeat our experiments at different confidence levels and different degrees of risk aversion, and the results are robust. It suggests that fireworks algorithm has more advantages than genetic algorithm in solving the portfolio optimization problem, and it is feasible and promising to apply it into this field.

  17. Myosin Va is developmentally regulated and expressed in the human cerebellum from birth to old age

    PubMed Central

    Souza, C.C.R.; Dombroski, T.C.D.; Machado, H.R.; Oliveira, R.S.; Rocha, L.B.; Rodrigues, A.R.A.; Neder, L.; Chimelli, L.; Corrêa, V.M.A.; Larson, R.E.; Martins, A.R.

    2013-01-01

    Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence. PMID:23558932

  18. VA Health Care: VA Spends Millions on Post-Traumatic Stress Disorder Research and Incorporates Research Outcomes into Guidelines and Policy for Post-Traumatic Stress Disorder Services

    DTIC Science & Technology

    2011-01-01

    summarized VA data on the funding of PTSD research from its medical and prosthetic research appropriation through its intramural research program...2009, intramural PTSD research funding ranged from 2.5 percent to 4.8 percent of VA’s medical and prosthetic research appropriation. In addition, the...Veterans Affairs (VA) Intramural Post- Traumatic Stress Disorder (PTSD) Research Funding and VA’s Medical and Prosthetic Research Appropriation

  19. Reduced pathology and improved behavioral performance in Alzheimer’s disease mice vaccinated with HSV amplicons expressing amyloid-beta and interleukin-4

    PubMed Central

    Frazer, Maria E.; Hughes, Jennifer E.; Mastrangelo, Michael A.; Tibbens, Jennifer L.; Federoff, Howard J.; Bowers, William J.

    2008-01-01

    Immunotherapeutics designed to dissolve existing amyloid plaques or to interrupt amyloid-beta (Aβ) accumulation may be feasible for treatment and/or prevention of Alzheimer’s disease (AD). “Shaping” immune responses elicited against Aβ is requisite to generate an efficacious and safe outcome by minimizing the possibility of deleterious inflammatory reactions in the brain as observed in clinical testing of Aβ peptide/adjuvant-based modalities. Herpes Simplex Virus (HSV)-based amplicons can co-express multiple antigens and/or immunomodulatory genes due to their large genetic size capacity, thereby facilitating antigen-specific immune response shaping. We have constructed an amplicon (HSVIEAβCMVIL-4) that co-delivers Aβ1-42 with interleukin-4, a cytokine that promotes the generation of Th2-like T cell responses, which are favored in the setting of AD immunotherapy. Triple-transgenic AD (3xTg-AD) mice, which progressively develop both amyloid and neurofibrillary tangle pathology, were vaccinated thrice with HSVIEAβCMVIL-4, or a set of control amplicon vectors. Increased Th2-related, Aβ-specific antibodies, improved learning and memory functioning, and prevention of AD-related amyloid and tau pathological progression were observed in HSVIEAβCMVIL-4 vaccinated mice as compared to the other experimental groups. Our study underscores the potential of Aβ immunotherapy for AD and highlights the potency of amplicons to facilitate immune response modulation to a disease-relevant antigen. PMID:18388924

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

    PubMed

    Weber, Axel; Taube, Sylvia; Zur Stadt, Udo; Horstmann, Martin; Krohn, Knut; Bradtke, Jutta; Teigler-Schlegel, Andrea; Leiblein, Sabine; Christiansen, Holger

    2012-11-09

    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.

  1. 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 of...

  2. 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... Events, Department of Veterans Affairs. ACTION: Notice. SUMMARY: The Office of National Veterans Sports Programs and Special Events (NVSP), Department of Veterans Affairs (VA), is announcing an opportunity...

  3. 78 FR 27882 - VA Veteran-Owned Small Business (VOSB) Verification Guidelines

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-13

    ... AFFAIRS 38 CFR Part 74 RIN 2900-AO63 VA Veteran-Owned Small Business (VOSB) Verification Guidelines AGENCY... and Disadvantaged Business Utilization (OSDBU) is currently reviewing its regulations governing the Department of Veterans Affairs (VA) Veteran-Owned Small Business (VOSB) Verification Program. OSDBU...

  4. 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 any...

  5. 76 FR 44288 - Establishment of Class E Airspace; New Market, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-25

    ... Federal Aviation Administration 14 CFR Part 71 Establishment of Class E Airspace; New Market, VA AGENCY... action proposes to establish Class E Airspace at New Market, VA, to accommodate the additional airspace needed for the Standard Instrument Approach Procedures developed for New Market Airport. This action...

  6. 33 CFR 334.130 - Atlantic Ocean off Wallops Island and Chincoteague Inlet, Va.; danger zone.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 3 2013-07-01 2013-07-01 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 area...

  7. 33 CFR 334.130 - Atlantic Ocean off Wallops Island and Chincoteague Inlet, Va.; danger zone.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 3 2012-07-01 2012-07-01 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 area...

  8. 33 CFR 334.130 - Atlantic Ocean off Wallops Island and Chincoteague Inlet, Va.; danger zone.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 3 2010-07-01 2010-07-01 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 area...

  9. 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 area...

  10. 33 CFR 334.130 - Atlantic Ocean off Wallops Island and Chincoteague Inlet, Va.; danger zone.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 3 2011-07-01 2011-07-01 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 area...

  11. 76 FR 27970 - Safety Zone; Cape Charles Fireworks, Cape Charles Harbor, Cape Charles, VA.

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-13

    ... in Cape Charles, VA in support of the Fourth of July Fireworks event. This action is necessary to... notice. Basis and Purpose On July 3, 2011 the Town of Cape Charles will sponsor a fireworks display on... established in the vicinity of Cape Charles, VA from 9 p.m. to 10 p.m. on July 3, 2011, with a rain date of...

  12. 38 CFR 17.96 - Medication prescribed by non-VA physicians.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2010-07-01 2010-07-01 false Medication prescribed by non-VA physicians. 17.96 Section 17.96 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS MEDICAL Outpatient Treatment § 17.96 Medication prescribed by non-VA physicians. Any...

  13. VA Construction: Improved Processes Needed to Monitor Contract Modifications, Develop Schedules, and Estimate Costs

    DTIC Science & Technology

    2017-03-01

    Abbreviations ACBM Activation Cost Budget Model CFM Office of Construction and Facilities Management eCMS Electronic Contract ...VA CONSTRUCTION Improved Processes Needed to Monitor Contract Modifications, Develop Schedules, and Estimate Costs...Monitor Contract Modifications, Develop Schedules, and Estimate Costs What GAO Found The Department of Veterans Affairs (VA) has taken steps to

  14. 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 collect...

  15. 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

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF COMMERCE Foreign-Trade Zones Board Foreign-Trade Zone 20--Suffolk, VA; Authorization of Production Activity; Usui International Corporation (Diesel Engine Fuel Lines); Chesapeake, VA On June 28, 2012, the Virginia...

  16. 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…

  17. 75 FR 54771 - Safety Zone; Thunder on the Bay, Chesapeake Bay, Buckroe Beach Park, Hampton, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-09

    ... Beach Park, Hampton, VA AGENCY: Coast Guard, DHS. ACTION: Temporary final rule. SUMMARY: The Coast Guard... a fireworks display on the Buckroe Beach Park Fishing Pier over the navigable waters of the...). This safety zone will be established in the vicinity of Buckroe Beach Park in Hampton, VA from 9:15 p.m...

  18. 77 FR 13519 - Safety Zone; Virginia Beach Oceanfront Air Show, Atlantic Ocean, Virginia Beach, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-07

    ... 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: Notice of proposed rulemaking. SUMMARY... Virginia Beach, VA. This action is necessary to provide for the safety of life on navigable waters during...

  19. 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

    ... 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: The... Beach, VA to support the Virginia Beach Oceanfront Air Show. This action is necessary to provide for the...

  20. 78 FR 56151 - Safety Zone, North Atlantic Ocean; Virginia Beach, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-12

    ... 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 Virginia... Delegation No. 0170.1. ] On September 12, 2013 the City of Virginia Beach will host a fireworks display...

  1. 30 CFR 57.22315 - Self-contained breathing apparatus (V-A mines).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Self-contained breathing apparatus (V-A mines... NONMETAL MINES Safety Standards for Methane in Metal and Nonmetal Mines Equipment § 57.22315 Self-contained breathing apparatus (V-A mines). Self-contained breathing apparatus of a duration to allow for escape...

  2. 30 CFR 57.22315 - Self-contained breathing apparatus (V-A mines).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Self-contained breathing apparatus (V-A mines... NONMETAL MINES Safety Standards for Methane in Metal and Nonmetal Mines Equipment § 57.22315 Self-contained breathing apparatus (V-A mines). Self-contained breathing apparatus of a duration to allow for escape...

  3. 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 836...

  4. 38 CFR 26.9 - Information on and public participation in VA environmental process.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) ACTIONS § 26.9 Information on and public participation in VA environmental process. (a) During the... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Information on and public participation in VA environmental process. 26.9 Section 26.9 Pensions, Bonuses, and Veterans' Relief DEPARTMENT...

  5. 38 CFR 26.9 - Information on and public participation in VA environmental process.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...) ACTIONS § 26.9 Information on and public participation in VA environmental process. (a) During the... 38 Pensions, Bonuses, and Veterans' Relief 2 2013-07-01 2013-07-01 false Information on and public participation in VA environmental process. 26.9 Section 26.9 Pensions, Bonuses, and Veterans' Relief DEPARTMENT...

  6. 38 CFR 26.9 - Information on and public participation in VA environmental process.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...) ACTIONS § 26.9 Information on and public participation in VA environmental process. (a) During the... 38 Pensions, Bonuses, and Veterans' Relief 2 2012-07-01 2012-07-01 false Information on and public participation in VA environmental process. 26.9 Section 26.9 Pensions, Bonuses, and Veterans' Relief DEPARTMENT...

  7. 38 CFR 26.9 - Information on and public participation in VA environmental process.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) ACTIONS § 26.9 Information on and public participation in VA environmental process. (a) During the... 38 Pensions, Bonuses, and Veterans' Relief 2 2011-07-01 2011-07-01 false Information on and public participation in VA environmental process. 26.9 Section 26.9 Pensions, Bonuses, and Veterans' Relief DEPARTMENT...

  8. 38 CFR 26.9 - Information on and public participation in VA environmental process.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...) ACTIONS § 26.9 Information on and public participation in VA environmental process. (a) During the... 38 Pensions, Bonuses, and Veterans' Relief 2 2014-07-01 2014-07-01 false Information on and public participation in VA environmental process. 26.9 Section 26.9 Pensions, Bonuses, and Veterans' Relief DEPARTMENT...

  9. 30 CFR 57.22315 - Self-contained breathing apparatus (V-A mines).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Self-contained breathing apparatus (V-A mines). 57.22315 Section 57.22315 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF... breathing apparatus (V-A mines). Self-contained breathing apparatus of a duration to allow for escape...

  10. 30 CFR 57.22315 - Self-contained breathing apparatus (V-A mines).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Self-contained breathing apparatus (V-A mines). 57.22315 Section 57.22315 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF... breathing apparatus (V-A mines). Self-contained breathing apparatus of a duration to allow for escape...

  11. 30 CFR 57.22315 - Self-contained breathing apparatus (V-A mines).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Self-contained breathing apparatus (V-A mines). 57.22315 Section 57.22315 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF... breathing apparatus (V-A mines). Self-contained breathing apparatus of a duration to allow for escape...

  12. 76 FR 64236 - Establishment of Class E Airspace; New Market, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-18

    ... Federal Aviation Administration 14 CFR Part 71 Establishment of Class E Airspace; New Market, VA AGENCY... Airspace at New Market, VA, to accommodate the new Standard Instrument Approach Procedures serving New Market Airport. This action enhances the safety and airspace management of Instrument Flight Rules...

  13. 77 FR 38396 - Agency Information Collection (One-VA Identification Verification Card) Activities Under OMB Review

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-27

    ... AFFAIRS Agency Information Collection (One-VA Identification Verification Card) Activities Under OMB... No. 2900-0673.'' SUPPLEMENTARY INFORMATION: Title: Request for One-VA Identification Card. OMB... personalize, print, and issue a PIV card. An agency may not conduct or sponsor, and a person is not required...

  14. 75 FR 61858 - Proposed Information Collection (VA Request for Determination of Reasonable Value) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-06

    ... AFFAIRS Proposed Information Collection (VA Request for Determination of Reasonable Value) Activity... solicits comments for information needed to determine the reasonable value of properties for guaranteed or... Request for Determination of Reasonable Value, VA Form 26-1805 and 26-1805-1. OMB Control Number: 2900...

  15. 75 FR 61249 - Proposed Information Collection (VA Request for Determination of Reasonable Value) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-04

    ... AFFAIRS Proposed Information Collection (VA Request for Determination of Reasonable Value) Activity... solicits comments for information needed to determine the reasonable value of properties for guaranteed or... Request for Determination of Reasonable Value, VA Form 26-1805 and 26-1805-1. OMB Control Number: 2900...

  16. 75 FR 20774 - Establishment of Class E Airspace; Fort A.P. Hill, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-21

    ... 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 Date..., Eastern Service Center, Federal Aviation Administration, P.O. Box 20636, Atlanta, Georgia 30320;...

  17. 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 technology...

  18. 76 FR 70831 - Proposed Information Collection (Survey of Veteran Enrollees (Quality and Efficiency of VA Health...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-15

    ... Care)) Activity; Comment Request AGENCY: Veterans Health Administration, Department of Veterans Affairs... Efficiency of VA Health Care), VA Form 10-21088. OMB Control Number: 2900-0725. Type of Review: Extension of... necessary to promote quality and efficient delivery of health care through the use of health information...

  19. 48 CFR 852.219-9 - VA Small business subcontracting plan minimum requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 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 Requirements... plan, the minimum goals for award of subcontracts to service-disabled veteran-owned small...

  20. 48 CFR 852.219-9 - VA Small business subcontracting plan minimum requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 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 Requirements... plan, the minimum goals for award of subcontracts to service-disabled veteran-owned small...

  1. VA/DOD Federal Health Care Center: Costly Information Technology Delays Continue and Evaluation Plan Lacking

    DTIC Science & Technology

    2012-06-01

    portability, which would allow VA and DOD clinicians to place, manage, and update clinical orders from either VA or DOD electronic health records systems...on, which includes five dedicated, full-time pharmacists to conduct manual checks of patient records to reconcile allergy information and identify

  2. 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…

  3. 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

    ... 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 has specifically contracted, this rulemaking will only affect home health and hospice care providers who do...

  4. 76 FR 71920 - Payment for Home Health Services and Hospice Care by Non-VA Providers

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-21

    ... care and home health aide services. In those states that reimburse separately for homemaker services... AFFAIRS 38 CFR Part 17 RIN 2900-AN98 Payment for Home Health Services and Hospice Care by Non-VA Providers... methodology for non-VA providers of home health services and hospice care. The proposed rulemaking would...

  5. 75 FR 17832 - Proposed Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-07

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF VETERANS AFFAIRS Proposed Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity... techniques or the use of other forms of information technology. Title: VA Loan Electronic Reporting Interface...

  6. 30 CFR 57.22312 - Distribution boxes (II-A and V-A mines).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Distribution boxes (II-A and V-A mines). 57... MINES Safety Standards for Methane in Metal and Nonmetal Mines Equipment § 57.22312 Distribution boxes (II-A and V-A mines). Distribution boxes containing short circuit protection for trailing cables...

  7. VA AND DoD Health Care: Factors Contributing to Reduced Pharmacy Costs and Continuing Challenges

    DTIC Science & Technology

    2007-11-02

    statement. Concluding Observations Contacts and Acknowledgments Page 11 GAO-02-969T VA Health Care: Implementation of Prescribing Guideline for Atypical ... Antipsychotic Drugs Generally Sound. GAO-02-579. Washington D.C.: April 29, 2002. VA Health Care: Continuing Oversight Needed to Achieve Formulary

  8. 38 CFR 74.26 - What types of business information will VA collect?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... VETERANS AFFAIRS (CONTINUED) VETERANS SMALL BUSINESS REGULATIONS Records Management § 74.26 What types of business information will VA collect? VA will examine a variety of business records. See § 74.12, “What is... 38 Pensions, Bonuses, and Veterans' Relief 2 2012-07-01 2012-07-01 false What types of business...

  9. 38 CFR 74.26 - What types of business information will VA collect?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... VETERANS AFFAIRS (CONTINUED) VETERANS SMALL BUSINESS REGULATIONS Records Management § 74.26 What types of business information will VA collect? VA will examine a variety of business records. See § 74.12, “What is... 38 Pensions, Bonuses, and Veterans' Relief 2 2013-07-01 2013-07-01 false What types of business...

  10. 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 to...

  11. 33 CFR 80.515 - Cape Henry, VA to Cape Hatteras, NC.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 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 to...

  12. 33 CFR 80.515 - Cape Henry, VA to Cape Hatteras, NC.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 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 to...

  13. 33 CFR 80.515 - Cape Henry, VA to Cape Hatteras, NC.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 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 to...

  14. 33 CFR 80.515 - Cape Henry, VA to Cape Hatteras, NC.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 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 to...

  15. 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…

  16. 76 FR 3017 - VA Veteran-Owned Small Business Verification Guidelines

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-19

    ... to one business has no foundation in law and that there is no compelling reason to limit... AFFAIRS 38 CFR Part 74 RIN 2900-AM78 VA Veteran-Owned Small Business Verification Guidelines AGENCY..., and Information Technology Act of 2006. This law requires the Department of Veterans Affairs (VA)...

  17. 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...

  18. 38 CFR 74.25 - What types of personally identifiable information will VA collect?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... identifiable information will VA collect? 74.25 Section 74.25 Pensions, Bonuses, and Veterans' Relief... What types of personally identifiable information will VA collect? In order to establish owner eligibility, the Department will collect individual names and Social Security numbers for veterans,...

  19. VA Health Care: Processes to Evaluate, Implement, and Monitor Organizational Structure Changes Needed

    DTIC Science & Technology

    2016-09-01

    VA HEALTH CARE Processes to Evaluate, Implement, and Monitor Organizational Structure Changes Needed Report to...Accountability Office Highlights of GAO-16-803, a report to congressional requesters September 2016 VA HEALTH CARE Processes to Evaluate, Implement, and...Monitor Organizational Structure Changes Needed What GAO Found Recent internal and external reviews of Veterans Health Administration (VHA

  20. 77 FR 38181 - VA Veteran-Owned Small Business Verification Guidelines

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-27

    ... AFFAIRS 38 CFR Part 74 RIN 2900-AO49 VA Veteran-Owned Small Business Verification Guidelines AGENCY... Department of Veterans Affairs (VA) to verify ownership and control of veteran-owned small businesses (VOSBs), including service-disabled veteran-owned small businesses (SDVOSBs) in order for these firms to...

  1. 33 CFR 334.350 - Chesapeake Bay off Fort Monroe, Va.; firing range danger zone.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 3 2010-07-01 2010-07-01 false Chesapeake Bay off Fort Monroe, Va.; firing range danger zone. 334.350 Section 334.350 Navigation and Navigable Waters CORPS OF....350 Chesapeake Bay off Fort Monroe, Va.; firing range danger zone. (a) The danger zone. All of the...

  2. 33 CFR 334.220 - Chesapeake Bay, south of Tangier Island, Va.; naval firing range.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 3 2010-07-01 2010-07-01 false Chesapeake Bay, south of Tangier 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...

  3. 33 CFR 334.220 - Chesapeake Bay, south of Tangier Island, Va.; naval firing range.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 3 2014-07-01 2014-07-01 false Chesapeake Bay, south of Tangier 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...

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

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 3 2011-07-01 2011-07-01 false Chesapeake Bay, south of Tangier 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...

  5. 33 CFR 334.350 - Chesapeake Bay off Fort Monroe, Va.; firing range danger zone.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 3 2014-07-01 2014-07-01 false Chesapeake Bay off Fort Monroe, Va.; firing range danger zone. 334.350 Section 334.350 Navigation and Navigable Waters CORPS OF....350 Chesapeake Bay off Fort Monroe, Va.; firing range danger zone. (a) The danger zone. All of the...

  6. 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 836...

  7. 76 FR 26607 - Safety Zone; Air Power Over Hampton Roads, Back River, Hampton, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-09

    ..., Hampton, VA AGENCY: Coast Guard, DHS. ACTION: Temporary final rule. SUMMARY: The Coast Guard is establishing a temporary safety zone on the navigable waters of the Back River in the vicinity of Hampton, VA... Friday, except Federal holidays. FOR FURTHER INFORMATION CONTACT: If you have questions on this temporary...

  8. 38 CFR 3.1701 - Deceased veterans for whom VA may provide burial benefits.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... whom VA may provide burial benefits. 3.1701 Section 3.1701 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS ADJUDICATION Burial Benefits Burial Benefits: General § 3.1701 Deceased veterans for whom VA may provide burial benefits. For purposes of providing burial benefits under subpart...

  9. 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...

  10. Myosin-Va binds to and mechanochemically couples microtubules to actin filaments.

    PubMed

    Cao, Tracy T; Chang, Wakam; Masters, Sarah E; Mooseker, Mark S

    2004-01-01

    Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (approximately 1:24 Myosin-Va:tubulin; Kd = 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca2+, ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 microM Ca2+, ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments.

  11. 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 notificatio...

  12. 48 CFR 819.602-3 - Resolving differences between VA and the Small Business Administration.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... between VA and the Small Business Administration. 819.602-3 Section 819.602-3 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS SOCIOECONOMIC PROGRAMS SMALL BUSINESS PROGRAMS Certificates... Small Business Administration. The Director, OSDBU, is the VA liaison with the SBA. Information...

  13. Expanded Access to Non-VA Care Through the Veterans Choice Program. Interim final rule.

    PubMed

    2015-12-01

    The Department of Veterans Affairs (VA) revises its medical regulations that implement section 101 of the Veterans Access, Choice, and Accountability Act of 2014 (hereafter referred to as "the Choice Act"), which requires VA to establish a program to furnish hospital care and medical services through eligible non-VA health care providers to eligible veterans who either cannot be seen within the wait-time goals of the Veterans Health Administration (VHA) or who qualify based on their place of residence (hereafter referred to as the "Veterans Choice Program" or the "Program"). These regulatory revisions are required by the most recent amendments to the Choice Act made by the Construction Authorization and Choice Improvement Act of 2014, and by the Surface Transportation and Veterans Health Care Choice Improvement Act of 2015. The Construction Authorization and Choice Improvement Act of 2014 amended the Choice Act to define additional criteria that VA may use to determine that a veteran's travel to a VA medical facility is an "unusual or excessive burden," and the Surface Transportation and Veterans Health Care Choice Improvement Act of 2015 amended the Choice Act to cover all veterans enrolled in the VA health care system, remove the 60-day limit on an episode of care, modify the wait-time and 40-mile distance eligibility criteria, and expand provider eligibility based on criteria as determined by VA. This interim final rule revises VA regulations consistent with the changes made to the Choice Act as described above.

  14. 38 CFR 74.26 - What types of business information will VA collect?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... VETERANS AFFAIRS (CONTINUED) VETERANS SMALL BUSINESS REGULATIONS Records Management § 74.26 What types of business information will VA collect? VA will examine a variety of business records. See § 74.12, “What is... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false What types of...

  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... 38 Pensions, Bonuses, and Veterans' Relief 2 2013-07-01 2013-07-01 false VA environmental decision...

  16. 38 CFR 26.7 - VA environmental decision making and documents.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 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... 38 Pensions, Bonuses, and Veterans' Relief 2 2014-07-01 2014-07-01 false VA environmental decision...

  17. 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... 38 Pensions, Bonuses, and Veterans' Relief 2 2012-07-01 2012-07-01 false VA environmental decision...

  18. 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... 38 Pensions, Bonuses, and Veterans' Relief 2 2011-07-01 2011-07-01 false VA environmental decision...

  19. 78 FR 60379 - Proposed Information Collection (Credit Underwriting Standards and Procedures for Processing VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-01

    ... specific information concerning a Veteran's credit history in order to properly underwrite the veteran's loan. VA loans may not be guaranteed unless the Veteran is a satisfactory credit risk. The data... AFFAIRS Proposed Information Collection (Credit Underwriting Standards and Procedures for Processing VA...

  20. 75 FR 76082 - Agency Information Collection (Credit Underwriting Standards and Procedures for Processing VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-07

    ... a veteran's credit history in order to properly underwrite the veteran's loan. VA loans may not be guaranteed unless the veteran is a satisfactory credit risk. The data collected on the following forms are... AFFAIRS Agency Information Collection (Credit Underwriting Standards and Procedures for Processing VA...

  1. 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.

  2. 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.

  3. Visualized detection of single-base difference in multiplexed loop-mediated isothermal amplification amplicons by invasive reaction coupled with oligonucleotide probe-modified gold nanoparticles.

    PubMed

    Lu, Yan; Ma, Xueping; Wang, Jianping; Sheng, Nan; Dong, Tianhui; Song, Qinxin; Rui, Jianzhong; Zou, Bingjie; Zhou, Guohua

    2017-04-15

    Loop-mediated isothermal amplification (LAMP) is a well-developed DNA amplification method with an ultra-high sensitivity, but it is difficult to recognize a single-base difference (like genotyping) in target-specific amplicons by conventional detection ways, such as the intercalation of dyes into dsDNA amplicons or the increase of solution turbidity along with the polymerization process. To allow genotyping based on LAMP suitable for POCT (point-of-care testing) or on-site testing, here we proposed a highly specific and cost-effective method for detecting a single-base difference in LAMP amplicons. The method includes three key steps, sequence amplifier to amplify multiple fragments containing the single nucleotide polymorphisms (SNPs) of interest, allele identifier to recognize a targeted base in the amplicons by invasive reaction, and signal generator to yield signals by hybridization-induced assembly of oligonucleotide probe-modified gold nanoparticles. Because the allele identifier is sensitive to one base difference, it is possible to use multiplexed LAMP (mLAMP) to generate amplicon mixtures for multiple SNP typing. Genotyping of 3 different SNPs (CYP2C19*2, CYP2C19*3 and MDR1-C3435T) for guiding the dosage of clopidogrel is successfully carried out in a 3-plex LAMP on real clinical samples. As our method relies on the naked-eye detection and constant-temperature reaction, no expensive instrument is required for both target amplification and sequence identification, thus much suitable for inexpensive gene-guided personalized medicine in source-limited regions.

  4. Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR.

    PubMed

    Kleiboeker, Steven B

    2005-04-11

    Although PCR and RT-PCR provided a valuable approach for detection of pathogens, the high level of sensitivity of these assays also makes them prone to false positive results. In addition to cross-contamination with true positive samples, false positive results are also possible due to "carry-over" contamination of samples with amplicon DNA generated by previous reactions. To reduce this source of false positives, amplicon generated by reactions in which dUTP was substituted for dTTP can be degraded by uracil DNA glycosylase (UNG). UNG does not degrade RNA but will cleave contaminating uracil-containing DNA while leaving thymine-containing DNA intact. The availability of heat-labile UNG makes use of this approach feasible for RT-PCR. In this study, real-time RT-PCR was used to quantify UNG degradation of amplicon DNA and the effect of UNG on RNA detection. Using the manufacturers' recommended conditions, complete degradation of DNA was not observed for samples containing 250 copies of amplicon DNA. Doubling the UNG concentration resulted in degradation of the two lowest concentrations of DNA tested, but also resulted in an increase of 1.94 cycles in the CT for RNA detection. To improve DNA degradation while minimizing the effect on RNA detection, a series of time, temperature and enzyme concentrations were evaluated. Optimal conditions were found to be 0.25 U UNG per 25 microl reaction with a 20 min, 30 degrees C incubation prior to RT-PCR. Under these conditions, high concentrations of amplicon DNA could be degraded while the CT for RNA detection was increased by 1.2 cycles.

  5. 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

  6. 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

  7. The use of VA Disability Compensation and Social Security Disability Insurance among working-aged veterans.

    PubMed

    Wilmoth, Janet M; London, Andrew S; Heflin, Colleen M

    2015-07-01

    Although there is substantial disability among veterans, relatively little is known about working-aged veterans' uptake of Department of Veterans Affairs (VA) Disability Compensation and Social Security Disability Insurance (DI). This study identifies levels of veteran participation in VA disability and/or DI benefit programs, examines transitions into and out of VA and DI programs among veterans, and estimates the size and composition of the veteran population receiving VA and/or DI benefits over time. Data from the 1992, 1993, 1996, 2001, 2004, and 2008 Survey of Income and Program Participation (SIPP) are used to describe VA and DI program participation among veterans under the age of 65. The majority of working-aged veterans do not receive VA or DI benefits and joint participation is low, but use of these programs has increased over time. A higher percentage of veterans receive VA compensation, which ranges from 4.9% in 1992 to 13.2% in 2008, than DI compensation, which ranges from 2.9% in 1992 to 6.7% in 2008. The rate of joint participation ranges from less than 1% in 1992 to 3.6% in 2008. Veterans experience few transitions between VA and DI programs during the 36-48 months they are observed. The number of veterans receiving benefits from VA and/or DI nearly doubled between 1992 and 2008. There have been substantial shifts in the composition of veterans using these programs, as cohorts who served prior to 1964 are replaced by those who served after 1964. The findings suggest potential gaps in veterans' access to disability programs that might be addressed through improved coordination of VA and DI benefits. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Identifying Homelessness among Veterans Using VA Administrative Data: Opportunities to Expand Detection Criteria.

    PubMed

    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.

  9. 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

  10. [Amplicon density-weighted algorithms for analyzing dissimilarity and dynamic alterations of RAPD polymorphisms of Cordyceps sinensis].

    PubMed

    Yao, Yi-sang; Gao, Ling; Li, Yu-ling; Ma, Shao-li; Wu, Zi-mei; Tan, Ning-zhi; Wu, Jian-yong; Ni, Lu-qun; Zhu, Jia-shi

    2014-08-18

    To examine the dynamic maturational alterations of random amplified polymorphic DNA (RAPD) molecular marker polymorphism resulted from differential expressions of multiple fungi in the caterpillar body, stroma and ascocarp portion of Cordyceps sinensis (Cs). Used the fuzzy, integral RAPD molecular marker polymorphism method with 20 random primers; used density-weighted cluster algorithms and ZUNIX similarity equations; compared RAPD polymorphisms of the caterpillar body, stroma and ascocarp of Cs during maturation; and compared RAPD polymorphisms of Cs and Hirsutella sinensis (Hs). Density-unweighted algorithms neglected the differences in density of the DNA amplicons. Use of the density-weighted ZUNIX similarity equations and the clustering method integrated components of the amplicon density differences in similarity computations and clustering construction and prevented from the loss of the information of fungal genomes. An overall similarity 0.42 (< the overall dissimilarity 0.58) was observed for all compartments of Cs at different maturation stages. The similarities for the stromata or caterpillar bodies of Cs at 3 maturational stages were 0.57 or 0.50, respectively. During Cs maturation, there were dynamic Low→High→Low alterations of the RAPD polymorphisms between stromata and caterpillar bodies dissected from the same pieces of Cs. The polymorphic similarity was the highest (0.87) between the ascocarp and mature stroma, forming a clustering clade, while the premature stroma and caterpillar body formed another clade. These 2 clades merged into one cluster. Another clade containing the maturing stroma and caterpillar body merged with mature caterpillar body, forming another cluster. The RAPD polymorphic similarities between Hs and Cs samples were 0.55-0.69. Hs were separated from Cs clusters by the out-group control Paecilomyces militaris. The wealthy RAPD polymorphisms change dynamically in the Cs compartments with maturation. The different RAPD

  11. Variations of the magnetic field at CNSC, VA

    NASA Astrophysics Data System (ADS)

    Gilstrap, Tatiana; Keane, James

    2010-02-01

    The current work had the goal to map the magnetic field at Claytor Nature Study Center (CNSC) near Bedford, VA. Magnetic ground measurements of the total intensity of the magnetic field were conducted over a period of two years. The data were obtained using a Geometrics G-856 proton precession magnetometer and were interpreted using the Mag2dc algorithm. The magnetometer provides a repeatable absolute total field magnetic reading. It has resolution of 0.1 nanotesla (nT), and accuracy of 0.5 nT. Readings were taken along several survey lines. Magnetic anomalies due to metal fences, buried pipes, well casings, and power lines were eliminated. The Mag2dc algorithm calculates the magnetic anomaly over 2.5 dimensional bodies. Each body can be represented by a polygon with up to 50 sides. The magnetic susceptibility for each body is assumed to be constant. Magnetic anomalies on the order of a few hundred to over a thousand nT were observed. The results were interpolated to obtain a continuous map of the magnetic field at CNSC. )

  12. 38 CFR 17.56 - VA payment for inpatient and outpatient health care professional services at non-departmental...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... the non-VA provider may have within the commercial health care industry. (iii) The amount that the... Health Insurance Portability and Accountability Act mandated national standard coding sets. VA will pay...

  13. 38 CFR 17.56 - VA payment for inpatient and outpatient health care professional services at non-departmental...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... the non-VA provider may have within the commercial health care industry. (iii) The amount that the... Health Insurance Portability and Accountability Act mandated national standard coding sets. VA will pay...

  14. 38 CFR 17.56 - VA payment for inpatient and outpatient health care professional services at non-departmental...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... the non-VA provider may have within the commercial health care industry. (iii) The amount that the... Health Insurance Portability and Accountability Act mandated national standard coding sets. VA will pay...

  15. VA Pipeline for Future Nurse Leaders: an Exploration of Current Nurse Leadership Development in the Veteran’s Health Administration

    DTIC Science & Technology

    2016-06-10

    organization. All of our resources—high- quality and safe health care, disability compensation and pensions, education and training, home mortgages, life...VA’s Budget Submission—www.va.gov/budget/products.asp VA Directives and Handbooks—www.va.gov/vapubs/ VA Health Quality and Safety Performance...and R. Adams Dudley. 2003. “Effect of the Transformation of the Veterans Affairs Health Care System on the Quality of Care.” New England Journal

  16. Variation in Utilization of Health Care Services for Rural VA Enrollees With Mental Health-Related Diagnoses.

    PubMed

    Johnson, Christopher E; Bush, Ruth L; Harman, Jeffrey; Bolin, Jane; Evans Hudnall, Gina; Nguyen, Ann M

    2015-01-01

    Rural-dwelling Department of Veterans Affairs (VA) enrollees are at high risk for a wide variety of mental health-related disorders. The objective of this study is to examine the variation in the types of mental and nonmental health services received by rural VA enrollees who have a mental health-related diagnosis. The Andersen and Aday behavioral model of health services use and the Agency for Healthcare Research and Quality Medical Expenditure Panel Survey (MEPS) data were used to examine how VA enrollees with mental health-related diagnoses accessed places of care from 1999 to 2009. Population survey weights were applied to the MEPS data, and logit regression was conducted to model how predisposing, enabling, and need factors influence rural veteran health services use (measured by visits to different places of care). Analyses were performed on the subpopulations: rural VA, rural non-VA, urban VA, and urban non-VA enrollees. For all types of care, both rural and urban VA enrollees received care from inpatient, outpatient, office-based, and emergency room settings at higher odds than urban non-VA enrollees. Rural VA enrollees also received all types of care from inpatient, office-based, and emergency room settings at higher odds than urban VA enrollees. Rural VA enrollees had higher odds of a mental health visit of any kind compared to urban VA and non-VA enrollees. Based on these variations, the VA may want to develop strategies to increase screening efforts in inpatient settings and emergency rooms to further capture rural VA enrollees who have undiagnosed mental health conditions. © 2015 National Rural Health Association.

  17. 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.

  18. 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.

  19. 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.

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

    PubMed Central

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

    2016-01-01

    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 multicompartment 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

  1. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons

    PubMed Central

    Fischer, Sandra; Kumar, Neeraj

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea. PMID:28097056

  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.

  3. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons.

    PubMed

    Lagkouvardos, Ilias; Fischer, Sandra; Kumar, Neeraj; Clavel, Thomas

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.

  4. Amplicon-pyrosequencing-based detection of compositional shifts in bryophyte-associated fungal communities along an elevation gradient.

    PubMed

    Davey, Marie L; Heegaard, Einar; Halvorsen, Rune; Kauserud, Håvard; Ohlson, Mikael

    2013-01-01

    Although bryophytes are a dominant vegetation component of boreal and alpine ecosystems, little is known about their associated fungal communities. HPLC assays of ergosterol (fungal biomass) and amplicon pyrosequencing of the ITS2 region of rDNA were used to investigate how the fungal communities associated with four bryophyte species changed across an elevational gradient transitioning from conifer forest to the low-alpine. Fungal biomass and OTU richness associated with the four moss hosts did not vary significantly across the gradient (P > 0.05), and both were more strongly affected by host and tissue type. Despite largely constant levels of fungal biomass, distinct shifts in community composition of fungi associated with Hylocomium, Pleurozium and Polytrichum occurred between the elevation zones of the gradient. This likely is a result of influence on fungal communities by major environmental factors such as temperature, directly or indirectly mediated by, or interacting with, the response of other components of the vegetation (i.e. the dominant trees). Fungal communities associated with Dicranum were an exception, exhibiting spatial autocorrelation between plots, and no significant structuring by elevation. Nevertheless, the detection of distinct fungal assemblages associated with a single host growing in different elevation zones along an elevational gradient is of particular relevance in the light of the ongoing changes in vegetation patterns in boreal and alpine systems due to global climate warming.

  5. High-throughput amplicon sequencing and stream benthic bacteria: identifying the best taxonomic level for multiple-stressor research

    PubMed Central

    Salis, R. K.; Bruder, A.; Piggott, J. J.; Summerfield, T. C.; Matthaei, C. D.

    2017-01-01

    Disentangling the individual and interactive effects of multiple stressors on microbial communities is a key challenge to our understanding and management of ecosystems. Advances in molecular techniques allow studying microbial communities in situ and with high taxonomic resolution. However, the taxonomic level which provides the best trade-off between our ability to detect multiple-stressor effects versus the goal of studying entire communities remains unknown. We used outdoor mesocosms simulating small streams to investigate the effects of four agricultural stressors (nutrient enrichment, the nitrification inhibitor dicyandiamide (DCD), fine sediment and flow velocity reduction) on stream bacteria (phyla, orders, genera, and species represented by Operational Taxonomic Units with 97% sequence similarity). Community composition was assessed using amplicon sequencing (16S rRNA gene, V3-V4 region). DCD was the most pervasive stressor, affecting evenness and most abundant taxa, followed by sediment and flow velocity. Stressor pervasiveness was similar across taxonomic levels and lower levels did not perform better in detecting stressor effects. Community coverage decreased from 96% of all sequences for abundant phyla to 28% for species. Order-level responses were generally representative of responses of corresponding genera and species, suggesting that this level may represent the best compromise between stressor sensitivity and coverage of bacterial communities. PMID:28327636

  6. High-throughput amplicon sequencing and stream benthic bacteria: identifying the best taxonomic level for multiple-stressor research

    NASA Astrophysics Data System (ADS)

    Salis, R. K.; Bruder, A.; Piggott, J. J.; Summerfield, T. C.; Matthaei, C. D.

    2017-03-01

    Disentangling the individual and interactive effects of multiple stressors on microbial communities is a key challenge to our understanding and management of ecosystems. Advances in molecular techniques allow studying microbial communities in situ and with high taxonomic resolution. However, the taxonomic level which provides the best trade-off between our ability to detect multiple-stressor effects versus the goal of studying entire communities remains unknown. We used outdoor mesocosms simulating small streams to investigate the effects of four agricultural stressors (nutrient enrichment, the nitrification inhibitor dicyandiamide (DCD), fine sediment and flow velocity reduction) on stream bacteria (phyla, orders, genera, and species represented by Operational Taxonomic Units with 97% sequence similarity). Community composition was assessed using amplicon sequencing (16S rRNA gene, V3-V4 region). DCD was the most pervasive stressor, affecting evenness and most abundant taxa, followed by sediment and flow velocity. Stressor pervasiveness was similar across taxonomic levels and lower levels did not perform better in detecting stressor effects. Community coverage decreased from 96% of all sequences for abundant phyla to 28% for species. Order-level responses were generally representative of responses of corresponding genera and species, suggesting that this level may represent the best compromise between stressor sensitivity and coverage of bacterial communities.

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

    PubMed Central

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

    2015-01-01

    ABSTRACT 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

  8. Molecular Epidemiology of Plasmodium falciparum kelch13 Mutations in Senegal Determined by Using Targeted Amplicon Deep Sequencing.

    PubMed

    Talundzic, Eldin; Ndiaye, Yaye D; Deme, Awa B; Olsen, Christian; Patel, Dhruviben S; Biliya, Shweta; Daniels, Rachel; Vannberg, Fredrik O; Volkman, Sarah K; Udhayakumar, Venkatachalam; Ndiaye, Daouda

    2017-03-01

    The emergence of Plasmodium falciparum resistance to artemisinin in Southeast Asia threatens malaria control and elimination activities worldwide. Multiple polymorphisms in the P. falciparum kelch gene found in chromosome 13 (Pfk13) have been associated with artemisinin resistance. Surveillance of potential drug resistance loci within a population that may emerge under increasing drug pressure is an important public health activity. In this context, P. falciparum infections from an observational surveillance study in Senegal were genotyped using targeted amplicon deep sequencing (TADS) for Pfk13 polymorphisms. The results were compared to previously reported Pfk13 polymorphisms from around the world. A total of 22 Pfk13 propeller domain polymorphisms were identified in this study, of which 12 have previously not been reported. Interestingly, of the 10 polymorphisms identified in the present study that were also previously reported, all had a different amino acid substitution at these codon positions. Most of the polymorphisms were present at low frequencies and were confined to single isolates, suggesting they are likely transient polymorphisms that are part of naturally evolving parasite populations. The results of this study underscore the need to identify potential drug resistance loci existing within a population, which may emerge under increasing drug pressure.

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

    PubMed

    Stiebens, Victor A; Merino, Sonia E; Chain, Frédéric J J; Eizaguirre, Christophe

    2013-04-30

    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. 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. 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. Multiple modes of evolution seem to maintain MHC diversity in the loggerhead turtles, with relatively high polymorphism for an endangered species.

  10. A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

    PubMed

    Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman

    2016-12-15

    The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. High-resolution melting analysis using unlabeled probe and amplicon scanning simultaneously detects several lactase persistence variants.

    PubMed

    Janukonyté, Jurgita; Vestergaard, Else M; Ladefoged, Søren A; Nissen, Peter H

    2010-12-01

    Lactase persistence and thereby tolerance to lactose is a common trait in people of Northern European descent. It is linked to the LCT -13910C>T variant located in intron 13 of the MCM6 gene 13.9 kb upstream of the lactase (LCT) gene. In people of African and Middle Eastern descent, lactase persistence can be associated with other variants nearby the -13910C>T variant, limiting the use of the -13910C>T-based SNP analysis, e.g. TaqMan assays for the diagnosis of lactose intolerance. Using high-resolution melting analysis, we identified five samples that were heterozygous for the -13915T>G variant among 78 patients genotyped as -13910C/C by a TaqMan assay. All samples originated from patients of probable Middle Eastern descent. In order to detect the -13910 and -13915 variants simultaneously, we developed a new high-resolution melting (HRM) analysis assay based on unlabeled probe genotyping and simultaneous amplicon scanning analysis. By using this assay we were able to distinguish the -13910 and -13915 genotypes clearly. Furthermore, we identified two rare variants, the -13907C>G and -13913T>C. With this method, based on an inexpensive unlabeled probe, it is possible to simultaneously detect the -13910C>T and -13915T>G variants in addition to rarer variants surrounding the -13910 site. This new method may contribute to improve the diagnostic performance of the genetic analysis for lactose intolerance.

  12. 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.

  13. The molecular motor Myosin Va interacts with the cilia-centrosomal protein RPGRIP1L

    PubMed Central

    Assis, L. H. P.; Silva-Junior, R. M. P.; Dolce, L. G.; Alborghetti, M. R.; Honorato, R. V.; Nascimento, A. F. Z.; Melo-Hanchuk, T. D.; Trindade, D. M.; Tonoli, C. C. C.; Santos, C. T.; Oliveira, P. S. L.; Larson, R. E.; Kobarg, J.; Espreafico, E. M.; Giuseppe, P. O.; Murakami, M. T.

    2017-01-01

    Myosin Va (MyoVa) is an actin-based molecular motor abundantly found at the centrosome. However, the role of MyoVa at this organelle has been elusive due to the lack of evidence on interacting partners or functional data. Herein, we combined yeast two-hybrid screen, biochemical studies and cellular assays to demonstrate that MyoVa interacts with RPGRIP1L, a cilia-centrosomal protein that controls ciliary signaling and positioning. MyoVa binds to the C2 domains of RPGRIP1L via residues located near or in the Rab11a-binding site, a conserved site in the globular tail domain (GTD) from class V myosins. According to proximity ligation assays, MyoVa and RPGRIP1L can interact near the cilium base in ciliated RPE cells. Furthermore, we showed that RPE cells expressing dominant-negative constructs of MyoVa are mostly unciliated, providing the first experimental evidence about a possible link between this molecular motor and cilia-related processes. PMID:28266547

  14. 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.

  15. Methods for estimating and comparing VA outpatient drug benefits with the private sector.

    PubMed

    Render, Marta L; Nowak, John; Hammond, Emmett K; Roselle, Gary

    2003-06-01

    To estimate and compare Veterans Health Administration (VA) expenditures for outpatient pharmaceuticals for veterans at six VA facilities with hypothetical private sector costs. Using the VA Pharmacy Benefits Management Strategic Health Care Group (PBM) database, we extracted data for all dispensed outpatient prescriptions from the six study sites over federal fiscal year 1999. After extensive data validation, we converted prescriptions to the same units and merged relevant VA pricing information by National Drug Code to Redbook listed average wholesale price and the Medicaid maximal allowable charge, where available. We added total VA drug expenditures to personnel cost from the pharmacy portion of that medical center's cost distribution report. Hypothetical private sector payments were $200.8 million compared with an aggregate VA budget of $118.8 million. Using National Drug Code numbers, 97% of all items dispensed from the six facilities were matched to private sector price data. Nonmatched pharmaceuticals were largely generic over-the-counter pain relievers and commodities like alcohol swabs. The most commonly prescribed medications reflect the diseases and complaints of an older male population: pain, cardiovascular problems, diabetes, and depression or other psychiatric disorders. Use of the VA PBM database permits researchers to merge expenditure and prescription data to patient diagnoses and sentinel events. A critical element in its use is creating similar units among the systems. Such data sets permit a deeper view of the variability in drug expenditures, an important sector of health care whose inflation has been disproportionate to that of the economy and even health care.

  16. 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

  17. Myosin Va Is Required for P Body but Not Stress Granule Formation*

    PubMed Central

    Lindsay, Andrew J.; McCaffrey, Mary W.

    2011-01-01

    In the present study we demonstrate an association between mammalian myosin Va and cytoplasmic P bodies, microscopic ribonucleoprotein granules that contain components of the 5′–3′ mRNA degradation machinery. Myosin Va colocalizes with several P body markers and its RNAi-mediated knockdown results in the disassembly of P bodies. Overexpression of a dominant-negative mutant of myosin Va reduced the motility of P bodies in living cells. Co-immunoprecipitation experiments demonstrate that myosin Va physically associates with eIF4E, an mRNA binding protein that localizes to P bodies. In contrast, we find that myosin Va does not play a role in stress granule formation. Stress granules are ribonucleoprotein structures that are involved in translational silencing and are spatially, functionally, and compositionally linked to P bodies. Myosin Va is found adjacent to stress granules in stressed cells but displays minimal localization within stress granules, and myosin Va knockdown has no effect on stress granule assembly or disassembly. Combined with recently published reports demonstrating a role for Drosophila and mammalian class V myosins in mRNA transport and the involvement of the yeast myosin V orthologue Myo2p in P body assembly, our results provide further evidence that the class V myosins serve an important role in the transport and turnover of mRNA. PMID:21245139

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. Neuroimaging overuse is more common in Medicare compared with the VA.

    PubMed

    Burke, James F; Kerr, Eve A; McCammon, Ryan J; Holleman, Rob; Langa, Kenneth M; Callaghan, Brian C

    2016-08-23

    To inform initiatives to reduce overuse, we compared neuroimaging appropriateness in a large Medicare cohort with a Department of Veterans Affairs (VA) cohort. Separate retrospective cohorts were established in Medicare and in VA for headache and neuropathy from 2004 to 2011. The Medicare cohorts included all patients enrolled in the Health and Retirement Study (HRS) with linked Medicare claims (HRS-Medicare; n = 1,244 for headache and 998 for neuropathy). The VA cohorts included all patients receiving services in the VA (n = 93,755 for headache and 183,642 for neuropathy). Inclusion criteria were age over 65 years and an outpatient visit for incident neuropathy or a primary headache. Neuroimaging use was measured with Current Procedural Terminology codes and potential overuse was defined using published criteria for use with administrative data. Increasingly specific appropriateness criteria excluded nontarget conditions for which neuroimaging may be appropriate. For both peripheral neuropathy and headache, potentially inappropriate imaging was more common in HRS-Medicare compared with the VA. Forty-nine percentage of all headache patients received neuroimaging in HRS-Medicare compared with 22.1% in the VA (p < 0.001) and differences persist when analyzing more specific definitions of overuse. A total of 23.7% of all HRS-Medicare incident neuropathy patients received neuroimaging compared with 9.0% in the VA (p < 0.001), and the difference persisted after excluding nontarget conditions. Overuse of neuroimaging is likely less common in the VA than in a Medicare population. Better understanding the reasons for the more selective use of neuroimaging in the VA could help inform future initiatives to reduce overuse of diagnostic testing. © 2016 American Academy of Neurology.

  3. 75 FR 51331 - Draft Tier II Environmental Impact Statement: Southeast High Speed Rail Corridor-Richmond, VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-19

    ... Corridor--Richmond, VA (Main Street Station) to Raleigh, NC (Boylan Wye) AGENCY: Federal Railroad... the Tier II Draft Environmental Impact Statement for the Southeast High Speed Rail, Richmond, VA to... the Draft Tier II Environmental Impact Statement for the Southeast High Speed Rail, Richmond, VA...

  4. 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%…

  5. 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

    ... 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: 2900-0427. Type of Review: Extension of a currently approved collection. Abstract: VA Form 10-0048 is completed...

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  11. 38 CFR 17.1000 - Payment or reimbursement for emergency services for nonservice-connected conditions in non-VA...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... for emergency services for nonservice-connected conditions in non-VA facilities. 17.1000 Section 17... Reimbursement for Emergency Services for Nonservice-Connected Conditions in Non-Va Facilities § 17.1000 Payment or reimbursement for emergency services for nonservice-connected conditions in non-VA...

  12. 38 CFR 17.1000 - Payment or reimbursement for emergency services for nonservice-connected conditions in non-VA...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... for emergency services for nonservice-connected conditions in non-VA facilities. 17.1000 Section 17... Reimbursement for Emergency Services for Nonservice-Connected Conditions in Non-Va Facilities § 17.1000 Payment or reimbursement for emergency services for nonservice-connected conditions in non-VA...

  13. 38 CFR 17.1000 - Payment or reimbursement for emergency services for nonservice-connected conditions in non-VA...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... for emergency services for nonservice-connected conditions in non-VA facilities. 17.1000 Section 17... Reimbursement for Emergency Services for Nonservice-Connected Conditions in Non-Va Facilities § 17.1000 Payment or reimbursement for emergency services for nonservice-connected conditions in non-VA...

  14. 38 CFR 17.1000 - Payment or reimbursement for emergency services for nonservice-connected conditions in non-VA...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... for emergency services for nonservice-connected conditions in non-VA facilities. 17.1000 Section 17... Reimbursement for Emergency Services for Nonservice-Connected Conditions in Non-Va Facilities § 17.1000 Payment or reimbursement for emergency services for nonservice-connected conditions in non-VA...

  15. Molecular genetic dissection of mouse unconventional myosin-VA: tail region mutations.

    PubMed Central

    Huang, J D; Mermall, V; Strobel, M C; Russell, L B; Mooseker, M S; Copeland, N G; Jenkins, N A

    1998-01-01

    We used an RT-PCR-based sequencing approach to identify the mutations responsible for 17 viable dilute alleles, a mouse-coat-color locus encoding unconventional myosin-VA. Ten of the mutations mapped to the MyoVA tail and are reported here. These mutations represent the first extensive collection of tail mutations reported for any unconventional mammalian myosin. They identify sequences important for tail function and identify domains potentially involved in cargo binding and/or proper folding of the MyoVA tail. Our results also provide support for the notion that different myosin tail isoforms produced by alternative splicing encode important cell-type-specific functions. PMID:9560409

  16. A quantitative and qualitative comparison of illumina MiSeq and 454 amplicon sequencing for genotyping the highly polymorphic major histocompatibility complex (MHC) in a non-model species.

    PubMed

    Razali, Haslina; O'Connor, Emily; Drews, Anna; Burke, Terry; Westerdahl, Helena

    2017-07-28

    High-throughput sequencing enables high-resolution genotyping of extremely duplicated genes. 454 amplicon sequencing (454) has become the standard technique for genotyping the major histocompatibility complex (MHC) genes in non-model organisms. However, illumina MiSeq amplicon sequencing (MiSeq), which offers a much higher read depth, is now superseding 454. The aim of this study was to quantitatively and qualitatively evaluate the performance of MiSeq in relation to 454 for genotyping MHC class I alleles using a house sparrow (Passer domesticus) dataset with pedigree information. House sparrows provide a good study system for this comparison as their MHC class I genes have been studied previously and, consequently, we had prior expectations concerning the number of alleles per individual. We found that 454 and MiSeq performed equally well in genotyping amplicons with low diversity, i.e. amplicons from individuals that had fewer than 6 alleles. Although there was a higher rate of failure in the 454 dataset in resolving amplicons with higher diversity (6-9 alleles), the same genotypes were identified by both 454 and MiSeq in 98% of cases. We conclude that low diversity amplicons are equally well genotyped using either 454 or MiSeq, but the higher coverage afforded by MiSeq can lead to this approach outperforming 454 in amplicons with higher diversity.

  17. Combined amplicon pyrosequencing assays reveal presence of the apicomplexan "type-N" (cf. Gemmocystis cylindrus) and Chromera velia on the Great Barrier Reef, Australia.

    PubMed

    Slapeta, Jan; Linares, Marjorie C

    2013-01-01

    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. 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. 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 important not only to understand the evolution of these

  18. 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

  19. 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

    2015-02-03

    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.

  20. 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

  1. 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 Fry...

  2. 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...

  3. 75 FR 3541 - Agency Information Collection (Verification of VA Benefits) Activity Under OMB Review

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-21

    ... AFFAIRS Agency Information Collection (Verification of VA Benefits) Activity Under OMB Review AGENCY: Veterans Benefits Administration, Department of Veterans Affairs. ACTION: Notice. SUMMARY: In compliance... Veterans Benefits Administration, Department of Veterans Affairs will submit the collection of information...

  4. 76 FR 40453 - Agency Information Collection (Application for VA Education Benefits) Activity Under OMB Review

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-08

    ... AFFAIRS Agency Information Collection (Application for VA Education Benefits) Activity Under OMB Review AGENCY: Veterans Benefits Administration, Department of Veterans Affairs. ACTION: Notice. SUMMARY: In... the Veterans Benefits Administration (VBA), Department of Veterans Affairs, will submit the collection...

  5. 76 FR 24570 - Proposed Information Collection (Application for VA Education Benefits) Activity; Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-02

    ... Entitlement (TOE), Basic Educational Assistance Under the Montgomery GI Bill, VA Form 22-1990E. c. Application... claimants who wish to transfer his or her Montgomery GI Bill entitlement their dependents. Affected Public...

  6. 76 FR 67561 - Proposed Information Collection (VA Enrollment Certification) Activity: Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-01

    ...: Veterans Benefits Administration, Department of Veterans Affairs. ACTION: Notice. SUMMARY: The Veterans Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public... programs of education. DATES: Written comments and recommendations on the proposed collection of...

  7. 38 CFR 17.96 - Medication prescribed by non-VA physicians.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... and medicines ordered by a private or non-Department of Veterans Affairs doctor of medicine or doctor... contract with VA for filling prescriptions for patients in state homes, provided: (a) The prescription...

  8. 38 CFR 17.96 - Medication prescribed by non-VA physicians.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... and medicines ordered by a private or non-Department of Veterans Affairs doctor of medicine or doctor... contract with VA for filling prescriptions for patients in state homes, provided: (a) The prescription...

  9. 38 CFR 17.96 - Medication prescribed by non-VA physicians.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... and medicines ordered by a private or non-Department of Veterans Affairs doctor of medicine or doctor... contract with VA for filling prescriptions for patients in state homes, provided: (a) The prescription...

  10. 38 CFR 17.96 - Medication prescribed by non-VA physicians.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... and medicines ordered by a private or non-Department of Veterans Affairs doctor of medicine or doctor... contract with VA for filling prescriptions for patients in state homes, provided: (a) The prescription...

  11. 75 FR 52989 - Specialty Minerals, Inc., Franklin, VA; Notice of Negative Determination Regarding Application...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-30

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF LABOR Employment and Training Administration Specialty Minerals, Inc., Franklin, VA; Notice of Negative Determination Regarding Application for Reconsideration By applications dated July 9, 2010 and July 16,...

  12. AdvanSix Resins & Chemicals LLC – Chester, VA VAD023690183

    EPA Pesticide Factsheets

    The EPA is announcing its environmental remediation under the Resource Conservation and Recovery Act, as amended (RCRA), as complete with Controls for the AdvanSix Resins & Chemicals LLC facility, Chester, VA (EPA ID: VAD023690183).

  13. 75 FR 60171 - Proposed Information Collection (Credit Underwriting Standards and Procedures for Processing VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-29

    ...-0521. Type of Review: Extension of a currently approved collection. Abstracts: Lenders must obtain... guaranteed and insured loans under the automatic or prior approval procedures. b. VA Form 26-8497 is used by...

  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. 78 FR 34892 - Drawbridge Operation Regulation; Elizabeth River, Eastern Branch, Norfolk, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-11

    ..., VA. This deviation is necessary to facilitate thermite welding on rail joints on the Norfolk Southern... 33 CFR 117.5 to facilitate thermite welding on the rails. Under the regular operating schedule,...

  16. 75 FR 54003 - Imperial Savings and Loan Association, Martinsville, VA; Notice of Appointment of Receiver

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-02

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF THE TREASURY Office of Thrift Supervision Imperial Savings and Loan Association, Martinsville, VA; Notice of... Insurance Corporation as sole Receiver for Imperial Savings and Loan Association, Martinsville, Virginia...

  17. 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... providers who provide temporary residential assistance for homeless persons with serious mental illness,...

  18. 38 CFR 21.1031 - VA responsibilities when a claim is filed.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... VETERANS AFFAIRS (CONTINUED) VOCATIONAL REHABILITATION AND EDUCATION Claims and Applications for... any supporting evidence required upon receipt of an informal claim. (Authority: 38 U.S.C. 5102) (b) VA...

  19. 75 FR 77664 - Vaughan Furniture Company, Galax, VA; Notice of Affirmative Determination Regarding Application...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-13

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF LABOR Employment and Training Administration Vaughan Furniture Company, Galax, VA; Notice of Affirmative Determination Regarding Application for Reconsideration By application dated November 4, 2010, a...

  20. 78 FR 33223 - Drawbridge Operation Regulation; York River, Between Yorktown and Gloucester Point, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-04

    ... Gloucester Point and Yorktown, VA. This deviation is necessary to facilitate electrical motor maintenance on... operating regulations set out in 33 CFR 117.1025, to facilitate electric motor maintenance on the structure...

  1. 38 CFR 74.29 - When will VA dispose of records?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... (CONTINUED) VETERANS SMALL BUSINESS REGULATIONS Records Management § 74.29 When will VA dispose of records? The records, including those pertaining to businesses not determined to be eligible for the program...

  2. EPA brownfields grants support jobs, property assessments in Huntington and 4 southern W. Va. counties

    EPA Pesticide Factsheets

    HUNTINGTON, W. VA. (July 7, 2015) -- The U.S. Environmental Protection Agency today announced $592,300 in brownfields grants that will provide job training and environmental property assessments in Huntington and southern West Virginia.

  3. Molecular genetic dissection of mouse unconventional myosin-VA: head region mutations.

    PubMed Central

    Huang, J D; Cope, M J; Mermall, V; Strobel, M C; Kendrick-Jones, J; Russell, L B; Mooseker, M S; Copeland, N G; Jenkins, N A

    1998-01-01

    The mouse dilute (d) locus encodes unconventional myosin-VA (MyoVA). Mice carrying null alleles of dilute have a lightened coat color and die from a neurological disorder resembling ataxia and opisthotonus within three weeks of birth. Immunological and ultrastructural studies suggest that MyoVA is involved in the transport of melanosomes in melanocytes and smooth endoplasmic reticulum in cerebellar Purkinje cells. In studies described here, we have used an RT-PCR-based sequencing approach to identify the mutations responsible for 17 viable dilute alleles that vary in their effects on coat color and the nervous system. Seven of these mutations mapped to the MyoVA motor domain and are reported here. Crystallographic modeling and mutant expression studies were used to predict how these mutations might affect motor domain function and to attempt to correlate these effects with the mutant phenotype. PMID:9560408

  4. 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

    ... Patriotic Festival Air Show. This action is intended to restrict vessel traffic movement in the restricted... navigable waters of the Atlantic Ocean in Virginia Beach, VA in order to restrict vessel traffic movement to...

  5. 38 CFR 17.66 - Notice of noncompliance with VA standards.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 20 calendar days after the date of VA notice of noncompliance, unless the hearing official determines that noncompliance with the standards threatens the lives of community residential care residents in...

  6. 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...

  7. 77 FR 60746 - Proposed Information Collection (VA/DOD Joint Disability Evaluation Board Claim) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-04

    ... 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 members are evaluated...

  8. 38 CFR 74.29 - When will VA dispose of records?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... (CONTINUED) VETERANS SMALL BUSINESS REGULATIONS Records Management § 74.29 When will VA dispose of records? The records, including those pertaining to businesses not determined to be eligible for the...

  9. 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

  10. 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

  11. Integration of active human beta-galactosidase gene (100 kb) into genome using HSV/AAV amplicon vector.

    PubMed

    Oehmig, A; Cortés, M L; Perry, K F; Sena-Esteves, M; Fraefel, Cornel; Breakefield, X O

    2007-07-01

    Vectors based on herpes simplex virus type-1 (HSV-1) permit delivery of transgenes of up to 150 kb, while the inverted terminal repeats and Rep of the adeno-associated virus (AAV) can confer site-specific integration into the AAVS1 site, which allows sustained expression of a transgene. In this study, combination of the viral elements in HSV/AAV hybrid vectors has been applied for the infectious transfer of the human lysosomal beta-galactosidase (BGAL) gene of 100 kb. Temporary expression and functional activity of beta-galactosidase (beta-gal) could be detected in human beta-gal-deficient patient and glioblastoma (Gli36) cells upon infection with the basic BGAL amplicon vector. Sustained expression of beta-gal was achieved in Gli36 cells infected with rep-plus, but not rep-minus, HSV/AAV hybrid vectors. None of five clones isolated after rep-minus hybrid vector infection showed elevated beta-gal activity or site-specific integration. In contrast, 80% of the rep-plus clones possessed beta-gal activity at least twofold greater than normal levels for up to 4 months of continuous growth, and 33% of the clones exhibited AAVS1-specific integration of the ITR-flanked transgene. One of the rep-plus clones displayed integration of the ITR cassette only at the AAVS1 site, with no sequences outside the cassette detectable and beta-gal activity fourfold above normal levels. These data demonstrate AAVS1-specific integration of an entire genomic locus and expression of the transgene from the endogenous promoter mediated by an HSV/AAV hybrid vector.

  12. 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

  13. Use of propidium monoazide and increased amplicon length reduce false-positive signals in quantitative PCR for bioburden analysis.

    PubMed

    Schnetzinger, Franz; Pan, Youwen; Nocker, Andreas

    2013-03-01

    Rapid microbiological methods (RMMs) as an alternative to conventional cultivation-based bioburden analysis are receiving increasing attention although no single technology is currently able to satisfy the needs of the health care industry. Among the RMMs, quantitative PCR (qPCR) seems particularly suited. Its implementation is, however, hampered by false-positive signals originating from free DNA in PCR reagents or from dead cells in the samples to be analysed. In this study, we assessed the capability of propidium monoazide (PMA) to inactivate exogenous DNA in PCR reagents and thus to minimise its impact in bioburden analysis. PMA is a membrane-impermeant dye that intercalates into DNA and covalently binds to it upon photoactivation leading to strong inhibition of PCR amplification. PMA is currently used mainly for treatment of microbiological samples to exclude signals from membrane-compromised cells, but is also very useful for suppression of exogenous DNA signals. In addition to testing the effect of different PMA concentrations on non-template controls and target DNA, we demonstrate the effect of amplicon length on the exclusion of background amplification. Targeting a 1,108-bp 16S rRNA gene fragment using universal bacterial primers and PCR reagents treated with 5 μM PMA resulted in complete suppression of signals from exogenous DNA within 50 cycles of amplification, while a limit of detection of 10 copies of Escherichia coli genomic DNA per PCR reaction was achieved. A combined PMA treatment of sample and PCR reagents furthermore improved the selective detection of live cells making this method appear a highly attractive RMM.

  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. Quantifying sequence proportions in a DNA-based diet study using Ion Torrent amplicon sequencing: which counts count?

    PubMed

    Deagle, Bruce E; Thomas, Austen C; Shaffer, Amanda K; Trites, Andrew W; Jarman, Simon N

    2013-07-01

    A goal of many environmental DNA barcoding studies is to infer quantitative information about relative abundances of different taxa based on sequence read proportions generated by high-throughput sequencing. However, potential biases associated with this approach are only beginning to be examined. We sequenced DNA amplified from faeces (scats) of captive harbour seals (Phoca vitulina) to investigate whether sequence counts could be used to quantify the seals' diet. Seals were fed fish in fixed proportions, a chordate-specific mitochondrial 16S marker was amplified from scat DNA and amplicons sequenced using an Ion Torrent PGM™. For a given set of bioinformatic parameters, there was generally low variability between scat samples in proportions of prey species sequences recovered. However, proportions varied substantially depending on sequencing direction, level of quality filtering (due to differences in sequence quality between species) and minimum read length considered. Short primer tags used to identify individual samples also influenced species proportions. In addition, there were complex interactions between factors; for example, the effect of quality filtering was influenced by the primer tag and sequencing direction. Resequencing of a subset of samples revealed some, but not all, biases were consistent between runs. Less stringent data filtering (based on quality scores or read length) generally produced more consistent proportional data, but overall proportions of sequences were very different than dietary mass proportions, indicating additional technical or biological biases are present. Our findings highlight that quantitative interpretations of sequence proportions generated via high-throughput sequencing will require careful experimental design and thoughtful data analysis. © 2013 John Wiley & Sons Ltd.

  16. Amplicon-based profiling of bacteria in raw and secondary treated wastewater from treatment plants across Australia.

    PubMed

    Ahmed, Warish; Staley, Christopher; Sidhu, Jatinder; Sadowsky, Michael; Toze, Simon

    2017-02-01

    In this study, we investigated the use of Illumina high-throughput sequencing of 16S ribosomal RNA (rRNA) amplicons to explore microbial diversity and community structure in raw and secondary treated wastewater (WW) samples from four municipal wastewater treatment plants (WWTPs A-D) across Australia. Sequence reads were analyzed to determine the abundance and diversity of bacterial communities in raw and secondary treated WW samples across the four WWTPs. In addition, sequence reads were also characterized to phenotypic features and to estimate the abundance of potential pathogenic bacterial genera and antibiotic-resistant genes in total bacterial communities. The mean coverage, Shannon diversity index, observed richness (S obs), and abundance-based coverage estimate (ACE) of richness for raw and secondary treated WW samples did not differ significantly (P > 0.05) among the four WWTPs examined. Generally, raw and secondary treated WW samples were dominated by members of the genera Pseudomonas, Arcobacter, and Bacteroides. Evaluation of source contributions to secondary treated WW, done using SourceTracker, revealed that 8.80-61.4% of the bacterial communities in secondary treated WW samples were attributed to raw WW. Twenty-five bacterial genera were classified as containing potential bacterial pathogens. The abundance of potentially pathogenic genera in raw WW samples was higher than that found in secondary treated WW samples. Among the pathogenic genera identified, Pseudomonas and Arcobacter had the greatest percentage of the sequence reads. The abundances of antibiotic resistance genes were generally low (<0.5%), except for genes encoding ABC transporters, which accounted for approximately 3% of inferred genes. These findings provided a comprehensive profile of bacterial communities, including potential bacterial pathogens and antibiotic-resistant genes, in raw and secondary treated WW samples from four WWTPs across Australia and demonstrated that Illumina

  17. 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.

  18. 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

  19. Amplicon-based taxonomic characterization of bacteria in urban and peri-urban roof-harvested rainwater stored in tanks.

    PubMed

    Ahmed, W; Staley, C; Hamilton, K A; Beale, D J; Sadowsky, M J; Toze, S; Haas, C N

    2017-01-15

    Overall, 26% of Australian households use rainwater tanks as a source of potable and nonpotable water. Limited information is available on the total bacterial communities in tank water. Therefore, identification of dominant bacterial communities, diversity, and their distribution is important in understanding the microbial quality of tank water. In this study, the abundance and diversity of bacterial communities in 88 tank water samples collected from the urban areas of Brisbane (n=44) and the peri-urban center of Currumbin (n=44) in Southeast Queensland, Australia were determined using amplicon-based Illumina next-generation sequencing. In addition, the SourceTracker program was used to identify the sources of fecal contamination in tank water samples. Sequence reads were also analyzed to detect potential bacterial pathogenic genera in the tank water samples collected. Differences in sample coverage, alpha diversity, and richness did not differ significantly between the Brisbane and Currumbin tank water samples. Comamonadaceae and Planctomycetaceae were the most abundant families in all tank water samples. Curvibacter was the most abundant genus in all tank water samples. SourceTracker revealed that around 34% (Brisbane) and 43% (Currumbin) of tank water samples had a signature for bird fecal contamination. The potential opportunistic pathogenic genera including Burkholderia, Chromobacterium, Clostridium, Legionella, Mycobacterium, Nocardia, and Pseudomonas were most prevalent in tank water samples. Next-generation sequencing can be used as an initial screening tool to identify a wide array of potential pathogenic genera in tank water samples followed by quantifying specific pathogen(s) of interest using more sensitive molecular assays such as quantitative PCR (qPCR).

  20. HSV-1 Amplicon Vectors Launch the Production of Heterologous Rotavirus-like Particles and Induce Rotavirus-specific Immune Responses in Mice

    PubMed Central

    Laimbacher, Andrea S; Esteban, Laura E; Castello, Alejandro A; Abdusetir Cerfoglio, Juan C; Argüelles, Marcelo H; Glikmann, Graciela; D'Antuono, Alejandra; Mattion, Nora; Berois, Mabel; Arbiza, Juan; Hilbe, Monika; Schraner, Elisabeth M; Seyffert, Michael; Dresch, Christiane; Epstein, Alberto L; Ackermann, Mathias; Fraefel, Cornel

    2012-01-01

    Virus-like particles (VLPs) are promising vaccine candidates because they represent viral antigens in the authentic conformation of the virion and are therefore readily recognized by the immune system. As VLPs do not contain genetic material they are safer than attenuated virus vaccines. In this study, herpes simplex virus type 1 (HSV-1) amplicon vectors were constructed to coexpress the rotavirus (RV) structural genes VP2, VP6, and VP7 and were used as platforms to launch the production of RV-like particles (RVLPs) in vector-infected mammalian cells. Despite the observed splicing of VP6 RNA, full-length VP6 protein and RVLPs were efficiently produced. Intramuscular injection of mice with the amplicon vectors as a two-dose regimen without adjuvants resulted in RV-specific humoral immune responses and, most importantly, immunized mice were partially protected at the mucosal level from challenge with live wild-type (wt) RV. This work provides proof of principle for the application of HSV-1 amplicon vectors that mediate the efficient production of heterologous VLPs as genetic vaccines. PMID:22713696