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Sample records for 17q12 amplicon promotes

  1. MGC9753 gene, located within PPP1R1B-STARD3-ERBB2-GRB7 amplicon on human chromosome 17q12, encodes the seven-transmembrane receptor with extracellular six-cystein domain.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2003-06-01

    MYC, ERBB2, MET, FGFR2, CCNE1, MYCN, WNT2, CD44, MDM2, NCOA3, IQGAP1 and STK6 loci are amplified in human gastric cancer. It has been reported that the gene corresponding to EST H16094 is co-amplified with ERBB2 gene in human gastric cancer. Here, we identified and characterized the gene corresponding to EST H16094 by using bioinformatics. BLAST programs revealed that EST H16094 was derived from the uncharacterized MGC9753 gene. Two ORFs were predicted within human MGC9753 mRNA, and ORF1 (nucleotide position 18-980 of NM_033419.1) was predicted as the coding region of human MGC9753 mRNA based on comparative genomics. Nucleotide sequence of mouse Mgc9753 mRNA was next determined in silico by modification of AK052486 cDNA (deleting C at the nucleotide position 37). Human MGC9753 and mouse Mgc9753 proteins were 320-amino-acid seven-transmembrane receptors with the N-terminal six-cysteine domain and an N-glycosylation site (85.0% total-amino-acid identity). Human MGC9753 protein showed 90.6% total-amino-acid identity with human CAB2 aberrant protein, which lacked the third-transmembrane domain of MGC9753 due to frame shifts within ORF. Human MGC9753 gene, consisting of eight exons, were clustered with PPP1R1B, STARD3, TCAP, PNMT, ERBB2, MGC14832 and GRB7 genes within the 120-kb region. PPP1R1B, STARD3, MGC9753, ERBB2 and GRB7 genes are co-amplified in several cases of gastric cancer. This is the first report on comprehensive characterization of the amplicon around the PPP1R1B-STARD3-TCAP-PNMT-MGC9753-ERBB2-MGC14832-GRB7 locus on human chromosome 17q12.

  2. Congenital diaphragmatic hernia may be associated with 17q12 microdeletion syndrome.

    PubMed

    Goumy, Carole; Laffargue, Fanny; Eymard-Pierre, Eléonore; Kemeny, Stéphen; Gay-Bellile, Mathilde; Gouas, Laetiti; Gallot, Denis; Francannet, Christine; Tchirkov, Andrei; Pebrel-Richard, Céline; Vago, Philippe

    2015-01-01

    Microdeletions of 17q12 encompassing TCF2 are associated with maturity-onset of diabetes of the young type 5, cystic renal disease, pancreatic atrophy, Mullerian aplasia in females and variable cognitive impairment. We report on a patient with a de novo 17q12 microdeletion, 1.8 Mb in size, associated with congenital diaphragmatic hernia (CDH). The 5-year-old male patient presented multicystic renal dysplasia kidneys, minor facial dysmorphic features and skeletal anomalies, but neither developmental delay nor behavioral abnormalities. CDH has been previously associated with the 17q12 microdeletion syndrome only in one prenatal case. The present study reinforces the hypothesis that CDH is part of the phenotype for 17q12 microdeletion and that 17q12 encompasses candidate(s) gene(s) involved in diaphragm development. We suggest that PIGW, a gene involved in an early step of GPI biosynthesis, could be a strong candidate gene for CDH.

  3. Complex autism spectrum disorder in a patient with a 17q12 microduplication.

    PubMed

    Brandt, Tracy; Desai, Khyati; Grodberg, David; Mehta, Lakshmi; Cohen, Ninette; Tryfon, Ana; Kolevzon, Alexander; Soorya, Latha; Buxbaum, Joseph D; Edelmann, Lisa

    2012-05-01

    Autism spectrum disorders (ASDs) are phenotypically complex developmental neuropsychiatric disorders affecting approximately 0.6% of the population. About 30-70% of affected children are also considered to have intellectual disability (ID). The underlying genetic causes of ASDs are diverse with a defined etiology in 16-20%. Array comparative genomic hybridization (aCGH) has proven useful in identifying sub-microscopic chromosome aberrations in a subset of patients, some of which have been shown to be recurrent. One such aberration is the 1.4 Mb microdeletion at chromosome 17q12, which has been reported to be associated with renal disease, growth restriction, diabetes, cognitive impairment, seizures, and in some cases an ASD. Patients with the reciprocal chromosome 17q12 microduplication typically have also been identified with ID and in some cases seizures and behavioral abnormalities. Here we report a patient with a de novo, 1.4 Mb microduplication diagnosed with significant ID involving complex deficits and autism. To our knowledge, this is the first report of a patient with the 17q12 microduplication and a complex ASD phenotype.

  4. A complex microdeletion 17q12 phenotype in a patient with recurrent de novo membranous nephropathy

    PubMed Central

    2012-01-01

    Background Microdeletions on chromosome 17q12 cause of diverse spectrum of disorders and have only recently been identified as a rare cause of Mayer-Rokitansky-Kuester-Hauser-Syndrome (MRKH), which is characterized by uterus aplasia ± partial/complete vaginal aplasia in females with a regular karyotype. For the first time we report about a patient with a 17q12 microdeletion who is affected by MRKH in combination with a vascular and soft tissue disorder. Repeatedly she suffered from kidney transplant failure caused by consuming membranous nephropathy. Case presentation A 38-year-old female patient had been diagnosed with right kidney aplasia, left kidney dysplasia and significantly impaired renal function during infancy. Aged 16 she had to start hemodialysis. Three years later she received her first kidney transplant. Only then she was diagnosed with MRKH. The kidney transplant was lost due to consuming nephrotic syndrome caused by de novo membranous nephropathy, as was a second kidney transplant years later. In addition, a hyperelasticity syndrome affects the patient with congenital joint laxity, kyphoscoliosis, bilateral hip dysplasia, persistent hypermobility of both elbows, knees and hips. Her clinical picture resembles a combination of traits of a hypermobile and a vascular form of Ehlers-Danlos-Syndrome, but no mutations in the COL3A1 gene was underlying. Instead, array-based comparative genomic hybridisation (CGH) detected a heterozygous 1.43 Mb deletion on chromosome 17q12 encompassing the two renal developmental genes HNF1β and LHX1. Conclusions Deletions of HNF1β have recently drawn significant attention in pediatric nephrology as an important cause of prenatally hyperechogenic kidneys, renal aplasia and renal hypodysplasia. In contrast, membranous nephropathy represents an often-unaccounted cause of nephrotic syndrome in the adult population. A causative connection between theses two conditions has never been postulated, but is suggestive enough in

  5. Lymphocyte Activation Dynamics Is Shaped by Hereditary Components at Chromosome Region 17q12-q21

    PubMed Central

    Carreras-Sureda, Amado; Rubio-Moscardo, Fanny; Olvera, Alex; Argilaguet, Jordi; Kiefer, Kerstin; Mothe, Beatriz; Meyerhans, Andreas; Brander, Christian

    2016-01-01

    Single nucleotide polymorphisms (SNPs) located in the chromosome region 17q12-q21 are risk factors for asthma. Particularly, there are cis-regulatory haplotypes within this region that regulate differentially the expression levels of ORMDL3, GSDMB and ZPBP2 genes. Remarkably, ORMDL3 has been shown to modulate lymphocyte activation parameters in a heterologous expression system. In this context, it has been shown that Th2 and Th17 cytokine production is affected by SNPs in this region. Therefore, we aim to assess the impact of hereditary components within region 17q12-q21 on the activation profile of human T lymphocytes, focusing on the haplotype formed by allelic variants of SNPs rs7216389 and rs12936231. We measured calcium influx and activation markers, as well as the proliferation rate upon T cell activation. Haplotype-dependent differences in mRNA expression levels of IL-2 and INF-γ were observed at early times after activation. In addition, the allelic variants of these SNPs impacted on the extent of calcium influx in resting lymphocytes and altered proliferation rates in a dose dependent manner. As a result, the asthma risk haplotype carriers showed a lower threshold of saturation during activation. Finally, we confirmed differences in activation marker expression by flow cytometry using phytohemagglutinin, a strong polyclonal stimulus. Altogether, our data suggest that the genetic component of pro-inflammatory pathologies present in this chromosome region could be explained by different T lymphocyte activation dynamics depending on individual allelic heredity. PMID:27835674

  6. Cloning mammary cell cDNAs from 17q12-q23 using interspecific somatic cell hybrids and subtractive hybridization

    SciTech Connect

    Cerosaletti, K.M.; Shapero, M.H.; Fournier, R.E.K.

    1995-01-01

    We have cloned human genes that are encoded in the region 17q12-q23 and expressed in breast tissue using interspecific somatic cell hybrids and subtractive hybridization. Two mouse microcell hybrids containing fragments of human chromosome 17 with a nonoverlap region at 17q12-q23 were generated by microcell transfer. Radiolabeled cDNA was synthesized from the hybrid cell containing the 17q12-q23 interval and was subtracted with an excess of RNA from the hybrid cell lacking the interval. Resulting cDNA probes enriched for sequences from 17q12-q23 were used to screen a human premenopausal breast cDNA library, and 60 cDNAs were identified. Three of these cDNAs mapped to the hybrid cell nonoverlap region. These cDNAs were expressed in mammary epithelial cell hybrids, although none appeared to be breast-specific. Sequence analysis of the cDNAs revealed that clone 93A represents a previously unidentified gene, clone 98C has homology to an expressed sequence tag from goat mammary tissue, and clone 200A is identical to the human homologue of the Drosophila melanogaster flightless-I gene. These genes map outside a 1-cM region linked to early onset familial breast cancer but may be useful genetic markers in the 17q12-q23 region. 47 refs., 6 figs.

  7. Familial site-specific Ovarian cancer is linked to BRCA1 on 17q12-21

    SciTech Connect

    Steichen-Gersdorf, E.; Gallion, H.H.; Ponder, M.A.; Pye, C.; Mazoyer, S.; Smith, S.A.; Ponder, B.A.J.; Ford, D.; Easton, D.F.; Girodet, C.

    1994-11-01

    In a study of nine families with {open_quotes}site-specific{close_quotes} ovarian cancer (criterion: three or more cases of epithelial ovarian cancer and no cases of breast cancer diagnosed at age <50 years) we have obtained evidence of linkage to the breast-ovarian cancer susceptibility gene, BRCA1 on 17q12-21. If the risk of cancer in these families is assumed to be restricted to the ovary, the best estimate of the proportion of families linked to BRCA1 is .78 (95% confidence interval .32-1.0). If predisposition to both breast and ovarian cancer is assumed, the proportion linked is 1.0 (95% confidence interval .46-1.0). The linkage of familial site-specific ovarian cancer to BRCA1 indicates the possibility of predictive testing in such families; however, this is only appropriate in families where the evidence for linkage to BRCA1 is conclusive. 17 refs., 3 figs., 1 tab.

  8. NEUROD2 and NEUROD3 genes map to human chromosomes 17q12 and 5q23-q31 and mouse chromosomes 11 and 13, respectively

    SciTech Connect

    Tamimi, R.M.; Montgomery-Dyer, K.; Tapscott, S.J.

    1997-03-01

    NEUROD2 and NEUROD3 are transcription factors involved in neurogenesis that are related to the basic helix-loop-helix protein NEUROD. NEUROD2 maps to human chromosome 17q12 and mouse chromosome 11. NEUROD3 maps to human chromosome 5q23-q31 and mouse chromosome 13. 16 refs., 2 figs.

  9. Localization of the gene for sclerosteosis to the van Buchem disease-gene region on chromosome 17q12-q21.

    PubMed Central

    Balemans, W; Van Den Ende, J; Freire Paes-Alves, A; Dikkers, F G; Willems, P J; Vanhoenacker, F; de Almeida-Melo, N; Alves, C F; Stratakis, C A; Hill, S C; Van Hul, W

    1999-01-01

    Sclerosteosis is an uncommon, autosomal recessive, progressive, sclerosing, bone dysplasia characterized by generalized osteosclerosis and hyperostosis of the skeleton, affecting mainly the skull and mandible. In most patients this causes facial paralysis and hearing loss. Other features are gigantism and hand abnormalities. In the present study, linkage analysis in two consanguineous families with sclerosteosis resulted in the assignment of the sclerosteosis gene to chromosome 17q12-q21. This region was analyzed because of the recent assignment to this chromosomal region of the gene causing van Buchem disease, a rare autosomal recessive condition with a hyperostosis similar to sclerosteosis. Because of the clinical similarities between sclerosteosis and van Buchem disease, it has previously been suggested that both conditions might be caused by mutations in the same gene. Our study now provides genetic evidence for this hypothesis. PMID:10330353

  10. Assessment of association between common variants at 17q12 and prostate cancer risk-evidence from Serbian population and meta--analysis.

    PubMed

    Nikolić, Zorana Z; Branković, Ana S; Savić-Pavićević, Dušanka L J; Preković, Stefan M; Vukotić, Vinka D; Cerović, Snežana J; Filipović, Nataša N; Tomović, Saša M; Romac, Stanka P; Brajušković, Goran N

    2014-08-01

    This study aimed to evaluate possible association between genotypes and alleles of two 17q12 polymorphisms (rs3760511 and rs7501939) and prostate cancer (PCa) risk and progression. Two hundred seventy-one patients with PCa, 261 patients with benign prostatic hyperplasia (BPH), and 171 controls were included in the study. Single nucleotide polymorphisms (SNPs) were genotyped by using PCR followed by restriction fragment length (PCR-RFLP) analysis. We conducted meta-analysis of published studies regarding association of these SNPs with PCa risk. Evidence of positive association between the AC genotype of the SNP rs3760511 and BPH risk for the best-fitting overdominant model of association (BPH vs. controls comparison, p = 0.026; odds ratio [OR] = 1.58; 95% confidence interval [95%CI] 1.05-2.36) were obtained. The association between T allele of rs7501939 and PCa risk was determined in PCa versus controls comparison (p = 0.0032; OR = 0.66, 95%CI 0.50-0.87) with the best-fitting model of inheritance being log-additive. This variant was also found to be associated with the risk of BPH (p = 0.0023; OR = 0.65, 95%CI 0.49-0.86). We found no association between parameters of PCa progression and the analyzed SNPs. Meta-analysis showed strong association between these variants and PCa risk. Our study shows association between SNPs at locus 17q12 and the risk of prostatic diseases in Serbian population. At the same time, results of meta-analysis suggest the association of these SNPs with PCa risk.

  11. Isolation of a gene (DLG3) encoding a second member of the discs-large family on chromosome 17q12-q21

    SciTech Connect

    Smith, S.A.; Holik, P.; Stevens, J.

    1996-01-15

    The discs-large family is a collection of proteins that have a common structural organization and are thought to be involved in signal transduction and mediating protein-protein interactions at the cytoplasmic surface of the cell membrane. The defining member of this group of proteins is the gene product of the Drosophila lethal (1) discs large (dlg) 1 locus, which was originally identified by the analysis of recessive lethal mutants. Germline mutations in dlg result in loss of apical-basolateral polarity, disruption of normal cell-cell adhesion, and neoplastic overgrowth of the imaginal disc epithelium. We have isolated and characterized a novel human gene, DLG3, that encodes a new member of the discs-large family of proteins. The putative DLG3 gene product has a molecular weight of 66 kDa and contains a discs-large homologous region, an src oncogene homology motif 3, and a domain with homology to guanylate kinase. The DLG3 gene is located on chromosome 17, in the same segment, 17q12-q21, as the related gene, DLG2. The products of the DLG2 and DLG3 genes show 36% identity and 58% similarity to each other, and both show nearly 60% sequence similarity to p55, an erythroid phosphoprotein that is a component of the red cell membrane. We suggest that p55, DLG2, and DLG3 are closely related members of a gene family, whose protein products have a common structural organization and probably a similar function. 25 refs., 3 figs.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. Promotion

    PubMed Central

    Alam, Hasan B.

    2013-01-01

    This article gives an overview of the promotion process in an academic medical center. A description of different promotional tracks, tenure and endowed chairs, and the process of submitting an application is provided. Finally, some practical advice about developing skills and attributes that can help with academic growth and promotion is dispensed. PMID:24436683

  2. 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

  3. 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.

  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. 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.

  6. 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

  7. 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

  8. 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

  9. "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.

  10. 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

  11. 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/.

  12. Microbial diversity of a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment: integration of 16S rRNA gene amplicon and shotgun metagenomic sequencing.

    PubMed

    Delforno, Tiago Palladino; Lacerda Júnior, Gileno Vieira; Noronha, Melline F; Sakamoto, Isabel K; Varesche, Maria Bernadete A; Oliveira, Valéria M

    2017-02-23

    The 16S rRNA gene amplicon and whole-genome shotgun metagenomic (WGSM) sequencing approaches were used to investigate wide-spectrum profiles of microbial composition and metabolic diversity from a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment. The data were generated by using MiSeq 2 × 250 bp and HiSeq 2 × 150 bp Illumina sequencing platforms for 16S amplicon and WGSM sequencing, respectively. Each approach revealed a distinct microbial community profile, with Pseudomonas and Psychrobacter as predominant genus for the WGSM dataset and Clostridium and Methanosaeta for the 16S rRNA gene amplicon dataset. The virome characterization revealed the presence of two viral families with Bacteria and Archaea as host, Myoviridae, and Siphoviridae. A wide functional diversity was found with predominance of genes involved in the metabolism of acetone, butanol, and ethanol synthesis; and one-carbon metabolism (e.g., methanogenesis). Genes related to the acetotrophic methanogenesis pathways were more abundant than methylotrophic and hydrogenotrophic, corroborating the taxonomic results that showed the prevalence of the acetotrophic genus Methanosaeta. Moreover, the dataset indicated a variety of metabolic genes involved in sulfur, nitrogen, iron, and phosphorus cycles, with many genera able to act in all cycles. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) revealed that microbial community contained 43 different types of antibiotic resistance genes, some of them were associated with growth chicken promotion (e.g., bacitracin, tetracycline, and polymyxin).

  13. 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.

  14. Formation of Linear Amplicons with Inverted Duplications in Leishmania Requires the MRE11 Nuclease

    PubMed Central

    Laffitte, Marie-Claude N.; Genois, Marie-Michelle; Mukherjee, Angana; Légaré, Danielle; Masson, Jean-Yves; Ouellette, Marc

    2014-01-01

    Extrachromosomal DNA amplification is frequent in the protozoan parasite Leishmania selected for drug resistance. The extrachromosomal amplified DNA is either circular or linear, and is formed at the level of direct or inverted homologous repeated sequences that abound in the Leishmania genome. The RAD51 recombinase plays an important role in circular amplicons formation, but the mechanism by which linear amplicons are formed is unknown. We hypothesized that the Leishmania infantum DNA repair protein MRE11 is required for linear amplicons following rearrangements at the level of inverted repeats. The purified LiMRE11 protein showed both DNA binding and exonuclease activities. Inactivation of the LiMRE11 gene led to parasites with enhanced sensitivity to DNA damaging agents. The MRE11−/− parasites had a reduced capacity to form linear amplicons after drug selection, and the reintroduction of an MRE11 allele led to parasites regaining their capacity to generate linear amplicons, but only when MRE11 had an active nuclease activity. These results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment. PMID:25474106

  15. 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.

  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. 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.

  18. 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

  19. 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.

  20. 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

  1. Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap.

    PubMed Central

    Conway, J E; Zolotukhin, S; Muzyczka, N; Hayward, G S; Byrne, B J

    1997-01-01

    Recombinant adeno-associated virus (AAV) type 2 (rAAV) vectors have recently been shown to have great utility as gene transfer agents both in vitro and in vivo. One of the problems associated with the use of rAAV vectors has been the difficulty of large-scale vector production. Low-efficiency plasmid transfection of the rAAV vector and complementing AAV type 2 (AAV-2) functions (rep and cap) followed by superinfection with adenovirus has been the standard approach to rAAV production. The objectives of this study were to demonstrate the ability of a recombinant herpes simplex virus type 1 (HSV-1) amplicon expressing AAV-2 Rep and Cap to support replication and packaging of rAAV vectors. HSV-1 amplicon vectors were constructed which contain the AAV-2 rep and cap genes under control of their native promoters (p5, p19, and p40). An HSV-1 amplicon vector, HSV-RC/KOS or HSV-RC/d27, was generated by supplying helper functions with either wild-type HSV-1 (KOS strain) or the ICP27-deleted mutant of HSV-1, d27-1, respectively. Replication of the amplicon stocks is not inhibited by the presence of AAV-2 Rep proteins, which highlights important differences between HSV-1 and adenovirus replication and the mechanism of providing helper function for productive AAV infection. Coinfection of rAAV and HSV-RC/KOS resulted in the replication and amplification of rAAV genomes. Similarly, rescue and replication of rAAV genomes occurred when rAAV vector plasmids were transfected into cells followed by HSV-RC/KOS infection and when two rAAV proviral cell lines were infected with HSV-RC/KOS or HSV-RC/d27. Production of infectious rAAV by rescue from two rAAV proviral cell lines has also been achieved with HSV-RC/KOS and HSV-RC/d27. The particle titer of rAAV produced with HSV-RC/d27 is equal to that achieved by supplying rep and cap by transfection followed by adenovirus superinfection. Importantly, no detectable wild-type AAV-2 is generated with this approach. These results demonstrate

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. Association of BRCA1 promoter methylation with sporadic breast cancers: Evidence from 40 studies.

    PubMed

    Zhang, Li; Long, Xinghua

    2015-12-08

    Breast cancer susceptibility gene 1 (BRCA1) located at chromosome 17q12-21 is a classic tumor suppressor gene, and has been considered as a significant role in hereditary breast cancers. Moreover, numerous studies demonstrated the methylation status of CpG islands in the promoter regions of BRCA1 gene was aberrant in patients with sporadic breast tumors compared with healthy females or patients with benign diseases. However, these conclusions were not always consistent. Hence, a meta-analysis was performed to get a more precise estimate for these associations. Crude odds ratio with 95% confidence interval were used to assess the association of BRCA1 promoter methylation and the risk or clinicopathologic characteristics of breast cancers under fixed or random effect model. A total of 40 studies were eligible for this present study. We observed the frequency of BRCA1 promoter methylation was statistically significant higher in breast cancers than non-cancer controls. Furthermore, BRCA1 methylation was statistically associated with lymph node metastasis, histological grade 3, ER(-), PR(-), triple-negative phenotype, and decreased or lack levels of BRCA1 protein expression. In conclusion, this study indicated that BRCA1 promoter methylation appeared to be a useful predictive or prognostic biomarker for breast cancers in clinical assessment.

  7. 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.

  8. 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.

  9. 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

  10. Functional Analysis of Frequent Lung Cancer Amplicons Under Current Genome Annotation

    DTIC Science & Technology

    2015-12-01

    Exemplary groups of such genes include many non-protein-coding genes like long non-coding RNAs and microRNAs (miRNAs). In study, we examined the PVT1...lung tumorigenesis. The implication is that future studies of the 8q24 amplicon should concentrate on characterizing the PVT1 RNA transcript. 15...SUBJECT TERMS Noncoding RNA , microRNA, lung cancer, Genome annotation 16. SECURITY CLASSIFICATION OF: U 17. LIMITATION OF ABSTRACT 18. NUMBER OF

  11. 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.

  12. 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.

  13. Engineering an improved cell cycle-regulatable herpes simplex virus type 1 amplicon vector with enhanced transgene expression in proliferating cells yet attenuated activities in resting cells.

    PubMed

    Wang, Grace Y; Ho, Ivy A W; Sia, Kian C; Miao, L; Hui, Kam M; Lam, Paula Y P

    2007-03-01

    We previously generated a herpes simplex virus type 1 (HSV-1)-based amplicon vector (denoted pC8-36) in which gene expression from the minimal cyclin A promoter is repressed by preventing the binding of a trans-activating protein, Gal4-NF-YA, to it through selective interaction with the transcriptional repressor protein CDF-1. Because CDF-1 is absent in actively dividing cells, transgene expression conferred by the pC8-36 vector is therefore cell cycle dependent. As gene therapy evolves to become a promising therapeutic modality for many human diseases, there is an increasing need to further improve the kinetics of gene regulation. In the present study, we examined whether the availability of more binding sites for CDF-1 repressor proteins could enhance transgene expression. Using an overlap extension polymerase chain reaction (PCR) method, the CDE and CHR elements within the minimum cyclin A promoter were multimerized to contain two, three, and six copies of the designated CDE/CHR sequence. Interestingly, our results demonstrated that six-copy CDE/CHR sequence motifs (pC8-6CC-Luc) conferred an approximately 20-fold increase in the ratio of cell cycle regulation compared with the previous reported construct. Further, the overall transcriptional activities mediated by pC8-6CC-Luc were stronger compared with the native human survivin promoter, which consists of three copies of the CDE element and one copy of the CHR element. pC8-6CC-Luc contained, in essence, only the synthetic six-copy CDE/CHR sequence motif (about 262 bp). In comparison with other native endogenous promoters, which usually contain many other transcription binding sites, pC8-6CC-Luc amplicon vectors should confer better regulated and consistent transgene expression and may be considered a gene delivery vector of choice to target actively proliferating tumor cells.

  14. 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

  15. 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

  16. 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

  17. Structure and organization of amplicons containing the E4 esterase genes responsible for insecticide resistance in the aphid Myzus persicae (Sulzer).

    PubMed Central

    Field, L M; Devonshire, A L

    1997-01-01

    Insecticide resistance in the aphid Myzus persicae results primarily from the amplification of genes encoding the insecticide-detoxifying esterase, E4. Here we report the analysis of flanking DNA co-amplified with the E4 gene. The 5' end of this gene has an untranslated leader sequence interspersed by two introns, and the promoter region lacks TATA and CAAT boxes. The DNA breakpoint involved in the generation of the amplification is just upstream (approx. 250 bp) of the putative E4 transcription start site; thus the E4 gene is very close to the 5' end of the approx. 24 kb amplicon. PCR primers specific to the 'novel joint' generated during the amplification have been used to show that a wide range of aphid clones have the same amplicons, arranged as a series of head-to-tail direct repeats. Long-distance mapping has revealed the structure of these repeats. This has important implications for understanding both the generation of the amplified genes and the origin and spread of insecticide resistance in M. persicae. PMID:9148762

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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).

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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.

  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. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  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. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. Construction of recombinant pseudorabies viruses by using PRV BACs deficient in IE180 or pac sequences: Application of vBAC90D recombinant virus to production of PRV amplicons.

    PubMed

    Lerma, L; Muñoz, A L; Wagner, S; Dinu, M; Martín, B; Tabarés, E

    2016-02-02

    We describe a simple and efficient method to obtain recombinant pseudorabies virus (PRV) in mammalian cells by using the PRV BACs, PBAC80 deficient in pac sequences and PBAC90 deficient in the IE180 gene. These essential viral sequences were used as targets to obtain viable recombinant viruses. PBAC80 was constructed, confirmed to encode a copy of the IE180 gene regulated by the inducible Ptet promoter, and used to obtain recombinant attenuated PRV viruses that express the EGFP protein (PRV-BT80GF virus). PBAC90 was used to obtain the vBAC90D virus, deficient in IE180 and free of replication-competent revertants, and which can be used as a helper in the production of PRV amplicons.

  3. A point mutation in the putative TATA box, detected in nondiseased individuals and patients with hereditary breast cancer, decreases promoter activity of the 17{beta}-hydroxysteroid dehydrogenase type 1 gene 2 (EDH17B2) in vitro

    SciTech Connect

    Peltoketo, H.; Piao, Y.; Isomaa, V.

    1994-09-01

    EDH17B2, the gene encoding 17{beta}-hydroxysteroid dehydrogenase type 1, has been suggested as a candidate for the familial breast cancer gene, BRCA1, located on 17q12-q21. We analyzed the promoter region of EDH17B2 in DNA from 20 control individuals and 40 patients with familial breast cancer. Two frequent (designated vI and vIII) and two rare (vII and vIV) nucleotide variations were present in both the breast cancer patients and the controls, except the alteration vII, which was found only in one patient. Although the data do not support the identification of EDH17B2 as the BRCA1 gene, it is of interest that point mutation vIV (A {yields} C) was located in the putative TATA box of the EDH17B2 gene. Reporter gene analysis showed that the mutation vIV decreases EDH17B2 promoter activity by an average of 45% in in vitro assays, suggesting that nucleotide A at position -27 is significant for efficient transcription. 12 refs., 2 figs., 1 tab.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. Comprehensive analysis of human endogenous retrovirus group HERV-W locus transcription in multiple sclerosis brain lesions by high-throughput amplicon sequencing.

    PubMed

    Schmitt, Katja; Richter, Christin; Backes, Christina; Meese, Eckart; Ruprecht, Klemens; Mayer, Jens

    2013-12-01

    Human endogenous retroviruses (HERVs) of the HERV-W group comprise hundreds of loci in the human genome. Deregulated HERV-W expression and HERV-W locus ERVWE1-encoded Syncytin-1 protein have been implicated in the pathogenesis of multiple sclerosis (MS). However, the actual transcription of HERV-W loci in the MS context has not been comprehensively analyzed. We investigated transcription of HERV-W in MS brain lesions and white matter brain tissue from healthy controls by employing next-generation amplicon sequencing of HERV-W env-specific reverse transcriptase (RT) PCR products, thus revealing transcribed HERV-W loci and the relative transcript levels of those loci. We identified more than 100 HERV-W loci that were transcribed in the human brain, with a limited number of loci being predominantly transcribed. Importantly, relative transcript levels of HERV-W loci were very similar between MS and healthy brain tissue samples, refuting deregulated transcription of HERV-W env in MS brain lesions, including the high-level-transcribed ERVWE1 locus encoding Syncytin-1. Quantitative RT-PCR likewise did not reveal differences in MS regarding HERV-W env general transcript or ERVWE1- and ERVWE2-specific transcript levels. However, we obtained evidence for interindividual differences in HERV-W transcript levels. Reporter gene assays indicated promoter activity of many HERV-W long terminal repeats (LTRs), including structurally incomplete LTRs. Our comprehensive analysis of HERV-W transcription in the human brain thus provides important information on the biology of HERV-W in MS lesions and normal human brain, implications for study design, and mechanisms by which HERV-W may (or may not) be involved in MS.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Investigation of Microbial Diversity in Geothermal Hot Springs in Unkeshwar, India, Based on 16S rRNA Amplicon Metagenome Sequencing

    PubMed Central

    Mehetre, Gajanan T.; Paranjpe, Aditi; Dastager, Syed G.

    2016-01-01

    Microbial diversity in geothermal waters of the Unkeshwar hot springs in Maharashtra, India, was studied using 16S rRNA amplicon metagenomic sequencing. Taxonomic analysis revealed the presence of Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, Archeae, and OD1 phyla. Metabolic function prediction analysis indicated a battery of biological information systems indicating rich and novel microbial diversity, with potential biotechnological applications in this niche. PMID:26950332

  16. 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.

  17. 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.

  18. Amplicon-dependent CCNE1 expression is critical for clonogenic survival after cisplatin treatment and is correlated with 20q11 gain in ovarian cancer.

    PubMed

    Etemadmoghadam, Dariush; George, Joshy; Cowin, Prue A; Cullinane, Carleen; Kansara, Maya; Gorringe, Kylie L; Smyth, Gordon K; Bowtell, David D L

    2010-11-12

    Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers.

  19. Amplicon-Dependent CCNE1 Expression Is Critical for Clonogenic Survival after Cisplatin Treatment and Is Correlated with 20q11 Gain in Ovarian Cancer

    PubMed Central

    Etemadmoghadam, Dariush; George, Joshy; Cowin, Prue A.; Cullinane, Carleen; Kansara, Maya; Gorringe, Kylie L.; Smyth, Gordon K.; Bowtell, David D. L.

    2010-01-01

    Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers. PMID:21103391

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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).

  6. 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

  7. Exploring the Gastrointestinal “Nemabiome”: Deep Amplicon Sequencing to Quantify the Species Composition of Parasitic Nematode Communities

    PubMed Central

    Avramenko, Russell W.; Redman, Elizabeth M.; Lewis, Roy; Yazwinski, Thomas A.; Wasmuth, James D.; Gilleard, John S.

    2015-01-01

    Parasitic helminth infections have a considerable impact on global human health as well as animal welfare and production. Although co-infection with multiple parasite species within a host is common, there is a dearth of tools with which to study the composition of these complex parasite communities. Helminth species vary in their pathogenicity, epidemiology and drug sensitivity and the interactions that occur between co-infecting species and their hosts are poorly understood. We describe the first application of deep amplicon sequencing to study parasitic nematode communities as well as introduce the concept of the gastro-intestinal “nemabiome”. The approach is analogous to 16S rDNA deep sequencing used to explore microbial communities, but utilizes the nematode ITS-2 rDNA locus instead. Gastro-intestinal parasites of cattle were used to develop the concept, as this host has many well-defined gastro-intestinal nematode species that commonly occur as complex co-infections. Further, the availability of pure mono-parasite populations from experimentally infected cattle allowed us to prepare mock parasite communities to determine, and correct for, species representation biases in the sequence data. We demonstrate that, once these biases have been corrected, accurate relative quantitation of gastro-intestinal parasitic nematode communities in cattle fecal samples can be achieved. We have validated the accuracy of the method applied to field-samples by comparing the results of detailed morphological examination of L3 larvae populations with those of the sequencing assay. The results illustrate the insights that can be gained into the species composition of parasite communities, using grazing cattle in the mid-west USA as an example. However, both the technical approach and the concept of the ‘nemabiome’ have a wide range of potential applications in human and veterinary medicine. These include investigations of host-parasite and parasite-parasite interactions

  8. Combination vascular delivery of herpes simplex oncolytic viruses and amplicon mediated cytokine gene transfer is effective therapy for experimental liver cancer.

    PubMed Central

    Zager, J. S.; Delman, K. A.; Malhotra, S.; Ebright, M. I.; Bennett, J. J.; Kates, T.; Halterman, M.; Federoff, H.; Fong, Y.

    2001-01-01

    BACKGROUND: Herpes simplex type I (HSV)-based vectors have been used experimentally for suicide gene therapy, immunomodulatory gene delivery, and direct oncolytic therapy. The current study utilizes the novel concept of regional delivery of an oncolytic virus in combination with or serving as the helper virus for packaging herpes-based amplicon vectors carrying a cytokine transgene, with the goal of identifying if this combination is more efficacious than either modality alone. MATERIALS AND METHODS: A replication competent oncolytic HSV (G207) and a replication incompetent HSV amplicon carrying the gene for the immunomodulatory cytokine IL-2 (HSV-IL2) were tested in murine syngeneic colorectal carcinoma and in rat hepatocellular carcinoma models. Liver tumors were treated with vascular delivery of (1) phosphate-buffered saline (PBS), (2) G207, (3) HSV-IL2, (4) G207 and HSV-IL2 mixed in combination (mG207/HSV- IL2), and (5) G207 as the helper virus for packaging the construct HSV-IL2 (pG207/HSV-IL2). RESULTS: Tumor burden was significantly reduced in all treatment groups in both rats and mice treated with high-dose G207, HSV-IL2, or both (p < 0.02). When a low dose of virus was used in mice, anti-tumor efficacy was improved by use of G207 and HSV-IL2 in combination or with HSV-IL2 packaged by G207 (p < 0.001). This improvement was abolished when CD4(+) and CD8(+) lymphocytes were depleted, implying that the enhanced anti-tumor response to low-dose combined therapy is immune mediated. CONCLUSIONS: Vascular regional delivery of oncolytic and amplicon HSV vectors can be used to induce improved anti-tumor efficacy by combining oncolytic and immunostimulatory strategies. PMID:11591892

  9. The application of amplicon length heterogeneity PCR (LH-PCR) for monitoring the dynamics of soil microbial communities associated with cadaver decomposition.

    PubMed

    Moreno, Lilliana I; Mills, DeEtta; Fetscher, Jill; John-Williams, Krista; Meadows-Jantz, Lee; McCord, Bruce

    2011-03-01

    The placement of cadavers in shallow, clandestine graves may alter the microbial and geochemical composition of the underlying and adjacent soils. Using amplicon length heterogeneity-PCR (LH-PCR) the microbial community changes in these soils can be assessed. In this investigation, nine different grave sites were examined over a period of 16weeks. The results indicated that measurable changes occurred in the soil bacterial community during the decomposition process. In this study, amplicons corresponding to anaerobic bacteria, not indigenous to the soil, were shown to produce differences between grave sites and control soils. Among the bacteria linked to these amplicons are those that are most often part of the commensal flora of the intestines, mouth and skin. In addition, over the 16week sampling interval, the level of indicator organisms (i.e., nitrogen fixing bacteria) dropped as the body decomposed and after four weeks of environmental exposure they began to increase again; thus differences in the abundance of nitrogen fixing bacteria were also found to contribute to the variation between controls and grave soils. These results were verified using primers that specifically targeted the nifH gene coding for nitrogenase reductase. LH-PCR provides a fast, robust and reproducible method to measure microbial changes in soil and could be used to determine potential cadaveric contact in a given area. The results obtained with this method could ultimately provide leads to investigators in criminal or missing person scenarios and allow for further analysis using human specific DNA assays to establish the identity of the buried body.

  10. Characterization of the fecal bacteria communities of forage-fed horses by pyrosequencing of 16S rRNA V4 gene amplicons.

    PubMed

    Shepherd, Megan L; Swecker, William S; Jensen, Roderick V; Ponder, Monica A

    2012-01-01

    The diversity of the equine fecal bacterial community was evaluated using pyrosequencing of 16S rRNA gene amplicons. Fecal samples were obtained from horses fed cool-season grass hay. Fecal bacteria were characterized by amplifying the V4 region of bacterial 16S rRNA gene. Of 5898 mean unique sequences, a mean of 1510 operational taxonomic units were identified in the four fecal samples. Equine fecal bacterial richness was higher than that reported in humans, but lower than that reported in either cattle feces or soil. Bacterial classified sequences were assigned to 16 phyla, of which 10 were present in all samples. The largest number of reads belonged to Firmicutes (43.7% of total bacterial sequences), Verrucomicrobia (4.1%), Proteobacteria (3.8%), and Bacteroidetes (3.7%). The less abundant Actinobacteria, Cyanobacteria, and TM7 phyla presented here have not been previously described in the gut contents or feces of horses. Unclassified sequences represented 38.1% of total bacterial sequences; therefore, the equine fecal microbiome diversity is likely greater than that described. This is the first study to characterize the fecal bacterial community in horses by the use of 16S rRNA gene amplicon pyrosequencing, expanding our knowledge of the fecal microbiota of forage-fed horses.

  11. High-Throughput, Amplicon-Based Sequencing of the CREBBP Gene as a Tool to Develop a Universal Platform-Independent Assay

    PubMed Central

    Fuellgrabe, Marc W.; Herrmann, Dietrich; Knecht, Henrik; Kuenzel, Sven; Kneba, Michael; Pott, Christiane; Brüggemann, Monika

    2015-01-01

    High-throughput sequencing technologies are widely used to analyse genomic variants or rare mutational events in different fields of genomic research, with a fast development of new or adapted platforms and technologies, enabling amplicon-based analysis of single target genes or even whole genome sequencing within a short period of time. Each sequencing platform is characterized by well-defined types of errors, resulting from different steps in the sequencing workflow. Here we describe a universal method to prepare amplicon libraries that can be used for sequencing on different high-throughput sequencing platforms. We have sequenced distinct exons of the CREB binding protein (CREBBP) gene and analysed the output resulting from three major deep-sequencing platforms. platform-specific errors were adjusted according to the result of sequence analysis from the remaining platforms. Additionally, bioinformatic methods are described to determine platform dependent errors. Summarizing the results we present a platform-independent cost-efficient and timesaving method that can be used as an alternative to commercially available sample-preparation kits. PMID:26057250

  12. Within-host competition between Borrelia afzelii ospC strains in wild hosts as revealed by massively parallel amplicon sequencing.

    PubMed

    Strandh, Maria; Råberg, Lars

    2015-08-19

    Infections frequently consist of more than one strain of a given pathogen. Experiments have shown that co-infecting strains often compete, so that the infection intensity of each strain in mixed infections is lower than in single strain infections. Such within-host competition can have important epidemiological and evolutionary consequences. However, the extent of competition has rarely been investigated in wild, naturally infected hosts, where there is noise in the form of varying inoculation doses, asynchronous infections and host heterogeneity, which can potentially alleviate or eliminate competition. Here, we investigated the extent of competition between Borrelia afzelii strains (as determined by ospC genotype) in three host species sampled in the wild. For this purpose, we developed a protocol for 454 amplicon sequencing of ospC, which allows both detection and quantification of each individual strain in an infection. Each host individual was infected with one to six ospC strains. The infection intensity of each strain was lower in mixed infections than in single ones, showing that there was competition. Rank-abundance plots revealed that there was typically one dominant strain, but that the evenness of the relative infection intensity of the different strains in an infection increased with the multiplicity of infection. We conclude that within-host competition can play an important role under natural conditions despite many potential sources of noise, and that quantification by next-generation amplicon sequencing offers new possibilities to dissect within-host interactions in naturally infected hosts.

  13. A New Method for Rapid Screening of End-Point PCR Products: Application to Single Genome Amplified HIV and SIV Envelope Amplicons.

    PubMed

    Houzet, Laurent; Deleage, Claire; Satie, Anne-Pascale; Merlande, Laetitia; Mahe, Dominique; Dejucq-Rainsford, Nathalie

    2015-01-01

    PCR is the most widely applied technique for large scale screening of bacterial clones, mouse genotypes, virus genomes etc. A drawback of large PCR screening is that amplicon analysis is usually performed using gel electrophoresis, a step that is very labor intensive, tedious and chemical waste generating. Single genome amplification (SGA) is used to characterize the diversity and evolutionary dynamics of virus populations within infected hosts. SGA is based on the isolation of single template molecule using limiting dilution followed by nested PCR amplification and requires the analysis of hundreds of reactions per sample, making large scale SGA studies very challenging. Here we present a novel approach entitled Long Amplicon Melt Profiling (LAMP) based on the analysis of the melting profile of the PCR reactions using SYBR Green and/or EvaGreen fluorescent dyes. The LAMP method represents an attractive alternative to gel electrophoresis and enables the quick discrimination of positive reactions. We validate LAMP for SIV and HIV env-SGA, in 96- and 384-well plate formats. Because the melt profiling allows the screening of several thousands of PCR reactions in a cost-effective, rapid and robust way, we believe it will greatly facilitate any large scale PCR screening.

  14. Health Promotion

    DTIC Science & Technology

    1986-03-11

    Department of Defense DIRECTIVEAD-A269 638 , , AD-A29 638March 11, 1986 IIIIii!IN 111111111,11 Ii1111,111111[NUMBER 1010.10 SUBJECT: Health Promotion ...34 March 13, 1985 INC A. URPOSE SThis Directive establishes a health promotion policy within the Department of Defense to improve and maintain military...civilian employees. C. DEFINITIONS 1. Health Promotion . Any combination of health education and related organizational, social, economic or health care

  15. Two amplicon sequencing strategies revealed different facets of the prokaryotic community associated with the anaerobic treatment of vinasses from ethanol distilleries.

    PubMed

    Martínez, M A; Romero, H; Perotti, N I

    2014-02-01

    The prokaryotic consortium from a pilot-scale UASB reactor fed with vinasses from ethanol distilleries was evaluated by means of amplicon sequencing of the 16S rRNA gene. Two different sets of primers targeted to overlapping regions of the V4-16S region were used to gain a broad picture of such community and to perform a comparative analysis. From the two datasets obtained, prevalent phyla were Firmicutes, Verrucomicrobia and Thermotogae. Interestingly, one set of primers captured variability in both the bacterial and archaeal portions of the community, whilst the other one revealed a more diverse community structure, but only in the Bacteria domain. Although a certain level of agreement between the two strategies was observed, sharp differences indicate that different facets of the community were disclosed by each approach.

  16. Bacterial tag encoded FLX titanium amplicon pyrosequencing (bTEFAP) based assessment of prokaryotic diversity in metagenome of Lonar soda lake, India

    PubMed Central

    Dudhagara, Pravin; Ghelani, Anjana; Patel, Rajesh; Chaudhari, Rajesh; Bhatt, Shreyas

    2015-01-01

    Bacterial diversity and archaeal diversity in metagenome of the Lonar soda lake sediment were assessed by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). Metagenome comprised 5093 sequences with 2,531,282 bp and 53 ± 2% G + C content. Metagenome sequence data are available at NCBI under the Bioproject database with accession no. PRJNA218849. Metagenome sequence represented the presence of 83.1% bacterial and 10.5% archaeal origin. A total of 14 different bacteria demonstrating 57 species were recorded with dominating species like Coxiella burnetii (17%), Fibrobacter intestinalis (12%) and Candidatus Cloacamonas acidaminovorans (11%). Occurrence of two archaeal phyla representing 24 species, among them Methanosaeta harundinacea (35%), Methanoculleus chikugoensis (12%) and Methanolinea tarda (11%) were dominating species. Significant presence of 11% sequences as an unclassified indicated the possibilities for unknown novel prokaryotes from the metagenome. PMID:26484168

  17. Pyrosequencing of 16S rRNA gene amplicons to study the microbiota in the gastrointestinal tract of carp (Cyprinus carpio L.)

    PubMed Central

    2011-01-01

    The microbes in the gastrointestinal (GI) tract are of high importance for the health of the host. In this study, Roche 454 pyrosequencing was applied to a pooled set of different 16S rRNA gene amplicons obtained from GI content of common carp (Cyprinus carpio) to make an inventory of the diversity of the microbiota in the GI tract. Compared to other studies, our culture-independent investigation reveals an impressive diversity of the microbial flora of the carp GI tract. The major group of obtained sequences belonged to the phylum Fusobacteria. Bacteroidetes, Planctomycetes and Gammaproteobacteria were other well represented groups of micro-organisms. Verrucomicrobiae, Clostridia and Bacilli (the latter two belonging to the phylum Firmicutes) had fewer representatives among the analyzed sequences. Many of these bacteria might be of high physiological relevance for carp as these groups have been implicated in vitamin production, nitrogen cycling and (cellulose) fermentation. PMID:22093413

  18. 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

  19. 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.

  20. Simplified strategy for rapid first-line screening of fragile X syndrome: closed-tube triplet-primed PCR and amplicon melt peak analysis.

    PubMed

    Rajan-Babu, Indhu-Shree; Law, Hai-Yang; Yoon, Chui-Sheun; Lee, Caroline G; Chong, Samuel S

    2015-05-04

    Premutation and full-mutation hyperexpansion of CGG-triplets in the X-linked Fragile X Mental Retardation 1 (FMR1) gene have been implicated in fragile X-associated tremor/ataxia syndrome, fragile X-associated primary ovarian insufficiency, and fragile X syndrome (FXS), respectively. The currently available molecular diagnostic tests are either costly or labour-intensive, which prohibits their application as a first-line FMR1 test in large-scale population-based screening programs. In this study, we demonstrate the utility of a simplified closed-tube strategy for rapid first-line screening of FXS based on melt peak temperature (Tm) analysis of direct triplet-primed polymerase chain reaction amplicons (dTP-PCR MCA). In addition, we also evaluated the correlation between Tm and CGG-repeat size based on capillary electrophoresis (CE) of dTP-PCR amplicons. The assays were initially tested on 29 FMR1 reference DNA samples, followed by a blinded validation on 107 previously characterised patient DNA samples. The dTP-PCR MCA produced distinct melt profiles of higher Tm for samples carrying an expanded allele. Among the samples tested, we also observed a good correlation between Tm and CGG-repeat size. In the blinded validation study, dTP-PCR MCA accurately classified all normal and expansion carriers, and the FMR1 genotypic classification of all samples was completely concordant with the previously determined genotypes as well as the dTP-PCR CE results. This simple and cost-effective MCA-based assay may be useful as a first-line FXS screening tool that could rapidly screen out the large majority of unaffected individuals, thus minimising the number of samples that need to be analysed by Southern blot analysis.

  1. The Quantification of Representative Sequences pipeline for amplicon sequencing: case study on within-population ITS1 sequence variation in a microparasite infecting Daphnia.

    PubMed

    González-Tortuero, E; Rusek, J; Petrusek, A; Gießler, S; Lyras, D; Grath, S; Castro-Monzón, F; Wolinska, J

    2015-11-01

    Next generation sequencing (NGS) platforms are replacing traditional molecular biology protocols like cloning and Sanger sequencing. However, accuracy of NGS platforms has rarely been measured when quantifying relative frequencies of genotypes or taxa within populations. Here we developed a new bioinformatic pipeline (QRS) that pools similar sequence variants and estimates their frequencies in NGS data sets from populations or communities. We tested whether the estimated frequency of representative sequences, generated by 454 amplicon sequencing, differs significantly from that obtained by Sanger sequencing of cloned PCR products. This was performed by analysing sequence variation of the highly variable first internal transcribed spacer (ITS1) of the ichthyosporean Caullerya mesnili, a microparasite of cladocerans of the genus Daphnia. This analysis also serves as a case example of the usage of this pipeline to study within-population variation. Additionally, a public Illumina data set was used to validate the pipeline on community-level data. Overall, there was a good correspondence in absolute frequencies of C. mesnili ITS1 sequences obtained from Sanger and 454 platforms. Furthermore, analyses of molecular variance (amova) revealed that population structure of C. mesnili differs across lakes and years independently of the sequencing platform. Our results support not only the usefulness of amplicon sequencing data for studies of within-population structure but also the successful application of the QRS pipeline on Illumina-generated data. The QRS pipeline is freely available together with its documentation under GNU Public Licence version 3 at http://code.google.com/p/quantification-representative-sequences.

  2. Health Promotion

    PubMed Central

    Wilson, Ron

    1992-01-01

    How physicians address issues of disease prevention and health promotion is discussed and current standards of screening for disease and counseling practices are reviewed. Collaboration among all health professionals is necessary if preventive medicine is to be effective. PMID:21221259

  3. Health Promotion

    PubMed Central

    Karmali-Rawji, Shameela; Kassim-Lakha, Shaheen; Taylor, Karmel

    1992-01-01

    Perceived lack or loss of control, stress, a rapidly again population and rising costs of health care necessitate effective health promotion and disease prevention in the elderly. In a collaborative health promotion effort, the private sector, public sector, and community partners have joined to increase the South Asian elders' sense of control over the decisions and circumstances that affect their everyday lives. The project was designed to help elders come to terms with the fragmentation of their extended families, cultural alienation, decreased autonomy, need for information, and greater risk of cardiovascular disease. Imagesp622-a

  4. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

    PubMed Central

    Rubin, Benjamin E R; Sanders, Jon G; Hampton-Marcell, Jarrad; Owens, Sarah M; Gilbert, Jack A; Moreau, Corrie S

    2014-01-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied. PMID:25257543

  5. Characterization of bacterial community associated with phytoplankton bloom in a eutrophic lake in South Norway using 16S rRNA gene amplicon sequence analysis.

    PubMed

    Parulekar, Niranjan Nitin; Kolekar, Pandurang; Jenkins, Andrew; Kleiven, Synne; Utkilen, Hans; Johansen, Anette; Sawant, Sangeeta; Kulkarni-Kale, Urmila; Kale, Mohan; Sæbø, Mona

    2017-01-01

    Interactions between different phytoplankton taxa and heterotrophic bacterial communities within aquatic environments can differentially support growth of various heterotrophic bacterial species. In this study, phytoplankton diversity was studied using traditional microscopic techniques and the bacterial communities associated with phytoplankton bloom were studied using High Throughput Sequencing (HTS) analysis of 16S rRNA gene amplicons from the V1-V3 and V3-V4 hypervariable regions. Samples were collected from Lake Akersvannet, a eutrophic lake in South Norway, during the growth season from June to August 2013. Microscopic examination revealed that the phytoplankton community was mostly represented by Cyanobacteria and the dinoflagellate Ceratium hirundinella. The HTS results revealed that Proteobacteria (Alpha, Beta, and Gamma), Bacteriodetes, Cyanobacteria, Actinobacteria and Verrucomicrobia dominated the bacterial community, with varying relative abundances throughout the sampling season. Species level identification of Cyanobacteria showed a mixed population of Aphanizomenon flos-aquae, Microcystis aeruginosa and Woronichinia naegeliana. A significant proportion of the microbial community was composed of unclassified taxa which might represent locally adapted freshwater bacterial groups. Comparison of cyanobacterial species composition from HTS and microscopy revealed quantitative discrepancies, indicating a need for cross validation of results. To our knowledge, this is the first study that uses HTS methods for studying the bacterial community associated with phytoplankton blooms in a Norwegian lake. The study demonstrates the value of considering results from multiple methods when studying bacterial communities.

  6. Gastrointestinal Bacterial and Methanogenic Archaea Diversity Dynamics Associated with Condensed Tannin-Containing Pine Bark Diet in Goats Using 16S rDNA Amplicon Pyrosequencing.

    PubMed

    Min, Byeng R; Solaiman, Sandra; Shange, Raymon; Eun, Jong-Su

    2014-01-01

    Eighteen Kiko-cross meat goats (n = 6) were used to collect gastrointestinal (GI) bacteria and methanogenic archaea for diversity measures when fed condensed tannin-containing pine bark (PB). Three dietary treatments were tested: control diet (0% PB and 30% wheat straw (WS); 0.17% condensed tannins (CT) dry matter (DM)); 15% PB and 15% WS (1.6% CT DM), and 30% PB and 0% WS (3.2% CT DM). A 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing technique was used to characterize and elucidate changes in GI bacteria and methanogenic archaea diversity among the diets. Proteobacteria was the most dominant phylum in goats with mean relative abundance values ranging from 39.7 (30% PB) to 46.5% (control) and 47.1% (15% PB). Other phyla individually accounted for fewer than 25% of the relative abundance observed. Predominant methanogens were Methanobrevibacter (75, 72, and 49%), Methanosphaera (3.3, 2.3, and 3.4%), and Methanobacteriaceae (1.2, 0.6, and 0.7%) population in control, 15, and 30% PB, respectively. Among methanogens, Methanobrevibacter was linearly decreased (P = 0.05) with increasing PB supplementation. These results indicate that feeding PB selectively altered bacteria and methanogenic archaeal populations in the GI tract of goats.

  7. Gastrointestinal Bacterial and Methanogenic Archaea Diversity Dynamics Associated with Condensed Tannin-Containing Pine Bark Diet in Goats Using 16S rDNA Amplicon Pyrosequencing

    PubMed Central

    Min, Byeng R.; Solaiman, Sandra; Shange, Raymon

    2014-01-01

    Eighteen Kiko-cross meat goats (n = 6) were used to collect gastrointestinal (GI) bacteria and methanogenic archaea for diversity measures when fed condensed tannin-containing pine bark (PB). Three dietary treatments were tested: control diet (0% PB and 30% wheat straw (WS); 0.17% condensed tannins (CT) dry matter (DM)); 15% PB and 15% WS (1.6% CT DM), and 30% PB and 0% WS (3.2% CT DM). A 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing technique was used to characterize and elucidate changes in GI bacteria and methanogenic archaea diversity among the diets. Proteobacteria was the most dominant phylum in goats with mean relative abundance values ranging from 39.7 (30% PB) to 46.5% (control) and 47.1% (15% PB). Other phyla individually accounted for fewer than 25% of the relative abundance observed. Predominant methanogens were Methanobrevibacter (75, 72, and 49%), Methanosphaera (3.3, 2.3, and 3.4%), and Methanobacteriaceae (1.2, 0.6, and 0.7%) population in control, 15, and 30% PB, respectively. Among methanogens, Methanobrevibacter was linearly decreased (P = 0.05) with increasing PB supplementation. These results indicate that feeding PB selectively altered bacteria and methanogenic archaeal populations in the GI tract of goats. PMID:24669219

  8. Characterization of bacterial community associated with phytoplankton bloom in a eutrophic lake in South Norway using 16S rRNA gene amplicon sequence analysis

    PubMed Central

    Parulekar, Niranjan Nitin; Kolekar, Pandurang; Jenkins, Andrew; Kleiven, Synne; Utkilen, Hans; Johansen, Anette; Sawant, Sangeeta; Kulkarni-Kale, Urmila; Kale, Mohan; Sæbø, Mona

    2017-01-01

    Interactions between different phytoplankton taxa and heterotrophic bacterial communities within aquatic environments can differentially support growth of various heterotrophic bacterial species. In this study, phytoplankton diversity was studied using traditional microscopic techniques and the bacterial communities associated with phytoplankton bloom were studied using High Throughput Sequencing (HTS) analysis of 16S rRNA gene amplicons from the V1-V3 and V3-V4 hypervariable regions. Samples were collected from Lake Akersvannet, a eutrophic lake in South Norway, during the growth season from June to August 2013. Microscopic examination revealed that the phytoplankton community was mostly represented by Cyanobacteria and the dinoflagellate Ceratium hirundinella. The HTS results revealed that Proteobacteria (Alpha, Beta, and Gamma), Bacteriodetes, Cyanobacteria, Actinobacteria and Verrucomicrobia dominated the bacterial community, with varying relative abundances throughout the sampling season. Species level identification of Cyanobacteria showed a mixed population of Aphanizomenon flos-aquae, Microcystis aeruginosa and Woronichinia naegeliana. A significant proportion of the microbial community was composed of unclassified taxa which might represent locally adapted freshwater bacterial groups. Comparison of cyanobacterial species composition from HTS and microscopy revealed quantitative discrepancies, indicating a need for cross validation of results. To our knowledge, this is the first study that uses HTS methods for studying the bacterial community associated with phytoplankton blooms in a Norwegian lake. The study demonstrates the value of considering results from multiple methods when studying bacterial communities. PMID:28282404

  9. The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons

    PubMed Central

    Starke, Ingo C.; Vahjen, Wilfried; Pieper, Robert; Zentek, Jürgen

    2014-01-01

    In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2 × 105 sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis. PMID:25120931

  10. Size Matters: Assessing Optimum Soil Sample Size for Fungal and Bacterial Community Structure Analyses Using High Throughput Sequencing of rRNA Gene Amplicons

    DOE PAGES

    Penton, C. Ryan; Gupta, Vadakattu V. S. R.; Yu, Julian; ...

    2016-06-02

    We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5, and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI) were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungalmore » community structure, replicate dispersion and the number of operational taxonomic units (OTUs) retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation.« less

  11. Size Matters: Assessing Optimum Soil Sample Size for Fungal and Bacterial Community Structure Analyses Using High Throughput Sequencing of rRNA Gene Amplicons

    SciTech Connect

    Penton, C. Ryan; Gupta, Vadakattu V. S. R.; Yu, Julian; Tiedje, James M.

    2016-06-02

    We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5, and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI) were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungal community structure, replicate dispersion and the number of operational taxonomic units (OTUs) retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation.

  12. The Use of Genus-Specific Amplicon Pyrosequencing to Assess Phytophthora Species Diversity Using eDNA from Soil and Water in Northern Spain

    PubMed Central

    Català, Santiago; Pérez-Sierra, Ana; Abad-Campos, Paloma

    2015-01-01

    Phytophthora is one of the most important and aggressive plant pathogenic genera in agriculture and forestry. Early detection and identification of its pathways of infection and spread are of high importance to minimize the threat they pose to natural ecosystems. eDNA was extracted from soil and water from forests and plantations in the north of Spain. Phytophthora-specific primers were adapted for use in high-throughput Sequencing (HTS). Primers were tested in a control reaction containing eight Phytophthora species and applied to water and soil eDNA samples from northern Spain. Different score coverage threshold values were tested for optimal Phytophthora species separation in a custom-curated database and in the control reaction. Clustering at 99% was the optimal criteria to separate most of the Phytophthora species. Multiple Molecular Operational Taxonomic Units (MOTUs) corresponding to 36 distinct Phytophthora species were amplified in the environmental samples. Pyrosequencing of amplicons from soil samples revealed low Phytophthora diversity (13 species) in comparison with the 35 species detected in water samples. Thirteen of the MOTUs detected in rivers and streams showed no close match to sequences in international sequence databases, revealing that eDNA pyrosequencing is a useful strategy to assess Phytophthora species diversity in natural ecosystems. PMID:25775250

  13. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

    SciTech Connect

    2014-12-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.

  14. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

    DOE PAGES

    None

    2014-12-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illuminamore » 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.« less

  15. Eubacterial 16S-rDNA amplicon profiling: a rapid technique for comparison and differentiation of heterotrophic plate count communities from drinking water.

    PubMed

    Farnleitner, Andreas H; Zibuschka, Franziska; Burtscher, Martina M; Lindner, Gerhard; Reischer, Georg; Mach, Robert L

    2004-05-01

    Determination of the heterotrophic plate count (HPC) is commonly used as a surrogate to assess the general microbial water quality in drinking water. For routine monitoring applications, the HPC is investigated in a quantitative way. However, qualitative data about the HPC bacterial community composition and/or population dynamics are required for particular situations. In order to provide fast and efficient qualitative approaches, molecular biological DNA profiling techniques seem to be suitable tools for the analysis of the total HPC community composition. In this work a DNA profiling technique is presented, which was recently demonstrated by our group to have potential for the rapid qualitative comparison and differentiation of HPC communities from raw and drinking water. The presented approach consists of a polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) for the generation of 16S-rDNA amplicon fingerprints from whole HPC community DNA extracts. In the context of this proceeding, the methodical background is presented and possible scientific merits as well as potential water management applications are discussed. Selected examples of (i) the demonstration of selective growth of HPC populations on different media and the comparison to the total in situ drinking water eubacterial community, (ii) the screening for HPC community variations at different locations of a drinking water distribution system, and (iii) the influence assessment on groundwater HPC communities by an infiltrating treated sewage effluent (bacterial source tracking) are given.

  16. 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.

  17. Microbial metagenome profiling using amplicon length heterogeneity-polymerase chain reaction proves more effective than elemental analysis in discriminating soil specimens.

    PubMed

    Moreno, Lilliana I; Mills, DeEtta K; Entry, James; Sautter, Robert T; Mathee, Kalai

    2006-11-01

    The combination of soil's ubiquity and its intrinsic abiotic and biotic information can contribute greatly to the forensic field. Although there are physical and chemical characterization methods of soil comparison for forensic purposes, these require a level of expertise not always encountered in crime laboratories. We hypothesized that soil microbial community profiling could be used to discriminate between soil types by providing biological fingerprints that confer uniqueness. Three of the six Miami-Dade soil types were randomly selected and sampled. We compared the microbial metagenome profiles generated using amplicon length heterogeneity-polymerase chain reaction analysis of the 16S rRNA genes with inductively coupled plasma optical emission spectroscopy analysis of 13 elements (Al, B, Ca, Cu, Fe, K, Mg, Mn, Na, P, S, Si, and Zn) that are commonly encountered in soils. Bray-Curtis similarity index and analysis of similarity were performed on all data to establish differences within sites, among sites, and across two seasons. These data matrices were used to group samples that shared similar community patterns using nonmetric multidimensional scaling analysis. We concluded that while chemical characterization could provide some differentiation between soils, microbial metagenome profiling was better able to discriminate between the soil types and had a high degree of reproducibility, therefore proving to be a potential tool for forensic soil comparisons.

  18. The utility of diversity profiling using Illumina 18S rRNA gene amplicon deep sequencing to detect and discriminate Toxoplasma gondii among the cyst-forming coccidia.

    PubMed

    Cooper, Madalyn K; Phalen, David N; Donahoe, Shannon L; Rose, Karrie; Šlapeta, Jan

    2016-01-30

    Next-generation sequencing (NGS) has the capacity to screen a single DNA sample and detect pathogen DNA from thousands of host DNA sequence reads, making it a versatile and informative tool for investigation of pathogens in diseased animals. The technique is effective and labor saving in the initial identification of pathogens, and will complement conventional diagnostic tests to associate the candidate pathogen with a disease process. In this report, we investigated the utility of the diversity profiling NGS approach using Illumina small subunit ribosomal RNA (18S rRNA) gene amplicon deep sequencing to detect Toxoplasma gondii in previously confirmed cases of toxoplasmosis. We then tested the diagnostic approach with species-specific PCR genotyping, histopathology and immunohistochemistry of toxoplasmosis in a Risso's dolphin (Grampus griseus) to systematically characterise the disease and associate causality. We show that the Euk7A/Euk570R primer set targeting the V1-V3 hypervariable region of the 18S rRNA gene can be used as a species-specific assay for cyst-forming coccidia and discriminate T. gondii. Overall, the approach is cost-effective and improves diagnostic decision support by narrowing the differential diagnosis list with more certainty than was previously possible. Furthermore, it supplements the limitations of cryptic protozoan morphology and surpasses the need for species-specific PCR primer combinations.

  19. Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes

    PubMed Central

    Kariya, Satomi; Kurokawa, Junji

    2014-01-01

    Screening of gene-specific amplicons from metagenomes (S-GAM) has tremendous biotechnological potential. We used this approach to isolate alcohol dehydrogenase (adh) genes from metagenomes based on the Leifsonia species adh gene (lsadh), the enzyme product of which can produce various chiral alcohols. A primer combination was synthesized by reference to homologs of lsadh, and PCR was used to amplify nearly full-length adh genes from metagenomic DNAs. All adh preparations were fused with lsadh at the terminal region and used to construct Escherichia coli plasmid libraries. Of the approximately 2,000 colonies obtained, 1,200 clones were identified as adh positive (∼60%). Finally, 40 adh genes, Hladh-001 to Hladh-040 (for homologous Leifsonia adh), were identified from 223 clones with high efficiency, which were randomly sequenced from the 1,200 clones. The Hladh genes obtained via this approach encoded a wide variety of amino acid sequences (8 to 99%). After screening, the enzymes obtained (HLADH-012 and HLADH-021) were confirmed to be superior to LSADH in some respects for the production of anti-Prelog chiral alcohols. PMID:25085492

  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. Parallel tagged amplicon sequencing of transcriptome-based genetic markers for Triturus newts with the Ion Torrent next-generation sequencing platform

    PubMed Central

    Wielstra, B; Duijm, E; Lagler, P; Lammers, Y; Meilink, W R M; Ziermann, J M; Arntzen, J W

    2014-01-01

    Next-generation sequencing is a fast and cost-effective way to obtain sequence data for nonmodel organisms for many markers and for many individuals. We describe a protocol through which we obtain orthologous markers for the crested newts (Amphibia: Salamandridae: Triturus), suitable for analysis of interspecific hybridization. We use transcriptome data of a single Triturus species and design 96 primer pairs that amplify c. 180 bp fragments positioned in 3-prime untranslated regions. Next, these markers are tested with uniplex PCR for a set of species spanning the taxonomical width of the genus Triturus. The 52 markers that consistently show a single band of expected length at gel electrophoreses for all tested crested newt species are then amplified in five multiplex PCRs (with a plexity of ten or eleven) for 132 individual newts: a set of 84 representing the seven (candidate) species and a set of 48 from a presumed hybrid population. After pooling multiplexes per individual, unique tags are ligated to link amplicons to individuals. Subsequently, individuals are pooled equimolar and sequenced on the Ion Torrent next-generation sequencing platform. A bioinformatics pipeline identifies the alleles and recodes these to a genotypic format. Next, we test the utility of our markers. baps allocates the 84 crested newt individuals representing (candidate) species to their expected (candidate) species, confirming the markers are suitable for species delineation. newhybrids, a hybrid index and hiest confirm the 48 individuals from the presumed hybrid population to be genetically admixed, illustrating the potential of the markers to identify interspecific hybridization. We expect the set of markers we designed to provide a high resolving power for analysis of hybridization in Triturus. PMID:24571307

  2. Rapid Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates Directly from Clinical Samples by Use of Amplicon Sequencing: a Proof-of-Concept Study

    PubMed Central

    Anderson, Julia; Lemmer, Darrin; Lehmkuhl, Erik; Georghiou, Sophia B.; Heaton, Hannah; Wiggins, Kristin; Gillece, John D.; Schupp, James M.; Catanzaro, Donald G.; Crudu, Valeriu; Cohen, Ted; Rodwell, Timothy C.; Engelthaler, David M.

    2016-01-01

    Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing of Mycobacterium tuberculosis DNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool. PMID:27225403

  3. Parallel tagged amplicon sequencing of transcriptome-based genetic markers for Triturus newts with the Ion Torrent next-generation sequencing platform.

    PubMed

    Wielstra, B; Duijm, E; Lagler, P; Lammers, Y; Meilink, W R M; Ziermann, J M; Arntzen, J W

    2014-09-01

    Next-generation sequencing is a fast and cost-effective way to obtain sequence data for nonmodel organisms for many markers and for many individuals. We describe a protocol through which we obtain orthologous markers for the crested newts (Amphibia: Salamandridae: Triturus), suitable for analysis of interspecific hybridization. We use transcriptome data of a single Triturus species and design 96 primer pairs that amplify c. 180 bp fragments positioned in 3-prime untranslated regions. Next, these markers are tested with uniplex PCR for a set of species spanning the taxonomical width of the genus Triturus. The 52 markers that consistently show a single band of expected length at gel electrophoreses for all tested crested newt species are then amplified in five multiplex PCRs (with a plexity of ten or eleven) for 132 individual newts: a set of 84 representing the seven (candidate) species and a set of 48 from a presumed hybrid population. After pooling multiplexes per individual, unique tags are ligated to link amplicons to individuals. Subsequently, individuals are pooled equimolar and sequenced on the Ion Torrent next-generation sequencing platform. A bioinformatics pipeline identifies the alleles and recodes these to a genotypic format. Next, we test the utility of our markers. baps allocates the 84 crested newt individuals representing (candidate) species to their expected (candidate) species, confirming the markers are suitable for species delineation. newhybrids, a hybrid index and hiest confirm the 48 individuals from the presumed hybrid population to be genetically admixed, illustrating the potential of the markers to identify interspecific hybridization. We expect the set of markers we designed to provide a high resolving power for analysis of hybridization in Triturus.

  4. Promoting Retention

    PubMed Central

    Hall, LaToya N.; Ficker, Lisa J.; Chadiha, Letha A.; Green, Carmen R.; Jackson, James S.; Lichtenberg, Peter A.

    2016-01-01

    Objectives: The objectives of this study were to evaluate the capability of a research volunteer registry to retain community-dwelling African American older adults, and to explore demographic and health factors associated with retention. Method: A logistic regression model was used to determine the influence of demographics, health factors, and registry logic model activities on retention in a sample of 1,730 older African American adults. Results: Almost 80% of participants active in the volunteer research registry between January 2012 and June 2015 were retained. Employment, being referred to research studies, a higher number of medical conditions, and more follow-up contacts were associated with an increased likelihood of retention. Older age, more months in the registry, and more mobility problems decreased the likelihood of retention. Discussion: These results suggest the Michigan Center for Urban African American Aging Research logic model promotes retention through involving older African American adults in research through study referrals and intensive follow-up. The loss of participants due to age- and mobility-related issues indicate the registry may be losing its most vulnerable participants. PMID:28138501

  5. Illumina MiSeq Phylogenetic Amplicon Sequencing Shows a Large Reduction of an Uncharacterised Succinivibrionaceae and an Increase of the Methanobrevibacter gottschalkii Clade in Feed Restricted Cattle

    PubMed Central

    McCabe, Matthew Sean; Cormican, Paul; Keogh, Kate; O’Connor, Aaron; O’Hara, Eoin; Palladino, Rafael Alejandro; Kenny, David Anthony; Waters, Sinéad Mary

    2015-01-01

    Periodic feed restriction is used in cattle production to reduce feed costs. When normal feed levels are resumed, cattle catch up to a normal weight by an acceleration of normal growth rate, known as compensatory growth, which is not yet fully understood. Illumina Miseq Phylogenetic marker amplicon sequencing of DNA extracted from rumen contents of 55 bulls showed that restriction of feed (70% concentrate, 30% grass silage) for 125 days, to levels that caused a 60% reduction of growth rate, resulted in a large increase of relative abundance of Methanobrevibacter gottschalkii clade (designated as OTU-M7), and a large reduction of an uncharacterised Succinivibrionaceae species (designated as OTU-S3004). There was a strong negative Spearman correlation (ρ = -0.72, P = <1x10-20) between relative abundances of OTU-3004 and OTU-M7 in the liquid rumen fraction. There was also a significant increase in acetate:propionate ratio (A:P) in feed restricted animals that showed a negative Spearman correlation (ρ = -0.69, P = <1x10-20) with the relative abundance of OTU-S3004 in the rumen liquid fraction but not the solid fraction, and a strong positive Spearman correlation with OTU-M7 in the rumen liquid (ρ = 0.74, P = <1x10-20) and solid (ρ = 0.69, P = <1x10-20) fractions. Reduced A:P ratios in the rumen are associated with increased feed efficiency and reduced production of methane which has a global warming potential (GWP 100 years) of 28. Succinivibrionaceae growth in the rumen was previously suggested to reduce methane emissions as some members of this family utilise hydrogen, which is also utilised by methanogens for methanogenesis, to generate succinate which is converted to propionate. Relative abundance of OTU-S3004 showed a positive Spearman correlation with propionate (ρ = 0.41, P = <0.01) but not acetate in the liquid rumen fraction. PMID:26226343

  6. Enhanced cytotoxicity with a novel system combining the paclitaxel-2'-ethylcarbonate prodrug and an HSV amplicon with an attenuated replication-competent virus, HF10 as a helper virus.

    PubMed

    Ishida, Daisuke; Nawa, Akihiro; Tanino, Tadatoshi; Goshima, Fumi; Luo, Chen Hong; Iwaki, Masahiro; Kajiyama, Hiroaki; Shibata, Kiyosumi; Yamamoto, Eiko; Ino, Kazuhiko; Tsurumi, Tatsuya; Nishiyama, Yukihiro; Kikkawa, Fumitaka

    2010-02-01

    We previously demonstrated that HF10, which is a natural, non-engineered HSV-1, has potent oncolytic activity in the treatment of solid malignant tumors in vitro and in vivo [H. Takakuwa, F. Goshima, N. Nozawa, T. Yoshikawa, H. Kimata, A. Nakao, et al., Oncolytic viral therapy using a spontaneously generated herpes simplex virus type 1 variant for disseminated peritoneal tumor in immunocompetent mice, Arch. Virol. 148 (2003) 813-825; S. Kohno, C. Lou, F. Goshima, Y. Nishiyama, T. Sata, Y. Ono, Herpes simplex virus type 1 mutant HF10 oncolytic viral therapy for bladder cancer, Urology 66 (2005) 1116-1121; D. Watanabe, F. Goshima, I. Mori, Y. Tamada, Y. Matsumoto, Y. Nishiyama, Oncolytic virotherapy for malignant melanoma with herpes simplex virus type 1 mutant HF10, J. Dermatol. Sci. 50 (2008) 185-196; A. Nawa, C. Luo, L. Zhang, Y. Ushijima, D. Ishida, M. Kamakura, et al., Non-engineered, naturally oncolytic herpes simplex virus HSV1 HF10: applications for cancer gene therapy, Curr. Gene. Ther. 8 (2008) 208-221]. Previous reports have also shown that a combination of HF10 and paclitaxel (TAX) was more efficacious than either regimen alone for some types of malignant tumors [S. Shimoyama, F. Goshima, O. Teshigahara, H. Kasuya, Y. Kodera, A. Nakao, et al., Enhanced efficacy of herpes simplex virus mutant HF10 combined with paclitaxel in peritoneal cancer dissemination models, Hepatogastroenterology 54 (2007) 1038-1042]. In this study, we investigated the efficacy of gene-directed enzyme prodrug therapy (GDEPT) using a novel system that combines the paclitaxel-2'-ethylcarbonate prodrug (TAX-2'-Et) and an HSV amplicon expressing rabbit-carboxylesterase (CES) with HF10 as a helper virus. This GDEPT system aims to produce high level of CES at the tumor site, resulting in efficient local conversion of the TAX-2'-Et prodrug into the active drug TAX [A. Nawa, T. Tanino, C. Lou, M. Iwaki, H. Kajiyama, K. Shibata, et al., Gene directed enzyme prodrug therapy for ovarian cancer

  7. Targeted Amplicon Sequencing for Single-Nucleotide-Polymorphism Genotyping of Attaching and Effacing Escherichia coli O26:H11 Cattle Strains via a High-Throughput Library Preparation Technique

    PubMed Central

    Delannoy, Sabine; Bugarel, Marie; Nagaraja, Tiruvoor G.; Renter, David G.; den Bakker, Henk C.; Nightingale, Kendra K.; Fach, Patrick; Loneragan, Guy H.

    2015-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O26:H11, a serotype within Shiga toxin-producing E. coli (STEC) that causes severe human disease, has been considered to have evolved from attaching and effacing E. coli (AEEC) O26:H11 through the acquisition of a Shiga toxin-encoding gene. Targeted amplicon sequencing using next-generation sequencing technology of 48 phylogenetically informative single-nucleotide polymorphisms (SNPs) and three SNPs differentiating Shiga toxin-positive (stx-positive) strains from Shiga toxin-negative (stx-negative) strains were used to infer the phylogenetic relationships of 178 E. coli O26:H11 strains (6 stx-positive strains and 172 stx-negative AEEC strains) from cattle feces to 7 publically available genomes of human clinical strains. The AEEC cattle strains displayed synonymous SNP genotypes with stx2-positive sequence type 29 (ST29) human O26:H11 strains, while stx1 ST21 human and cattle strains clustered separately, demonstrating the close phylogenetic relatedness of these Shiga toxin-negative AEEC cattle strains and human clinical strains. With the exception of seven stx-negative strains, five of which contained espK, three stx-related SNPs differentiated the STEC strains from non-STEC strains, supporting the hypothesis that these AEEC cattle strains could serve as a potential reservoir for new or existing pathogenic human strains. Our results support the idea that targeted amplicon sequencing for SNP genotyping expedites strain identification and genetic characterization of E. coli O26:H11, which is important for food safety and public health. PMID:26567298

  8. Developing a Promotional Video

    ERIC Educational Resources Information Center

    Epley, Hannah K.

    2014-01-01

    There is a need for Extension professionals to show clientele the benefits of their program. This article shares how promotional videos are one way of reaching audiences online. An example is given on how a promotional video has been used and developed using iMovie software. Tips are offered for how professionals can create a promotional video and…

  9. Promoting uranium immobilization by the activities of microbial phophatases

    SciTech Connect

    Sobecky, Patricia A.; Martial Taillefert

    2006-06-01

    The following is a summary of progress in our project ''Promoting uranium immobilization by the activities of microbial phosphatases'' during the second year of the project. (1). Assignment of microbial phosphatases to molecular classes. One objective of this project is to determine the relationship of phosphatase activity to metal resistance in subsurface strains and possible contributions of horizontal gene transfer (HGT) to the dissemination of nonspecific acid phosphatase genes. Non-specific acid phosphohydrolases are a broad group of secreted microbial phosphatases that function in acidic-to-neutral pH ranges and utilize a wide range of organophosphate substrates. To address this objective we have designed a collection of PCR primer sets based on known microbial acid phosphatase sequences. Genomic DNA is extracted from subsurface FRC isolates and amplicons of the expected sizes are sequenced and searched for conserved signature motifs. During this reporting period we have successfully designed and tested a suite of PCR primers for gram-positive and gram-negative groups of the following phosphatase classes: (1) Class A; (2) Class B; and (3) Class C (gram negative). We have obtained specific PCR products for each of the classes using the primers we have designed using control strains as well as with subsurface isolates.

  10. Strain-promoted "click" chemistry for terminal labeling of DNA.

    PubMed

    Marks, Isaac S; Kang, Jun Sung; Jones, Brady T; Landmark, Kevin J; Cleland, Andrew J; Taton, T Andrew

    2011-07-20

    1,3-Dipolar [3 + 2] cycloaddition between azides and alkynes--an archetypal "click" chemistry--has been used increasingly for the functionalization of nucleic acids. Copper(I)-catalyzed 1,3-dipolar cycloaddition reactions between alkyne-tagged DNA molecules and azides work well, but they require optimization of multiple reagents, and Cu ions are known to mediate DNA cleavage. For many applications, it would be preferable to eliminate the Cu(I) catalyst from these reactions. Here, we describe the solid-phase synthesis and characterization of 5'-dibenzocyclooctyne (DIBO)-modified oligonucleotides, using a new DIBO phosphoramidite, which react with azides via copper-free, strain-promoted alkyne-azide cycloaddition (SPAAC). We found that the DIBO group not only survived the standard acidic and oxidative reactions of solid-phase oligonucleotide synthesis (SPOS), but that it also survived the thermal cycling and standard conditions of the polymerase chain reaction (PCR). As a result, PCR with DIBO-modified primers yielded "clickable" amplicons that could be tagged with azide-modified fluorophores or immobilized on azide-modified surfaces. Given its simplicity, SPAAC on DNA could streamline the bioconjugate chemistry of nucleic acids in a number of modern biotechnologies.

  11. Health promotion in Brazil.

    PubMed

    Buss, Paulo Marchiori; de Carvalho, Antonio Ivo

    2007-01-01

    The evolution of health promotion within the Brazilian health system is examined, including an assessment of the intersectoral and development policies that have influenced the process. Particular attention is paid to the legal characteristics of the Unified Health System. Human resources formation and research initiatives in health promotion are outlined, with a summary of the obstacles that need to be overcome in order to ensure the effective implementation of health promotion in the future. Up to the end of the 20th Century health promotion was not used as a term in the Brazilian public heath context. Health promoting activities were concentrated in the area of health education, although targeting the social determinants of health and the principle of intersectoral action were part of the rhetoric. The situation has changed during the last decade, with the publication of a national policy of health promotion, issued by the Ministry of Health and jointly implemented with the States and Municipals Health Secretaries. More recently there has been a re-emergence of the discourse on the social determinants of health and the formation of intersectoral public policies as the basis of a comprehensive health promotion. Health promotion infrastructure, particularly around human resources and financing, requires strengthening in order to ensure capacity and sustainability in health promotion practice.

  12. High expression Zymomonas promoters

    DOEpatents

    Viitanen, Paul V.; Tao, Luan; Zhang, Yuying; Caimi, Perry G.; McCole, Laura : Zhang, Min; Chou, Yat-Chen; McCutchen, Carol M.; Franden, Mary Ann

    2011-08-02

    Identified are mutants of the promoter of the Z. mobilis glyceraldehyde-3-phosphate dehydrogenase gene, which direct improved expression levels of operably linked heterologous nucleic acids. These are high expression promoters useful for expression of chimeric genes in Zymomonas, Zymobacter, and other related bacteria.

  13. Health Promotion Program.

    ERIC Educational Resources Information Center

    McClary, Cheryl

    The Health Promotion Program began with establishment of a one-credit course in health promotion and wellness and the training of family practice residents at the Mountain Area Health Education Center to serve as lab leaders in the course. The course later became part of the university's general education requirements. In addition, a health…

  14. Promoting Resilience in Children.

    ERIC Educational Resources Information Center

    Rolfe, Sharne A.

    2002-01-01

    This booklet invites reflection on ways in which childhood resilience can be promoted, thereby helping children to adapt effectively in the face of adversity. The attributes of resilient children are described, as is the importance of protective factors in building or promoting resilience. The booklet discusses the complex interplay between risk…

  15. Dynein axonemal heavy chain 8 promotes androgen receptor activity and associates with prostate cancer progression

    PubMed Central

    Wang, Yu; Ledet, Russell J.; Imberg-Kazdan, Keren; Logan, Susan K.; Garabedian, Michael J.

    2016-01-01

    To gain insight into cellular factors regulating AR action that could promote castration resistant prostate cancer (CRPC), we performed a genome-wide RNAi screen for factors that promote ligand-independent AR transcriptional activity and integrated clinical databases for candidate genes that are positively associated with prostate cancer metastasis and recurrence. From this analysis, we identified Dynein Axonemal Heavy Chain 8 (DNAH8) as an AR regulator that displayed higher mRNA expression in metastatic than in primary tumors, and showed high expression in patients with poor prognosis. Axonemal dyneins function in cellular motility, but the function of DNAH8 in prostate cancer or other cell types has not been reported. DNAH8 is on chromosome 6q21.2, a cancer-associated amplicon, and is primarily expressed in prostate and testis. Its expression is higher in primary tumors compared to normal prostate, and is further increased in metastatic prostate cancers. Patients expressing high levels of DNAH8 have a greater risk of relapse and a poor prognosis after prostatectomy. Depletion of DNAH8 in prostate cancer cells suppressed AR transcriptional activity and proliferation. Androgen treatment increased DNAH8 mRNA expression, and AR bound the DNAH8 promoter sequence indicating DNAH8 is an AR target gene. Thus, DNAH8 is a new regulator of AR associated with metastatic tumors and poor prognosis. PMID:27363033

  16. 77 FR 47820 - Invention Promoters/Promotion Firms Complaints

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-10

    ... United States Patent and Trademark Office Invention Promoters/Promotion Firms Complaints ACTION: Proposed... participate in any legal proceedings against invention promoters or promotion firms. Complaints submitted to... promotion firm, explain the basis for the complaint, and include the signature of the complainant....

  17. Novelty and Promotional Items

    EPA Pesticide Factsheets

    Small novelty or promotional products, primarily used for outreach and educational purposes, must effectively convey a message, and their purchase will only be allowed if the item will contribute to the accomplishment of the Agency's mission.

  18. Promoting "Global" Attitudes.

    ERIC Educational Resources Information Center

    Case, Roland

    1996-01-01

    Discusses and illustrates three ways to promote prosocial attitudes towards global issues among students. Includes classroom environments that reinforce desired attitudes; facilitating direct "emotional" experiences that influence attitudes; and engaging students in thoughtful deliberation about global issues. Offers illustrative…

  19. Glycomacropeptide Sustains Microbiota Diversity and Promotes Specific Taxa in an Artificial Colon Model of Elderly Gut Microbiota.

    PubMed

    Ntemiri, Alexandra; Chonchúir, Fodhla Ní; O'Callaghan, Tom F; Stanton, Catherine; Ross, R Paul; O'Toole, Paul W

    2017-03-01

    The potential of milk-derived glycomacropeptide (GMP) and lactose for modulating the human gut microbiota of older people, in whom loss of diversity correlates with inferior health, was investigated. We used an in vitro batch fermentation (artificial colon model) to simulate colonic fermentation processes of two GMP products, i.e., a commercially available GMP concentrate and a semipurified GMP concentrate, and lactose. Faecal samples were collected from healthy and frail older people. Samples were analyzed by Illumina Miseq sequencing of rRNA gene amplicons. The commercial GMP preparation had a positive effect on the growth of Coprococcus and Clostridium cluster XIVb and sustained a higher faecal microbiota diversity compared to control substrates or lactose. Lactose fermentation promoted the growth of Proteobacteria including Escherichia/Shigella. This work provides an in-depth insight on the potential of GMP and lactose for modulating the gut microbiota and contributes more evidence confirming the prebiotic activity of GMP.

  20. The Dark Side of the Mushroom Spring Microbial Mat: Life in the Shadow of Chlorophototrophs. I. Microbial Diversity Based on 16S rRNA Gene Amplicons and Metagenomic Sequencing.

    PubMed

    Thiel, Vera; Wood, Jason M; Olsen, Millie T; Tank, Marcus; Klatt, Christian G; Ward, David M; Bryant, Donald A

    2016-01-01

    Microbial-mat communities in the effluent channels of Octopus and Mushroom Springs within the Lower Geyser Basin at Yellowstone National Park have been studied for nearly 50 years. The emphasis has mostly focused on the chlorophototrophic bacterial organisms of the phyla Cyanobacteria and Chloroflexi. In contrast, the diversity and metabolic functions of the heterotrophic community in the microoxic/anoxic region of the mat are not well understood. In this study we analyzed the orange-colored undermat of the microbial community of Mushroom Spring using metagenomic and rRNA-amplicon (iTag) analyses. Our analyses disclosed a highly diverse community exhibiting a high degree of unevenness, strongly dominated by a single taxon, the filamentous anoxygenic phototroph, Roseiflexus spp. The second most abundant organisms belonged to the Thermotogae, which have been hypothesized to be a major source of H2 from fermentation that could enable photomixotrophic metabolism by Chloroflexus and Roseiflexus spp. Other abundant organisms include two members of the Armatimonadetes (OP10); Thermocrinis sp.; and phototrophic and heterotrophic members of the Chloroflexi. Further, an Atribacteria (OP9/JS1) member; a sulfate-reducing Thermodesulfovibrio sp.; a Planctomycetes member; a member of the EM3 group tentatively affiliated with the Thermotogae, as well as a putative member of the Arminicenantes (OP8) represented ≥1% of the reads. Archaea were not abundant in the iTag analysis, and no metagenomic bin representing an archaeon was identified. A high microdiversity of 16S rRNA gene sequences was identified for the dominant taxon, Roseiflexus spp. Previous studies demonstrated that highly similar Synechococcus variants in the upper layer of the mats represent ecological species populations with specific ecological adaptations. This study suggests that similar putative ecotypes specifically adapted to different niches occur within the undermat community, particularly for Roseiflexus

  1. The Dark Side of the Mushroom Spring Microbial Mat: Life in the Shadow of Chlorophototrophs. I. Microbial Diversity Based on 16S rRNA Gene Amplicons and Metagenomic Sequencing

    PubMed Central

    Thiel, Vera; Wood, Jason M.; Olsen, Millie T.; Tank, Marcus; Klatt, Christian G.; Ward, David M.; Bryant, Donald A.

    2016-01-01

    Microbial-mat communities in the effluent channels of Octopus and Mushroom Springs within the Lower Geyser Basin at Yellowstone National Park have been studied for nearly 50 years. The emphasis has mostly focused on the chlorophototrophic bacterial organisms of the phyla Cyanobacteria and Chloroflexi. In contrast, the diversity and metabolic functions of the heterotrophic community in the microoxic/anoxic region of the mat are not well understood. In this study we analyzed the orange-colored undermat of the microbial community of Mushroom Spring using metagenomic and rRNA-amplicon (iTag) analyses. Our analyses disclosed a highly diverse community exhibiting a high degree of unevenness, strongly dominated by a single taxon, the filamentous anoxygenic phototroph, Roseiflexus spp. The second most abundant organisms belonged to the Thermotogae, which have been hypothesized to be a major source of H2 from fermentation that could enable photomixotrophic metabolism by Chloroflexus and Roseiflexus spp. Other abundant organisms include two members of the Armatimonadetes (OP10); Thermocrinis sp.; and phototrophic and heterotrophic members of the Chloroflexi. Further, an Atribacteria (OP9/JS1) member; a sulfate-reducing Thermodesulfovibrio sp.; a Planctomycetes member; a member of the EM3 group tentatively affiliated with the Thermotogae, as well as a putative member of the Arminicenantes (OP8) represented ≥1% of the reads. Archaea were not abundant in the iTag analysis, and no metagenomic bin representing an archaeon was identified. A high microdiversity of 16S rRNA gene sequences was identified for the dominant taxon, Roseiflexus spp. Previous studies demonstrated that highly similar Synechococcus variants in the upper layer of the mats represent ecological species populations with specific ecological adaptations. This study suggests that similar putative ecotypes specifically adapted to different niches occur within the undermat community, particularly for Roseiflexus

  2. The Dark Side of the Mushroom Spring Microbial Mat: Life in the Shadow of Chlorophototrophs. I. Microbial Diversity Based on 16S rRNA Gene Amplicons and Metagenomic Sequencing

    DOE PAGES

    Thiel, Vera; Wood, Jason M.; Olsen, Millie T.; ...

    2016-06-17

    Microbial-mat communities in the effluent channels of Octopus and Mushroom Springs within the Lower Geyser Basin at Yellowstone National Park have been studied for nearly 50 years. The emphasis has mostly focused on the chlorophototrophic bacterial organisms of the phyla Cyanobacteria and Chloroflexi. In contrast, the diversity and metabolic functions of the heterotrophic community in the microoxic/anoxic region of the mat are not well understood. In this study we analyzed the orange-colored undermat of the microbial community of Mushroom Spring using metagenomic and rRNA-amplicon (iTag) analyses. Our analyses disclosed a highly diverse community exhibiting a high degree of unevenness, stronglymore » dominated by a single taxon, the filamentous anoxygenic phototroph, Roseiflexus spp. The second most abundant organisms belonged to the Thermotogae, which have been hypothesized to be a major source of H-2 from fermentation that could enable photomixotrophic metabolism by Chloroflexus and Roseiflexus spp. Other abundant organisms include two members of the Armatimonadetes (OP10); Thermocrinis sp.; and phototrophic and heterotrophic members of the Chloroflexi. Further, an Atribacteria (OP9/JS1) member; a sulfate-reducing Therrnodesulfovibrio sp.; a Planctomycetes member; a member of the EM3 group tentatively affiliated with the Thermotogae, as well as a putative member of the Arrninicenantes (OP8) represented ≥ 1% of the reads. Archaea were not abundant in the iTag analysis, and no metagenomic bin representing an archaeon was identified. A high microdiversity of 16S rRNA gene sequences was identified for the dominant taxon, Roseiflexus spp. Previous studies demonstrated that highly similar Synechococcus variants in the upper layer of the mats represent ecological species populations with specific ecological adaptations. In conclusion, this study suggests that similar putative ecotypes specifically adapted to different niches occur within the undermat community

  3. TP0262 is a modulator of promoter activity of tpr Subfamily II genes of Treponema pallidum subsp. pallidum

    PubMed Central

    Giacani, Lorenzo; Godornes, Charmie; Puray-Chavez, Maritza; Guerra-Giraldez, Cristina; Tompa, Martin; Lukehart, Sheila A.; Centurion-Lara, Arturo

    2009-01-01

    Transcriptional regulation in Treponema pallidum subsp. pallidum is poorly understood, primarily because this organism cannot be cultivated in vitro or genetically manipulated. We have recently shown a phase variation mechanism controlling transcription initiation of Subfamily II tpr (T. pallidum repeat) genes (tprE, tprG, and tprJ), a group of virulence factor candidates. Furthermore, the same study suggested that additional mechanisms might influence the level of transcription of these tprs. The T. pallidum genome sequence has revealed a few open reading frames (ORFs) with similarity to known bacterial transcription factors (TFs), including four catabolite activator protein (CAP) homologs. In this work, sequences matching the E. coli cAMP receptor protein (CRP) binding motif were identified in silico upstream of tprE, tprG, and tprJ. Using elecrophoretic mobility shift assay (EMSA) and DNaseI footprinting assay, recombinant TP0262, a T. pallidum CRP homolog, was shown to bind specifically to amplicons obtained from the tpr promoters containing putative CRP binding motifs. Using a heterologous reporter system, binding of TP0262 to these promoters was shown to either increase (tprE and tprJ) or decrease (tprG) tpr promoter activity. This is the first characterization of a T. pallidum transcriptional modulator which influences tpr promoter activity. PMID:19432808

  4. Promoting La Cultura Hispana

    ERIC Educational Resources Information Center

    Pluviose, David

    2007-01-01

    Launched in 1985 at Arizona State University, the Hispanic Research Center's (HRC) efforts to promote Latino and Chicano art and issues have flourished in recent years. In 2004, the HRC hosted the Arizona International Latina/o Arts Festival in collaboration with the Mesa Southwest Museum. The HRC has also founded a mentoring institute for…

  5. The Ideal Promotion Effort.

    ERIC Educational Resources Information Center

    Morris, Edward L.

    The ideal promotional effort for an educational television (ETV) station is dependent on a professional approach to the problem. This means that each ETV station should employ a public relations manager and should keep him informed about all major station decisions. The Public Broadcasting Service (PBS) has a campaign of its own to bring attention…

  6. Health Promotion Interventions.

    ERIC Educational Resources Information Center

    Jason, Leonard A.; Curie, Carrie J.; Townsend, Stephanie M.; Pokorny, Steven B.; Katz, Richard B.; Sherk, Joseph L.

    2002-01-01

    Reviews four areas from the prevention science field, including: promoting healthy behavior; preventing substance abuse; preventing high-risk sexual behaviors; and preventing child abuse and sexual abuse. Recommendations are made regarding strategies for implementing empirically validated programs, supplementing school programs with ecological…

  7. Does Paid Promotion Pay?

    ERIC Educational Resources Information Center

    Scherer, Chris

    1980-01-01

    A study to determine the cost effectiveness of newspaper display advertising for cooperative extension programs showed such publicity to be more effective in large metropolitan areas. Though not a technique for increasing enrollment, this method provided long-range benefits in the overall promotion of extension educational services. (SK)

  8. Promoting Healthy Dietary Behaviors.

    ERIC Educational Resources Information Center

    Perry, Cheryl L.; Story, Mary; Lytle, Leslie A.

    This chapter reviews the research on promoting healthy dietary behaviors in all youth, not just those who exhibit problems such as obesity or eating disorders. The first section of this chapter presents a rationale for addressing healthy dietary behavior with children and adolescents, on the basis of the impact of these behaviors on short- and…

  9. Partners: Promoting Accessible Recreation.

    ERIC Educational Resources Information Center

    Sable, Janet; Gravink, Jill

    1995-01-01

    The Promoting Accessible Recreation through Networking, Education, Resources and Services (PARTNERS) Project, a partnership between Northeast Passage, the University of New Hampshire, and Granite State Independent Living Foundation, helps create barrier-free recreation for individuals with physical disabilities. The paper describes PARTNERS and…

  10. Promoting Continuing Education Programs.

    ERIC Educational Resources Information Center

    Hendrickson, Gayle A.

    This handbook is intended for use by institutions in marketing their continuing education programs. A section on "Devising Your Strategy" looks at identifying a target audience, determining the marketing approach, and developing a marketing plan and promotional techniques. A discussion of media options looks at the advantages and…

  11. 50 Practical Promotion Ideas.

    ERIC Educational Resources Information Center

    Madeyski, Tom

    1997-01-01

    Includes 50 cost-effective ideas for promoting camp in the areas of recruiting new campers, encouraging returning campers, advertising strategies, printing brochures and other written materials, using photographs, targeting groups for camp facility rental, and effectively using the media. (LP)

  12. Amplicon DNA melting analysis for the simultaneous detection of Brucella spp and Mycobacterium tuberculosis complex. Potential use in rapid differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.

    PubMed

    Sanjuan-Jimenez, Rocio; Colmenero, Juan D; Bermúdez, Pilar; Alonso, Antonio; Morata, Pilar

    2013-01-01

    Some sites of extrapulmonary tuberculosis and focal complications of brucellosis are very difficult to differentiate clinically, radiologically, and even histopathologically. Conventional microbiological methods for the diagnosis of extrapulmonary tuberculosis and complicated brucellosis not only lack adequate sensitivity, they are also time consuming, which could lead to an unfavourable prognosis. The aim of this work was to develop a multiplex real-time PCR assay based on SYBR Green I to simultaneously detect Brucella spp and Mycobacterium tuberculosis complex and evaluate the efficacy of the technique with different candidate genes. The IS711, bcsp31 and omp2a genes were used for the identification of Brucella spp and the IS6110, senX3-regX3 and cfp31 genes were targeted for the detection of the M. tuberculosis complex. As a result of the different combinations of primers, nine different reactions were evaluated. A test was defined as positive only when the gene combinations were capable of co-amplifying both pathogens in a single reaction tube and showed distinguishable melting temperatures for each microorganism. According to the melting analysis, only three combinations of amplicons (senX3-regX3+bcsp31, senX3-regX3+IS711 and IS6110+IS711) were visible. Detection limits of senX3-regX3+bcsp31 and senX3-regX3+IS711 were of 2 and 3 genome equivalents for M. tuberculosis complex and Brucella while for IS6110+IS711 they were of 200 and 300 genome equivalents, respectively. The three assays correctly identified all the samples, showing negative results for the control patients. The presence of multicopy elements and GC content were the components most influencing the efficiency of the test; this should be taken into account when designing a multiplex-based SYBR Green I assay. In conclusion, multiplex real time PCR assays based on the targets senX3-regX3+bcsp31 and senX3-regX3+IS711 using SYBR Green I are highly sensitive and reproducible. This may therefore be a

  13. The Dark Side of the Mushroom Spring Microbial Mat: Life in the Shadow of Chlorophototrophs. I. Microbial Diversity Based on 16S rRNA Gene Amplicons and Metagenomic Sequencing

    SciTech Connect

    Thiel, Vera; Wood, Jason M.; Olsen, Millie T.; Tank, Marcus; Klatt, Christian G.; Ward, David M.; Bryant, Donald A.

    2016-06-17

    Microbial-mat communities in the effluent channels of Octopus and Mushroom Springs within the Lower Geyser Basin at Yellowstone National Park have been studied for nearly 50 years. The emphasis has mostly focused on the chlorophototrophic bacterial organisms of the phyla Cyanobacteria and Chloroflexi. In contrast, the diversity and metabolic functions of the heterotrophic community in the microoxic/anoxic region of the mat are not well understood. In this study we analyzed the orange-colored undermat of the microbial community of Mushroom Spring using metagenomic and rRNA-amplicon (iTag) analyses. Our analyses disclosed a highly diverse community exhibiting a high degree of unevenness, strongly dominated by a single taxon, the filamentous anoxygenic phototroph, Roseiflexus spp. The second most abundant organisms belonged to the Thermotogae, which have been hypothesized to be a major source of H-2 from fermentation that could enable photomixotrophic metabolism by Chloroflexus and Roseiflexus spp. Other abundant organisms include two members of the Armatimonadetes (OP10); Thermocrinis sp.; and phototrophic and heterotrophic members of the Chloroflexi. Further, an Atribacteria (OP9/JS1) member; a sulfate-reducing Therrnodesulfovibrio sp.; a Planctomycetes member; a member of the EM3 group tentatively affiliated with the Thermotogae, as well as a putative member of the Arrninicenantes (OP8) represented ≥ 1% of the reads. Archaea were not abundant in the iTag analysis, and no metagenomic bin representing an archaeon was identified. A high microdiversity of 16S rRNA gene sequences was identified for the dominant taxon, Roseiflexus spp. Previous studies demonstrated that highly similar Synechococcus variants in the upper layer of the mats represent ecological species populations with specific ecological adaptations. In conclusion, this study suggests

  14. The promotion of breastfeeding.

    PubMed

    Tuluhungwa, R R; Yung, W

    1979-01-01

    To reverse the current trend of a significant decline worldwide in breast feeding means reeducation of medical and health personnel as well as the general public. Programs to promote breast feeding require the commitment of governments, with support from various ministries including health, education, labor, community development and judiciary. Examples of what 3 developing countries--Jamaica, Colombia and Thailand--are doing to promote breast feeding are reported. A large scale breast feeding campaign was launched in Jamaica in October 1977. The 3 phases of the campaign were: 1) preliminary surveys and research and motivation of professional, voluntary and extension groups through training seminars, panel discussions, and meetings; 2) promotion of breast feeding via mass media and motivation of target groups by trained personnel; and 3) evaluation of the campaign. A survey undertaken in 1978 showed that the breast feeding messages had achieved the desired effect--more mothers practiced breast feeding. In Colombia the breast feeding campaign emphasized non-formal education through the use of games and pictures. A game is used which is usually initiated by a health worker in the waiting room of a health center and involves the mothers, the general public, and sometimes the professional personnel. Through reading and interpreting rhymed breast feeding messages, the participants exchange opinions and experiences. Before starting a campaign to encourage low-income urban and semi-urban mothers to breast feed, the National Food and Nutrition Committee of Thailand pretested slogans and posters designed for the promotion of breast feeding. Posters develpoed in accordance with the suggestions made by the women were tested among 126 pregnant and lactating women. The Committee decided which picture to print for low-income and rural audiences and which to print for middle-class audiences.

  15. Democracy Promotion in Oman

    DTIC Science & Technology

    2013-01-01

    succession process . The “Ruling Family Council” will meet to designate a successor within 3 days of Qaboos’s death. If it fails to select someone, it will...the Gulf is to increase democracy promotion programs designed to encourage timely and peaceful transitions to more representative forms of govern...stronger efforts toward reform supported by the United States could foster a more peaceful political transformation. This process should begin with the

  16. Publishing and academic promotion.

    PubMed

    Dixon, A K

    2009-09-01

    Clearly, academic endeavour has to be the single most important criterion for appointment to an academic position and for subsequent promotion. It is rare for excellence either in teaching or clinical practice to offset a poor publication record. However, the pressure to publish and gain related grant income can lead to problems in the normal academic pursuits of a department or institution. These and other related issues will be explored in this editorial.

  17. Bicycle Promotion Plan

    SciTech Connect

    Simone, G. A.

    1981-03-09

    The objective of this Bicycle Promotion Plan is to outline a set of recommendations and supporting strategies for implementation by the US DOE toward increased use of the bicycle for energy conservation. The recommendations are designed in such a way as to function in concert with: (1) bicycle programs administered by other Federal government agencies; and (2) related programs and activities already sponsored by DOE. The approach to preparation of the Plan involved a review of all current and planned bicycle promotion programs at the Federal level as well as a review of the array of lierature on the subject. The UniWorld project staff also interacted with several DOE program offices, in order to determine the extent to which they might appropriately contribute to the implementation of bicycle promotional efforts. A synthesis of all the information gathered was published in January of 1981 as a part of the project (The Bicycle Program Review). Based upon this information and an examination of the barriers to bicycle use identified by bicycle transportation specialists in the field, UniWorld developed a series of the most potentially effective recommendations and program strategies for implementation by DOE. The recommendations address activities that could be undertaken in conjunction with existing DOE programs, new developments that might be considered to fulfill critical needs in the field, and interagency efforts that DOE could play a role in.

  18. The dynamics of mobile promoters: Enhanced stability in promoter regions.

    PubMed

    Rabbani, Mahnaz; Wahl, Lindi M

    2016-10-21

    Mobile promoters are emerging as a new class of mobile genetic elements, first identified by examining prokaryote genome sequences, and more recently confirmed by experimental observations in bacteria. Recent datasets have identified over 40,000 putative mobile promoters in sequenced prokaryote genomes, however only one-third of these are in regions of the genome directly upstream from coding sequences, that is, in promoter regions. The presence of many promoter sequences in non-promoter regions is unexplained. Here we develop a general mathematical model for the dynamics of mobile promoters, extending previous work to capture the dynamics both within and outside promoter regions. From this general model, we apply rigorous model selection techniques to identify which parameters are statistically justified in describing the available mobile promoter data, and find best-fit values of these parameters. Our results suggest that high rates of horizontal gene transfer maintain the population of mobile promoters in promoter regions, and that once established at these sites, mobile promoters are rarely lost, but are commonly copied to other genomic regions. In contrast, mobile promoter copies in non-promoter regions are more numerous and more volatile, experiencing substantially higher rates of duplication, loss and diversification.

  19. Human Promoters Are Intrinsically Directional

    PubMed Central

    Duttke, Sascha H.C.; Lacadie, Scott A.; Ibrahim, Mahmoud M.; Glass, Christopher K.; Corcoran, David L.; Benner, Christopher; Heinz, Sven; Kadonaga, James T.; Ohler, Uwe

    2015-01-01

    Divergent transcription, in which reverse-oriented transcripts occur upstream of eukaryotic promoters in regions devoid of annotated genes, has been suggested to be a general property of active promoters. Here we show that the human basal RNA polymerase II transcriptional machinery and core promoter are inherently unidirectional, and that reverse-oriented transcripts originate from their own cognate reverse-directed core promoters. In vitro transcription analysis and mapping of nascent transcripts in cells revealed that sequences at reverse start sites are similar to those of their forward counterparts. The use of DNase I accessibility to define proximal promoter borders revealed that up to half of promoters are unidirectional and that unidirectional promoters are depleted at their upstream edges of reverse core promoter sequences and their associated chromatin features. Divergent transcription is thus not an inherent property of the transcription process, but rather the consequence of the presence of both forward- and reverse-directed core promoters. PMID:25639469

  20. PROMOTIONS: PROper MOTION Software

    NASA Astrophysics Data System (ADS)

    Caleb Wherry, John; Sahai, R.

    2009-05-01

    We report on the development of a software tool (PROMOTIONS) to streamline the process of measuring proper motions of material in expanding nebulae. Our tool makes use of IDL's widget programming capabilities to design a unique GUI that is used to compare images of the objects from two epochs. The software allows us to first orient and register the images to a common frame of reference and pixel scale, using field stars in each of the images. We then cross-correlate specific morphological features in order to determine their proper motions, which consist of the proper motion of the nebula as a whole (PM-neb), and expansion motions of the features relative to the center. If the central star is not visible (quite common in bipolar nebulae with dense dusty waists), point-symmetric expansion is assumed and we use the average motion of high-quality symmetric pairs of features on opposite sides of the nebular center to compute PM-neb. This is then subtracted out to determine the individual movements of these and additional features relative to the nebular center. PROMOTIONS should find wide applicability in measuring proper motions in astrophysical objects such as the expanding outflows/jets commonly seen around young and dying stars. We present first results from using PROMOTIONS to successfully measure proper motions in several pre-planetary nebulae (transition objects between the red giant and planetary nebula phases), using images taken 7-10 years apart with the WFPC2 and ACS instruments on board HST. The authors are grateful to NASA's Undergradute Scholars Research Program (USRP) for supporting this research.

  1. Promoting healthy sleep.

    PubMed

    Price, Bob

    2016-03-09

    Nurses are accustomed to helping others with their sleep problems and dealing with issues such as pain that may delay or interrupt sleep. However, they may be less familiar with what constitutes a healthy night's sleep. This article examines what is known about the process and purpose of sleep, and examines the ways in which factors that promote wakefulness and sleep combine to help establish a normal circadian rhythm. Theories relating to the function of sleep are discussed and research is considered that suggests that sleep deficit may lead to metabolic risks, including heart disease, obesity, type 2 diabetes mellitus and several types of cancer.

  2. Students Promoted Despite Test Failure

    ERIC Educational Resources Information Center

    Wakefield, Dara V.

    2012-01-01

    "No Child Left Behind" (NCLB) dictates students in Grades 3, 5, and 8 pass state tests to be promoted. Accordingly, most state education codes require students to pass reading and math exams for promotion. The majority of those who fail, however, appear to be promoted anyway. This article addresses core questions concerning the paradigm…

  3. TWEAK Promotes Peritoneal Inflammation

    PubMed Central

    Sanz, Ana Belen; Aroeira, Luiz Stark; Bellon, Teresa; del Peso, Gloria; Jimenez-Heffernan, Jose; Santamaria, Beatriz; Sanchez-Niño, Maria Dolores; Blanco-Colio, Luis Miguel; Lopez-Cabrera, Manuel; Ruiz-Ortega, Marta; Egido, Jesus; Selgas, Rafael; Ortiz, Alberto

    2014-01-01

    Peritoneal dialysis (PD) is complicated by peritonitis episodes that cause loss of mesothelium and eventually sclerosing peritonitis. An improved understanding of the molecular contributors to peritoneal injury and defense may increase the therapeutic armamentarium to optimize peritoneal defenses while minimizing peritoneal injury. There is no information on the expression and function of the cytokine TWEAK and its receptor Fn14 during peritoneal injury. Fn14 expression and soluble TWEAK levels were measured in human PD peritoneal effluent cells or fluids with or without peritonitis. Fn14 expression was also analyzed in peritoneal biopsies from PD patients. Actions of intraperitoneal TWEAK were studied in mice in vivo. sTWEAK levels were increased in peritoneal effluent in PD peritonitis. Effluent sTWEAK levels correlated with the number of peritoneal macrophages (r = 0.491, p = 0.002). Potential TWEAK targets that express the receptor Fn14 include mesothelial cells and macrophages, as demonstrated by flow cytometry of peritoneal effluents and by analysis of peritoneal biopsies. Peritoneal biopsy Fn14 correlated with mesothelial injury, fibrosis and inflammation, suggesting a potential deleterious effect of TWEAK/Fn14. In this regard, intraperitoneal TWEAK administration to mice promoted peritoneal inflammation characterized by increased peritoneal effluent MCP-1, Fn14 and Gr1+ macrophages, increased mesothelial Fn14, MCP-1 and CCL21 expression and submesothelial tissue macrophage recruitment. Taken together these data suggest that the TWEAK/Fn14 system may promote inflammation and tissue injury during peritonitis and PD. PMID:24599047

  4. Leptin Promotes Glioblastoma

    PubMed Central

    Lawrence, Johnathan E.; Cook, Nicholas J.; Rovin, Richard A.; Winn, Robert J.

    2012-01-01

    The hormone leptin has a variety of functions. Originally known for its role in satiety and weight loss, leptin more recently has been shown to augment tumor growth in a variety of cancers. Within gliomas, there is a correlation between tumor grade and tumor expression of leptin and its receptor. This suggests that autocrine signaling within the tumor microenvironment may promote the growth of high-grade gliomas. Leptin does this through stimulation of cellular pathways that are also advantageous for tumor growth and recurrence: antiapoptosis, proliferation, angiogenesis, and migration. Conversely, a loss of leptin expression attenuates tumor growth. In animal models of colon cancer and melanoma, a decline in the expression and secretion of leptin resulted in a reduction of tumor growth. In these models, positive mental stimulation through environmental enrichment decreased leptin secretion and improved tumor outcome. This review explores the link between leptin and glioblastoma. PMID:22263109

  5. Love promotes health.

    PubMed

    Esch, Tobias; Stefano, George B

    2005-06-01

    Love has consequences for health and well-being. Engaging in joyful activities such as love may activate areas in the brain responsible for emotion, attention, motivation and memory (i.e., limbic structures), and it may further serve to control the autonomic nervous system, i.e., stress reduction. This specific CNS activity pattern appears to exert protective effects, even on the brain itself. Moreover, anxiolytic effects of pleasurable experiences may occur by promotion of an inhibitory tone in specific areas of the brain. Thus, love and pleasure clearly are capable of stimulating health, well-being and (re)productivity: This wonderful biological instrument makes procreation and maintenance of organisms and their species a deeply rewarding and pleasurable experience, thus ensuring survival, health, and perpetuation.

  6. Surface Protonics Promotes Catalysis

    NASA Astrophysics Data System (ADS)

    Manabe, R.; Okada, S.; Inagaki, R.; Oshima, K.; Ogo, S.; Sekine, Y.

    2016-12-01

    Catalytic steam reforming of methane for hydrogen production proceeds even at 473 K over 1 wt% Pd/CeO2 catalyst in an electric field, thanks to the surface protonics. Kinetic analyses demonstrated the synergetic effect between catalytic reaction and electric field, revealing strengthened water pressure dependence of the reaction rate when applying an electric field, with one-third the apparent activation energy at the lower reaction temperature range. Operando–IR measurements revealed that proton conduction via adsorbed water on the catalyst surface occurred during electric field application. Methane was activated by proton collision at the Pd–CeO2 interface, based on the inverse kinetic isotope effect. Proton conduction on the catalyst surface plays an important role in methane activation at low temperature. This report is the first describing promotion of the catalytic reaction by surface protonics.

  7. Surface Protonics Promotes Catalysis

    PubMed Central

    Manabe, R.; Okada, S.; Inagaki, R.; Oshima, K.; Ogo, S.; Sekine, Y.

    2016-01-01

    Catalytic steam reforming of methane for hydrogen production proceeds even at 473 K over 1 wt% Pd/CeO2 catalyst in an electric field, thanks to the surface protonics. Kinetic analyses demonstrated the synergetic effect between catalytic reaction and electric field, revealing strengthened water pressure dependence of the reaction rate when applying an electric field, with one-third the apparent activation energy at the lower reaction temperature range. Operando–IR measurements revealed that proton conduction via adsorbed water on the catalyst surface occurred during electric field application. Methane was activated by proton collision at the Pd–CeO2 interface, based on the inverse kinetic isotope effect. Proton conduction on the catalyst surface plays an important role in methane activation at low temperature. This report is the first describing promotion of the catalytic reaction by surface protonics. PMID:27905505

  8. Sales promotions and food consumption.

    PubMed

    Hawkes, Corinna

    2009-06-01

    Sales promotions are widely used to market food to adults, children, and youth. Yet, in contrast to advertising, practically no attention has been paid to their impacts on dietary behaviors, or to how they may be used more effectively to promote healthy eating. This review explores the available literature on the subject. The objective is to identify if and what literature exists, examine the nature of this literature, and analyze what can be learned from it about the effects of sales promotions on food consumption. The review finds that while sales promotions lead to significant sales increases over the short-term, this does not necessarily lead to changes in food-consumption patterns. Nevertheless, there is evidence from econometric modeling studies indicating that sales promotions can influence consumption patterns by influencing the purchasing choices of consumers and encouraging them to eat more. These effects depend on the characteristics of the food product, sales promotion, and consumer. The complexity of the effects means that sales promotions aiming to encourage consumption of nutritious foods need to be carefully designed. These conclusions are based on studies that use mainly sales data as a proxy for dietary intake. The nutrition (and economics) research communities should add to this existing body of research to provide evidence on the impact of sales promotions on dietary intake and related behaviors. This would help support the development of a sales promotion environment conducive to healthy eating.

  9. Environmental Health Promotion: Bridging Traditional Environmental Health and Health Promotion

    ERIC Educational Resources Information Center

    Howze, Elizabeth H.; Baldwin, Grant T.; Kegler, Michelle Crozier

    2004-01-01

    This article highlights the juncture between environmental health and health promotion and underscores the need for health promotion involvement in environmental health practice. It begins with a synopsis of current issues in environmental public health and deficiencies in environmental public health practice that could be partly ameliorated by an…

  10. 7 CFR 1219.22 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.22 Promotion. Promotion means any action to advance the image, desirability, or marketability of Hass...

  11. 7 CFR 1219.22 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.22 Promotion. Promotion means any action to advance the image, desirability, or marketability of Hass...

  12. 7 CFR 1219.22 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.22 Promotion. Promotion means any action to advance the image, desirability, or marketability of Hass...

  13. 7 CFR 1219.22 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.22 Promotion. Promotion means any action to advance the image, desirability, or marketability of Hass...

  14. 7 CFR 1217.22 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOFTWOOD LUMBER RESEARCH, PROMOTION, CONSUMER EDUCATION AND INDUSTRY INFORMATION ORDER Softwood Lumber Research, Promotion, Consumer Education, and Industry Information Order Definitions § 1217.22 Promotion. Promotion means any action...

  15. 7 CFR 1217.22 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOFTWOOD LUMBER RESEARCH, PROMOTION, CONSUMER EDUCATION AND INDUSTRY INFORMATION ORDER Softwood Lumber Research, Promotion, Consumer Education, and Industry Information Order Definitions § 1217.22 Promotion. Promotion means any action...

  16. 7 CFR 1217.22 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOFTWOOD LUMBER RESEARCH, PROMOTION, CONSUMER EDUCATION AND INDUSTRY INFORMATION ORDER Softwood Lumber Research, Promotion, Consumer Education, and Industry Information Order Definitions § 1217.22 Promotion. Promotion means any action...

  17. Health-promoting schools: an opportunity for oral health promotion.

    PubMed Central

    Kwan, Stella Y. L.; Petersen, Poul Erik; Pine, Cynthia M.; Borutta, Annerose

    2005-01-01

    Schools provide an important setting for promoting health, as they reach over 1 billion children worldwide and, through them, the school staff, families and the community as a whole. Health promotion messages can be reinforced throughout the most influential stages of children's lives, enabling them to develop lifelong sustainable attitudes and skills. Poor oral health can have a detrimental effect on children's quality of life, their performance at school and their success in later life. This paper examines the global need for promoting oral health through schools. The WHO Global School Health Initiative and the potential for setting up oral health programmes in schools using the health-promoting school framework are discussed. The challenges faced in promoting oral health in schools in both developed and developing countries are highlighted. The importance of using a validated framework and appropriate methodologies for the evaluation of school oral health projects is emphasized. PMID:16211159

  18. Promoting Leadership in Australian Universities

    ERIC Educational Resources Information Center

    Bradley, Andrew P.; Grice, Tim; Paulsen, Neil

    2017-01-01

    In this paper we review current practices for developing and promoting academic leadership in universities. We consider the forms of leadership that are appropriate for academic organisations, while exploring the types of leadership favoured by recruitment and promotion committees. Using the Australian higher education context as a case study, we…

  19. ACTFL's Accent! on Promoting FL's.

    ERIC Educational Resources Information Center

    Accent on ACTFL, 1975

    1975-01-01

    This insert in "Accent on ACTFL" is devoted to ways of promoting the study of foreign languages. The first section is a comic book, "The Continuing Story of Captain Fore Lang," created as an assignment by education students. The comic points out several benefits of language study. The second section is a language-specific promotion focusing on…

  20. Promoting Reading in Developing Countries.

    ERIC Educational Resources Information Center

    Greaney, Vincent, Ed.

    With the intention of illuminating the many obstacles involved with literacy promotion in the developing nations of Africa, Asia, and South America, the authors of the 10 articles in this collection share their knowledge and experience of literacy promotion in the developing world--including the unique challenges faced by those who publish, print,…

  1. Promoting Creativity in Young Children.

    ERIC Educational Resources Information Center

    Honig, Alice Sterling

    This paper discusses creativity in young children and what teachers can do to support and promote it. Topics addressed in the paper include: (1) teacher interest in promoting creativity; (2) defining creativity; (3) creativity in the socioemotional domain; (4) the relationship between creativity and empathy for others; (4) bibliotherapy; (5)…

  2. Information technology in health promotion.

    PubMed

    Lintonen, T P; Konu, A I; Seedhouse, D

    2008-06-01

    eHealth, the use of information technology to improve or enable health and health care, has recently been high on the health care development agenda. Given the vivid interest in eHealth, little reference has been made to the use of these technologies in the promotion of health. The aim of this present study was to conduct a review on recent uses of information technology in health promotion through looking at research articles published in peer-reviewed journals. Fifteen relevant journals with issues published between 2003 and June 2005 yielded altogether 1352 articles, 56 of which contained content related to the use of information technology in the context of health promotion. As reflected by this rather small proportion, research on the role of information technology is only starting to emerge. Four broad thematic application areas within health promotion were identified: use of information technology as an intervention medium, use of information technology as a research focus, use of information technology as a research instrument and use of information technology for professional development. In line with this rather instrumental focus, the concepts 'ePromotion of Health' or 'Health ePromotion' would come close to describing the role of information technology in health promotion.

  3. Health promotion: an ethical analysis.

    PubMed

    Carter, Stacy M

    2014-04-01

    Thinking and practising ethically requires reasoning systematically about the right thing to do. Health promotion ethics - a form of applied ethics - includes analysis of health promotion practice and how this can be ethically justified. Existing frameworks can assist in such evaluation. These acknowledge the moral value of delivering benefits. But benefits need to be weighed against burdens, harms or wrongs, and these should be minimised: they include invading privacy, breaking confidentiality, restraining liberty, undermining self-determination or people's own values, or perpetuating injustice. Thinking about the ethics of health promotion also means recognising health promotion as a normative ideal: a vision of the good society. This ideal society values health, sees citizens as active and includes them in decisions that affect them, and makes the state responsible for providing all of its citizens, no matter how advantaged or disadvantaged, with the conditions and resources they need to be healthy. Ethicists writing about health promotion have focused on this relationship between the citizen and the state. Comparing existing frameworks, theories and the expressed values of practitioners themselves, we can see common patterns. All oppose pursuing an instrumental, individualistic, health-at-all-costs vision of health promotion. And all defend the moral significance of just processes: those that engage with citizens in a transparent, inclusive and open way. In recent years, some Australian governments have sought to delegitimise health promotion, defining it as extraneous to the role of the state. Good evidence is not enough to counter this trend, because it is founded in competing visions of a good society. For this reason, the most pressing agenda for health promotion ethics is to engage with communities, in a procedurally just way, about the role and responsibilities of the citizen and the state in promoting and maintaining good health.

  4. Photovoltaic module with adhesion promoter

    SciTech Connect

    Xavier, Grace

    2013-10-08

    Photovoltaic modules with adhesion promoters and methods for fabricating photovoltaic modules with adhesion promoters are described. A photovoltaic module includes a solar cell including a first surface and a second surface, the second surface including a plurality of interspaced back-side contacts. A first glass layer is coupled to the first surface by a first encapsulating layer. A second glass layer is coupled to the second surface by a second encapsulating layer. At least a portion of the second encapsulating layer is bonded directly to the plurality of interspaced back-side contacts by an adhesion promoter.

  5. Zoning should promote public health.

    PubMed

    Hirschhorn, Joel S

    2004-01-01

    Legally, governments use their police powers to protect public health, safety, and welfare through zoning. This paper presents a case for revisiting zoning on the basis of increasing evidence that certain types of community design promote public health, as opposed to the dominant pattern of sprawl development, which does not. Zoning, and the land use planning linked to it, that prohibits or disfavors health-promoting community designs contradicts the inherent public policy goal on which it is based. If there is a paradigm shift underway, from traditional sprawl to health-promoting community designs, then health professionals and others should understand why zoning must be reassessed.

  6. Detection of p16INK4a promoter methylation status in non-small cell lung cancer by a fluorescence polarization assay.

    PubMed

    Song, Zujun; Zhou, Rongbin; Li, Ding; Chen, Yanan; Liang, Ping; Liu, Wenchao; Zhang, Ju

    2011-09-01

    The detection of the p16INK4a promoter methylation status has a good value for the prognosis, early detection, and individualized management of patients with non-small cell lung cancer. A novel method detecting the p16INK4a promoter methylation status of primary carcinoma tissue samples by a fluorescence polarization assay was developed in this research. A pair of general primers was used to amplify a 305-basepair fragment in the promoter region of p16INK4a. Two probes specific for either methylated p16INK4a or unmethylated p16INK4a DNA labeled with either tetramethyl 6-carboxyrhodamine or 6-carboxy-fluorescein hybridized, respectively, with their target amplicons, and the hybridization increased the fluorescence polarization values. The p16INK4a promoter methylation status was determined by the analysis of the fluorescence polarization values. One hundred and twenty-nine non-small cell lung cancer samples were analyzed in parallel with a fluorescence polarization assay and a gel-based methylation-specific polymerase chain reaction (PCR) assay. There was no significant difference between the results of the p16INK4a promoter methylation status obtained with the fluorescence polarization assay and the results obtained with the gel-based methylation-specific PCR assay. The minimum detection level of the fluorescence polarization assay was 25 copies/μL. The fluorescence polarization assay allowed the semiautomated detection of the methylated p16INK4a and unmethylated p16INK4a promoters directly in the solution with 1 PCR cycle, and it was much simpler than methylation-specific PCR and methylation-specific multiplex ligation-dependent probe amplification assays.

  7. Nutritional Recommendation Should Promote Sustainability.

    ERIC Educational Resources Information Center

    Reber, Robert J.

    1991-01-01

    Any process or event that disrupts the flow of nutrients and energy becomes a nutrition problem. Nutritionists should promote practices that protect the integrity, stability, and beauty of the land community (soil, water, air, all biological species). (Author)

  8. The Business of Radio Promotion.

    ERIC Educational Resources Information Center

    Hall, Jonathan

    This speech suggests that public radio stations should examine and use the techniques employed by commercial stations to increase their listening audience--creative promotion based on community involvement and participation. Some examples are included. (SC)

  9. [Five paradoxes in health promotion].

    PubMed

    López-Dicastillo, Olga; Canga-Armayor, Navidad; Mujika, Agurtzane; Pardavila-Belio, Miren Idoia; Belintxon, Maider; Serrano-Monzó, Inmaculada; Pumar-Méndez, María J

    2017-02-17

    The World Health Organization states that health promotion is a key strategy to improve health, and it is conceived as a global process of enabling people to increase control over, and to improve, their health. Health promotion does not focus solely on empowering individuals dealing with their knowledge, attitudes and skills, but it also takes political, social, economic and environmental aspects influencing health and wellbeing into account. The complexity of applying these concepts is reflected in the five paradoxes in health promotion; these arise in between the rhetoric in health promotion and implementation. The detected paradoxes which are described herein involve the patient versus the person, the individual versus the group, disease professionals versus health professionals, disease indicators versus health indicators, and health as an expense versus health as an investment. Making these contradictions explicit can help determine why it is so complex to put the concepts related to health promotion into practice. It can also help to put forward aspects that need further work if health promotion is to put into practice.

  10. 7 CFR 1260.122 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1260.122 Section 1260.122 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BEEF PROMOTION AND RESEARCH Beef Promotion and Research Order Definitions § 1260.122 Promotion. Promotion means any action, including...

  11. 7 CFR 1160.111 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 9 2010-01-01 2009-01-01 true Promotion. 1160.111 Section 1160.111 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE FLUID MILK PROMOTION PROGRAM Fluid Milk Promotion Order Definitions § 1160.111 Promotion. Promotion means the following activities: (a) Consumer Education,...

  12. 7 CFR 1250.310 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1250.310 Section 1250.310 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE EGG RESEARCH AND PROMOTION Egg Research and Promotion Order Definitions § 1250.310 Promotion. Promotion means any action, including...

  13. 7 CFR 1206.17 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1206.17 Section 1206.17 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MANGO PROMOTION, RESEARCH, AND INFORMATION Mango Promotion, Research, and Information Order Definitions § 1206.17 Promotion. Promotion...

  14. 7 CFR 1150.114 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 9 2010-01-01 2009-01-01 true Promotion. 1150.114 Section 1150.114 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE DAIRY PROMOTION PROGRAM Dairy Promotion and Research Order Definitions § 1150.114 Promotion. Promotion means actions such as paid advertising,...

  15. 7 CFR 1210.312 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1210.312 Section 1210.312 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE WATERMELON RESEARCH AND PROMOTION PLAN Watermelon Research and Promotion Plan Definitions § 1210.312 Promotion. Promotion means...

  16. Creating and Promoting a Natural History Collection.

    ERIC Educational Resources Information Center

    Belben, Cathy

    2003-01-01

    Discusses the value of developing and promoting a natural history library by school library media specialists. Topics include benefits to students; promoting outdoor education; recommended reading for high school students; using technology; and other aids to promote outdoor education. (LRW)

  17. DNA signals at isoform promoters

    PubMed Central

    Dai, Zhiming; Xiong, Yuanyan; Dai, Xianhua

    2016-01-01

    Transcriptional heterogeneity is extensive in the genome, and most genes express variable transcript isoforms. However, whether variable transcript isoforms of one gene are regulated by common promoter elements remain to be elucidated. Here, we investigated whether isoform promoters of one gene have separated DNA signals for transcription and translation initiation. We found that TATA box and nucleosome-disfavored DNA sequences are prevalent in distinct transcript isoform promoters of one gene. These DNA signals are conserved among species. Transcript isoform has a RNA-determined unstructured region around its start site. We found that these DNA/RNA features facilitate isoform transcription and translation. These results suggest a DNA-encoded mechanism by which transcript isoform is generated. PMID:27353836

  18. Introduction to Global Health Promotion.

    PubMed

    Torres, Jennifer

    2017-03-01

    Global health education is becoming increasingly prominent in universities throughout the country especially in programs focused on health and behavioral sciences, law, economics, and political science. Introduction to Global Health Promotion is a book that can be used by both instructors and students in the field of global health. The book provides theories and models, human rights, and technology relevant to the field. In addition the book is designed to share best evidence for promoting health and reducing morbidity and mortality in a variety of areas. The book can be used by health educators, public health practitioners, professors, and students as a resource for research and practice in the field of health promotion and disease prevention.

  19. Promoter Hypermethylation in Prostate Cancer

    PubMed Central

    Park, Jong Y.

    2011-01-01

    Background The prostate gland is the most common site of cancer and the second leading cause of cancer mortality in American men. It is well known that epigenetic alterations such as DNA methylation within the regulatory (promoter) regions of genes are associated with transcriptional silencing in cancer. Promoter hypermethylation of critical pathway genes could be potential biomarkers and therapeutic targets for prostate cancer. Methods This review discusses current information on methylated genes associated with prostate cancer development and progression. Results Over 30 genes have been investigated for promoter methylation in prostate cancer. These methylated genes are involved in critical pathways, such as DNA repair, metabolism, and invasion/metastasis. The role of hypermethylated genes in regulation of critical pathways in prostate cancer is reviewed. Conclusions These findings may provide new information of the pathogenesis of prostate cancer. Certain epigenetic alterations in prostate tumors are being translated into clinical practice for therapeutic use. PMID:20861812

  20. AAHD's Health Promotion and Wellness, Part 2: Health Promotion Programs

    ERIC Educational Resources Information Center

    Exceptional Parent, 2011

    2011-01-01

    This article is part 2 of a 4-part series on "Health Promotion and Wellness" from the American Association on Health and Disability (AAHD). According to the U.S. Census Bureau, more than 54 million people--one in five Americans--have a disability, and these Americans are more likely to report: (1) Being in poorer overall health; (2) Having less…

  1. Promoting recovery from ischemic stroke.

    PubMed

    Schmidt, Antje; Minnerup, Jens

    2016-01-01

    Over recent decades, experimental and clinical stroke studies have identified a number of neurorestorative treatments that stimulate neural plasticity and promote functional recovery. In contrast to the acute stroke treatments thrombolysis and endovascular thrombectomy, neurorestorative treatments are still effective when initiated days after stroke onset, which makes them applicable to virtually all stroke patients. In this article, selected physical, pharmacological and cell-based neurorestorative therapies are discussed, with special emphasis on interventions that have already been transferred from the laboratory to the clinical setting. We explain molecular and structural processes that promote neural plasticity, discuss potential limitations of neurorestorative treatments, and offer a speculative viewpoint on how neurorestorative treatments will evolve.

  2. Synthetic Core Promoters for Pichia pastoris

    PubMed Central

    2013-01-01

    Synthetic promoters are commonly used tools for circuit design or high level protein production. Promoter engineering efforts in yeasts, such as Saccharomyces cerevisiae and Pichia pastoris have mostly been focused on altering upstream regulatory sequences such as transcription factor binding sites. In higher eukaryotes synthetic core promoters, directly needed for transcription initiation by RNA Polymerase II, have been successfully designed. Here we report the first synthetic yeast core promoter for P. pastoris, based on natural yeast core promoters. Furthermore we used this synthetic core promoter sequence to engineer the core promoter of the natural AOX1 promoter, thereby creating a set of core promoters providing a range of different expression levels. As opposed to engineering strategies of the significantly longer entire promoter, such short core promoters can directly be added on a PCR primer facilitating library generation and are sufficient to obtain variable expression yields. PMID:24187969

  3. Genetic and phenotypic diversity of plant growth promoting rhizobacteria isolated from sugarcane plants growing in pakistan.

    PubMed

    Mehnaz, Samina; Baig, Deeba Noreen; Lazarovits, George

    2010-12-01

    Bacteria were isolated from roots of sugarcane varieties grown in the fields of Punjab. They were identified by using API20E/NE bacterial identification kits and from sequences of 16S rRNA and amplicons of the cpn60 gene. The majority of bacteria were found to belong to the genera of Enterobacter, Pseudomonas, and Klebsiella, but members of genera Azospirillum, Rhizobium, Rahnella, Delftia, Caulobacter, Pannonibacter, Xanthomonas, and Stenotrophomonas were also found. The community, however, was dominated by members of the Pseudomonadaceae and Enterobacteriaceae, as representatives of these genera were found in samples from every variety and location examined. All isolates were tested for the presence of five enzymes and seven factors known to be associated with plant growth promotion. Ten isolates showed lipase activity and eight were positive for protease activity. Cellulase, chitinase, and pectinase were not detected in any strain. Nine strains showed nitrogen fixing ability (acetylene reduction assay) and 26 were capable of solubilizing phosphate. In the presence of 100 mg/l tryptophan, all strains except one produced indole acetic acid in the growth medium. All isolates were positive for ACC deaminase activity. Six strains produced homoserine lactones and three produced HCN and hexamate type siderophores. One isolate was capable of inhibiting the growth of 24 pathogenic fungal strains of Colletotrichum, Fusarium, Pythium, and Rhizoctonia spp. In tests of their abilities to grow under a range of temperature, pH, and NaCl concentrations, all isolates grew well on plates with 3% NaCl and most of them grew well at 4 to 41degrees C and at pH 11.

  4. Health Promotion at the Ballpark.

    PubMed

    Hodges, Bonni C

    2017-03-01

    The arrival of a new summer collegiate baseball league franchise to a small central New York city was seen as an opportunity for health promotion. The initiative was set up to explore two overarching questions: (1) Are summer collegiate baseball events acceptable to local public health organizations as viable places for health promotion activities addressing local health issues? (2) Are summer collegiate baseball organizations amenable to health promotion activities built in to their fan and/or player experiences? Planning and implementation were guided by precede-proceed, social cognitive theory, social marketing, and diffusion of innovations constructs. Environmental changes were implemented to support healthy eating and nontobacco use by players and fans; four health awareness nights were implemented at home games corresponding to local public health priorities and included public service announcements, between inning quizzes, information dissemination at concession and team market locations, and special guests. Sales and fan feedback support mostly healthy concession offerings and a tobacco-free ballpark; postseason evaluations from team staff and public health partners support continuing the trials of this sports event as a venue for health promotion.

  5. Teachers Promoting Student Mathematical Reasoning

    ERIC Educational Resources Information Center

    Mueller, Mary; Yankelewitz, Dina; Maher, Carolyn

    2014-01-01

    During an informal, after-school, math program, a group of middle school students worked collaboratively on open-ended problems. The students co-constructed arguments, provided justifications for their solutions, and engaged in mathematical reasoning. This paper describes the specific teacher moves that promoted this phenomenon. The findings of…

  6. Women's Studies Online: Promoting Visibility.

    ERIC Educational Resources Information Center

    Hildenbrand, Suzanne

    1986-01-01

    Three problems in online searching in women's studies are type of indexing, database quality, and adequacy of coverage. Results of a recent study show there is significant end-user satisfaction and the searches done are described in order to promote greater and more effective use of online retrieval in women's studies. (Author/EM)

  7. Using Data to Promote Equity

    ERIC Educational Resources Information Center

    Shum, Brenda

    2016-01-01

    Data plays a starring role in promoting educational equity, and data-driven decision making begins with good state policies. With the recent passage of the Every Student Succeeds Act (ESSA) and a proposed federal rule to address racial disproportionality in special education, states will shoulder increased responsibility for eliminating…

  8. The Rhetoric of "Promoting Health."

    ERIC Educational Resources Information Center

    Hamilton, Margaret

    2002-01-01

    Uses Chaim Perelman's theories of argumentation to examine a recent Institute of Medicine report, "Promoting Health: Intervention Strategies from Social and Behavioral Research" (2000). Notes that it focuses on social, economic, behavioral, and political health as a means of assuring population health--and thereby expands the…

  9. Promoting Inclusive Education in Ghana

    ERIC Educational Resources Information Center

    Djietror, Beauty B. K.; Okai, Edward; Kwapong, Olivia A. T. Frimpong

    2011-01-01

    Inclusive education is critical for nation building. The government of Ghana has put in measures for promoting inclusion from basic through to tertiary level of education. Some of these measures include expansion of school facilities, implementation of the Free Compulsory Universal Basic Education (FCUBE); the change of policy on girls who drop…

  10. Promoting Discussions in ESL Students

    ERIC Educational Resources Information Center

    Navarro, Ann

    2010-01-01

    Background: Teachers who work with English as a Second Language (ESL) students, struggle with promoting discussion during guided reading. When ESL students are asked comprehension questions during group discussions and throughout the reading of a book, often teachers receive minimal feedback. Purpose: The purpose of this research is to identify…

  11. Promoting and Assessing Mathematical Generalising

    ERIC Educational Resources Information Center

    Hill, Tiffany; Lannin, John; van Garderen, Delinda

    2015-01-01

    Helping students generalise mathematical ideas is an essential component of teaching and learning of mathematics (Lannin, Ellis, Elliott & Zbiek, 2011). However, it can be challenging for primary teachers to assess and promote generalisation. Because generalisation is an essential part of mathematics instruction, the authors highlight the…

  12. Advertising and Sales Promotion Guide.

    ERIC Educational Resources Information Center

    North Carolina State Dept. of Public Instruction, Raleigh. Div. of Vocational Education.

    This document contains teacher materials for a 4-unit, 1-year marketing education course in advertising and sales promotion offered in grades 11 and 12 in North Carolina. The preface contains a rationale for the development of the course, a course description, course objectives, a list of the instructional units of the course, and a list of the…

  13. Promoting SETI in the UK

    NASA Astrophysics Data System (ADS)

    Penny, Alan

    2013-10-01

    MEETING REPORT What does the UK presently do in the search for extraterrestrial intelligence and what are the plans for the future? Alan Penny reports on a meeting of UK academics active in SETI, held as sessions in the recent National Astronomy Meeting in Scotland - and the formation of the UK SETI Research Network to promote UK academic work.

  14. University Festival Promotes STEM Education

    ERIC Educational Resources Information Center

    Quagliata, Andrew B.

    2015-01-01

    STEM education is argued as an essential ingredient in preparing our children for careers of the future. This study describes a university festival that includes the promotion of STEM-related career interests in young people among its goals. A total of 203 participants between the age of 7 and 17 completed both pre-event and post-event surveys. In…

  15. Promoting Multiculturalism in Developmental Education.

    ERIC Educational Resources Information Center

    Higbee, Jeanne L.

    2001-01-01

    Asserts that the teaching profession needs to recognize the natural connections between multicultural and developmental education. Presents eight steps developmental educators can take to promote pluralism, including (1) establishing a clear link between cultural pluralism and institutional and programmatic mission and goals; (2) striving for…

  16. Promoting science together with art

    NASA Astrophysics Data System (ADS)

    Peacock, John

    2011-08-01

    The divide between science and art will forever be fuel for discussion, as two articles in the June issue of Physics World show. In the first, Leonardo Colletti (p16) calls for physicists to invite poets to conferences, while in the second (p19) Robert P Crease urges us to take insights from the study of culture when we try to promote science.

  17. Promoting Community Cohesion in England

    ERIC Educational Resources Information Center

    Morris, Andrew B.; McDaid, Maggie; Potter, Hugh

    2011-01-01

    Following serious disturbances in some northern cities in England in 2001, concerns about possible rising inter-communal tension have led to a statutory duty to promote community cohesion being placed on schools. Inspectors from the Office for Standards in Education (Ofsted) are required to make judgements in the leadership and management section…

  18. Ecological Foundations of Health Promotion.

    ERIC Educational Resources Information Center

    Green, Lawrence W.; And Others

    1996-01-01

    In this article, human ecology is defined; its historical and intellectual roots are traced through various disciplines to its applications in public health and health promotion today; its strengths and limitations are described; some potential contributions to systems theory are suggested; and some emerging ecological models of health promotion…

  19. Promoting Metacognition in Music Classes

    ERIC Educational Resources Information Center

    Benton, Carol W.

    2013-01-01

    Metacognition is a type of thinking in which learners think about their own cognitive processes. Because it transcends disciplines and grade levels, metacognition is useful in many educational settings and can be transferred from the music classroom to other subject areas. Music educators can promote metacognition by designing and implementing…

  20. Plant-growth-promoting rhizobacteria.

    PubMed

    Lugtenberg, Ben; Kamilova, Faina

    2009-01-01

    Several microbes promote plant growth, and many microbial products that stimulate plant growth have been marketed. In this review we restrict ourselves to bacteria that are derived from and exert this effect on the root. Such bacteria are generally designated as PGPR (plant-growth-promoting rhizobacteria). The beneficial effects of these rhizobacteria on plant growth can be direct or indirect. This review begins with describing the conditions under which bacteria live in the rhizosphere. To exert their beneficial effects, bacteria usually must colonize the root surface efficiently. Therefore, bacterial traits required for root colonization are subsequently described. Finally, several mechanisms by which microbes can act beneficially on plant growth are described. Examples of direct plant growth promotion that are discussed include (a) biofertilization, (b) stimulation of root growth, (c) rhizoremediation, and (d) plant stress control. Mechanisms of biological control by which rhizobacteria can promote plant growth indirectly, i.e., by reducing the level of disease, include antibiosis, induction of systemic resistance, and competition for nutrients and niches.

  1. When Promoting Democracy Is Counterproductive

    ERIC Educational Resources Information Center

    Esfandiari, Haleh; Litwak, Robert S.

    2007-01-01

    The United States has begun a $75-million program to promote democracy by supporting Iranian nongovernmental organizations (NGOs). That program, coupled with loose talk about regime change from members of Congress, commentators close to the administration, and individuals within the administration, has fed a sense of vulnerability and paranoia…

  2. Mutation analysis of TRPS1 gene including core promoter, 5'UTR, and 3'UTR regulatory sequences with insight into their organization.

    PubMed

    Solc, Roman; Klugerova, Michaela; Vcelak, Josef; Baxova, Alice; Kuklik, Miloslav; Vseticka, Jan; Beharka, Rastislav; Hirschfeldova, Katerina

    2017-01-01

    The TRPS1 protein is a potent regulator of proliferation, differentiation, and apoptosis. The TRPS1 gene aberrations are strongly associated with rare trichorhinophalangeal syndrome (TRPS) development. We have conducted MLPA analysis to capture deletion within the crucial 8q24.1 chromosomal region in combination with mutation analysis of TRPS1 gene including core promoter, 5'UTR, and 3'UTR sequences in nine TRPS patients. Low complexity or extent of untranslated regulatory sequences avoided them from analysis in previous studies. Amplicon based next generation sequencing used in our study bridge over these technical limitations. Finally, we have made extended in silico analysis of TRPS1 gene regulatory sequences organization. Single contiguous deletion and an intragenic deletion intervening several exons were detected. Mutation analysis revealed five TRPS1 gene aberrations (two structural rearrangements, two nonsense mutations, and one missense substitution) reaching the overall detection rate of 78%. Several polymorphic variants were detected within the analysed regulatory sequences but without proposed pathogenic effect. In silico analysis suggested alternative promoter usage and diverse expression effectivity for different TRPS1 transcripts. Haploinsufficiency of TRPS1 gene was responsible for most of the TRPS phenotype. Structure of TRPS1 gene regulatory sequences is indicative of generally low single allele expression and its tight control.

  3. 7 CFR 1221.23 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.23 Promotion. Promotion means any action taken to present a favorable image of sorghum to the public and the...

  4. 7 CFR 1221.23 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.23 Promotion. Promotion means any action taken to present a favorable image of sorghum to the public and the...

  5. 7 CFR 1221.23 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.23 Promotion. Promotion means any action taken to present a favorable image of sorghum to the public and the...

  6. 7 CFR 1221.23 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.23 Promotion. Promotion means any action taken to present a favorable image of sorghum to the public and the...

  7. 7 CFR 1218.17 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.17 Promotion. Promotion means any action taken to present a favorable image of blueberries to the general public and...

  8. 7 CFR 1218.17 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.17 Promotion. Promotion means any action taken to present a favorable image of blueberries to the general public and...

  9. 7 CFR 1218.17 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.17 Promotion. Promotion means any action taken to present a favorable image of blueberries to the general public and...

  10. 7 CFR 1218.17 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.17 Promotion. Promotion means any action taken to present a favorable image of blueberries to the general public and...

  11. 7 CFR 1216.23 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.23 Promotion. Promotion... image of peanuts to the public to improve the competitive position of peanuts in the...

  12. 7 CFR 1216.23 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.23 Promotion. Promotion... image of peanuts to the public to improve the competitive position of peanuts in the...

  13. 7 CFR 1216.23 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.23 Promotion. Promotion... image of peanuts to the public to improve the competitive position of peanuts in the...

  14. 7 CFR 1216.23 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.23 Promotion. Promotion... image of peanuts to the public to improve the competitive position of peanuts in the...

  15. 7 CFR 1216.23 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1216.23 Section 1216.23 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.23 Promotion....

  16. 78 FR 24239 - Temporary Mailing Promotion

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-24

    ... Temporary Mailing Promotion AGENCY: Postal Regulatory Commission. ACTION: Notice. SUMMARY: The Commission is... with offering a Technology Credit Promotion. This notice informs the public of the Postal Service's... changes associated with offering a Technology Credit Promotion.\\1\\ The promotion is planned to begin...

  17. 29 CFR 541.503 - Promotion work.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 3 2010-07-01 2010-07-01 false Promotion work. 541.503 Section 541.503 Labor Regulations... Outside Sales Employees § 541.503 Promotion work. (a) Promotion work is one type of activity often.... Promotion activities directed toward consummation of the employee's own sales are exempt....

  18. 76 FR 34871 - Mobile Barcode Promotion

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-15

    ... 111 Mobile Barcode Promotion AGENCY: Postal Service TM . ACTION: Final rule. SUMMARY: The Postal... ) 709.4 to add a temporary promotion for First-Class Mail cards, letters and flats, and Standard Mail... barcode promotion, and the new mailing standards to implement the promotion. To be eligible,...

  19. 7 CFR 1280.118 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1280.118 Section 1280.118 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE LAMB PROMOTION, RESEARCH, AND INFORMATION ORDER Lamb Promotion, Research, and Information Order Definitions § 1280.118 Promotion....

  20. 5 CFR 532.407 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Promotion. 532.407 Section 532.407... Administration § 532.407 Promotion. (a) An employee who is promoted is entitled to be paid at the lowest.... (c) If the promotion is to a position in a different wage area, the agency shall determine...

  1. 7 CFR 1212.20 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1212.20 Section 1212.20 Agriculture..., PROMOTION, CONSUMER EDUCATION AND INDUSTRY INFORMATION ORDER Honey Packers and Importers Research, Promotion, Consumer Education, and Industry Information Order Definitions § 1212.20 Promotion. “Promotion” means...

  2. 7 CFR 1206.17 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MANGO PROMOTION, RESEARCH, AND INFORMATION Mango Promotion, Research, and Information Order Definitions § 1206.17 Promotion. Promotion means any action taken to present a favorable image of mangos to the general public and the food...

  3. 7 CFR 1206.17 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MANGO PROMOTION, RESEARCH, AND INFORMATION Mango Promotion, Research, and Information Order Definitions § 1206.17 Promotion. Promotion means any action taken to present a favorable image of mangos to the general public and the food...

  4. 7 CFR 1206.17 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MANGO PROMOTION, RESEARCH, AND INFORMATION Mango Promotion, Research, and Information Order Definitions § 1206.17 Promotion. Promotion means any action taken to present a favorable image of mangos to the general public and the food...

  5. 7 CFR 1206.17 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MANGO PROMOTION, RESEARCH, AND INFORMATION Mango Promotion, Research, and Information Order Definitions § 1206.17 Promotion. Promotion means any action taken to present a favorable image of mangos to the general public and the food...

  6. 7 CFR 1210.312 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE WATERMELON RESEARCH AND PROMOTION PLAN Watermelon Research and Promotion Plan Definitions § 1210.312 Promotion. Promotion means any action taken by the Board, pursuant to the Act, to present a favorable image for watermelons to...

  7. 7 CFR 1210.312 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE WATERMELON RESEARCH AND PROMOTION PLAN Watermelon Research and Promotion Plan Definitions § 1210.312 Promotion. Promotion means any action taken by the Board, pursuant to the Act, to present a favorable image for watermelons to...

  8. 7 CFR 1210.312 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE WATERMELON RESEARCH AND PROMOTION PLAN Watermelon Research and Promotion Plan Definitions § 1210.312 Promotion. Promotion means any action taken by the Board, pursuant to the Act, to present a favorable image for watermelons to...

  9. 7 CFR 1210.312 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE WATERMELON RESEARCH AND PROMOTION PLAN Watermelon Research and Promotion Plan Definitions § 1210.312 Promotion. Promotion means any action taken by the Board, pursuant to the Act, to present a favorable image for watermelons to...

  10. 21 CFR 601.45 - Promotional materials.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Promotional materials. 601.45 Section 601.45 Food... Promotional materials. For biological products being considered for approval under this subpart, unless... preapproval review period copies of all promotional materials, including promotional labeling as well...

  11. 21 CFR 601.45 - Promotional materials.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Promotional materials. 601.45 Section 601.45 Food... Promotional materials. For biological products being considered for approval under this subpart, unless... preapproval review period copies of all promotional materials, including promotional labeling as well...

  12. 21 CFR 601.45 - Promotional materials.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Promotional materials. 601.45 Section 601.45 Food... Promotional materials. For biological products being considered for approval under this subpart, unless... preapproval review period copies of all promotional materials, including promotional labeling as well...

  13. 21 CFR 601.94 - Promotional materials.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Promotional materials. 601.94 Section 601.94 Food... Promotional materials. For biological products being considered for approval under this subpart, unless... preapproval review period copies of all promotional materials, including promotional labeling as well...

  14. 21 CFR 601.94 - Promotional materials.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Promotional materials. 601.94 Section 601.94 Food... Promotional materials. For biological products being considered for approval under this subpart, unless... preapproval review period copies of all promotional materials, including promotional labeling as well...

  15. 21 CFR 601.45 - Promotional materials.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Promotional materials. 601.45 Section 601.45 Food... Promotional materials. For biological products being considered for approval under this subpart, unless... preapproval review period copies of all promotional materials, including promotional labeling as well...

  16. 21 CFR 601.94 - Promotional materials.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Promotional materials. 601.94 Section 601.94 Food... Promotional materials. For biological products being considered for approval under this subpart, unless... preapproval review period copies of all promotional materials, including promotional labeling as well...

  17. Electrochemical promotion of catalytic reactions

    NASA Astrophysics Data System (ADS)

    Imbihl, R.

    2010-05-01

    The electrochemical promotion of heterogeneously catalyzed reactions (EPOC) became feasible through the use of porous metal electrodes interfaced to a solid electrolyte. With the O 2- conducting yttrium stabilized zirconia (YSZ), the Na + conducting β″-Al 2O 3 (β-alumina), and several other types of solid electrolytes the EPOC effect has been demonstrated for about 100 reaction systems in studies conducted mainly in the mbar range. Surface science investigations showed that the physical basis for the EPOC effect lies in the electrochemically induced spillover of oxygen and alkali metal, respectively, onto the surface of the metal electrodes. For the catalytic promotion effect general concepts and mechanistic schemes were proposed but these concepts and schemes are largely speculative. Applying surface analytical tools to EPOC systems the proposed mechanistic schemes can be verified or invalidated. This report summarizes the progress which has been achieved in the mechanistic understanding of the EPOC effect.

  18. [Empowerment and health promotion programming].

    PubMed

    Laverack, G

    2008-12-01

    Health promotion often presents a tension between "bottom up" and "top down" programming. "Bottom-up" is associated with community empowerment and begins on issues of concern to particular groups or individuals and regards an increase in overall control as an important element of the health outcome. "Top-down" is associated with disease prevention efforts and begins by seeking to involve beneficiaries on issues defined by health agencies. It regards improvements in health behaviours or bio-medical indicators as the important outcome and community empowerment is viewed simply as a means to the end of health behaviour change. The tension between these two approaches is not unresolvable, and this article presents a framework, the "parallel-track", intended to assist health promotion practitioners to systematically accommodate community empowerment goals within "top-down" health programming.

  19. Promoting the exotic pet practice.

    PubMed

    Harris, Don J

    2005-09-01

    The marketing and promotion of an exotic pet veterinary practice allows the use of strategies that are not necessarily available in other veterinary disciplines. The advantage that an exotics practice enjoys is that it is able to capitalize not only on the unique nature of the species being attended but also on the specialized features of the hospital itself that make it specifically appropriate in caring for exotic pets. Before marketing, however, comes the responsibility that the practice live up to the claims made in promotional materials. A practice cannot ethically be presented as an "exotics" practice if it is nothing more than a dog and cat facility that is willing to attend to exotic pets. It is the competence of the veterinary staff and the appropriateness of the facility that determines the suitability of the practice for exotics management.

  20. Drugs that promote dental caries.

    PubMed

    2015-02-01

    Dental caries result from erosion of tooth enamel or cementum by acidic substances produced by bacteria found in dental plaque. Caries can lead to pulp necrosis and tooth loss. Risk factors include certain dietary habits, poor oral hygiene, and dry mouth. Diabetes and Sjogren's syndrome can also promote dental caries. Psychotropic substances such as cocaine, methamphetamine, heroin and cannabis can promote dental caries. Many medicinal drugs facilitate the formation of dental caries, through various mechanisms; they include formulations with a high sugar content; drugs that cause dry mouth (especially antimuscarinics); drugs that lower the buccal pH (inhaled powders, etc.); and drugs that cause demineralisation (tetracyclines, etc.). In practice, patients (and parents) should be informed that some drugs can increase the risk of dental caries. They should be encouraged to adapt and reinforce dental hygiene, and advised to visit a dentist regularly.

  1. South Asia's health promotion kaleidoscope.

    PubMed

    Mukhopadhyay, Alok

    2007-01-01

    South Asia has 22 percent of the world's population but only 1.3 percent of the global income. Consequently 40 percent of the population is living in absolute poverty. However the health transition in some of its countries including India and Sri Lanka is a testimony to the fact that there are proven solutions to the problems of health and development within the region. The countries of the region have much in common, including a democratic political system, four major religions, a vibrant and living tradition of voluntarism and an extensive health infrastructure which is operating well below par. Despite the underlying unity, South Asia enjoys enormous cultural, linguistic and ethnic diversity. In this large, complex and vibrant region, health promotion is a challenging task, but it also holds the key to a dramatic change in the global health situation. Many of these solutions lie in wider areas of socio-political action. There are much needed shifts in the health promotion and development efforts, particularly in the area of poverty and social justice; gender inequity; population stabilisation; health and environment; control of communicable and non-communicable diseases; and urban health strategies. The principle of cooperation, partnership and intersectoral collaboration for health will be explored. Developing an appropriate, sustainable and people centred health and development strategy in the coming decades is an enormous challenge. There has been an attempt to focus on the emerging needs of the region, which call for health promotion, and involvement of civil society, private sector and the governments bestowed with the increased responsibility of ensuring health security for people. Strengthening the existing health systems, allocating adequate resources for health development and ensuring community participation are all prerequisites to the success of health promotion in the region.

  2. PARP promoter-mediated activation of a VSG expression site promoter in insect form Trypanosoma brucei.

    PubMed

    Urményi, T P; Van der Ploeg, L H

    1995-03-25

    In trypanosomes the rRNA, PARP and VSG gene promoters mediate alpha-amanitin-resistant transcription of protein coding genes, presumably by RNA polymerase (pol) I. We compared the activity of PARP and VSG promoters integrated at one of the alleles of the largest subunit of pol II genes in insect form trypanosomes. Even though both promoters are roughly equally active in transient transformation assays in insect form trypanosomes, only the PARP promoter functioned effectively when integrated at the pol II largest subunit or other loci. Promoter activity in transient transformation assays is therefore not necessarily predictive of transcriptional activity once integrated into the trypanosome genome. The integrated fully active PARP promoter could upregulate in cis an otherwise poorly active integrated VSG promoter. The PARP promoter nucleotide sequence elements responsible for VSG promoter activation coincided with most of the important PARP promoter elements mapped previously by linker scanning mutagenesis, indicating that it is not a single unique promoter element that was responsible for VSG promoter activation. The data suggest that PARP promoter-mediated activation of the VSG promoter does not result from complementation of the VSG promoter with a single insect form-specific transcription factor whose binding site is missing from the VSG promoter and present in the PARP promoter. We favor a model in which chromatin structure at the locus is altered by the PARP promoter, allowing VSG promoter activation in insect form trypanosomes. We discuss the significance of these observations for the control of VSG promoters in insect form trypanosomes.

  3. Physical characteristics in eucaryotic promoters.

    PubMed Central

    Bensimhon, M; Gabarro-Arpa, J; Ehrlich, R; Reiss, C

    1983-01-01

    For a series of wild type and mutated eucaryotic gene prelude sequences (mainly "promoters" of SV40 early gene (Benoist and Chambon, Nature 290, 304 (1981); Moreau et al., Nuc. Acids Res. 9, 6047 (1982)) and of Herpes Simplex Virus TK gene (McKnight and Kingsbury, Science 217, 316 (1982)), in vivo promoter activity and local stability (denaturability) have been correlated. In agreement with the conclusions drawn in these papers, the correlation points to three major eucaryotic promoter elements and loci: (i) enzyme enabling by an enhancer sequence; SV40 and Moloney Sarcoma Virus enhancers have a striking stability homology; (ii) enzyme activation, occurring 50-70 b.p. upstream the cap site in a high stability domain; the enzyme apparently deactivates exponentially upon moving away to trap site; (iii) enzyme positioning at trap site, 30 +/- 5 b.p. upstream the cap site. The trap site contains the TATA box, or, when absent, other low stability domains downstream the activator. The number and occupancy of cap sites may depend on the stability and size of the trap site-cap site couple and its distance from the activator. PMID:6306592

  4. Incentives to promote family planning

    PubMed Central

    Heil, Sarah H.; Gaalema, Diann E.; Herrmann, Evan S.

    2012-01-01

    Objective Over the past 60 years, population control has become an increasingly urgent issue worldwide as a growing population strains already limited resources. The use of financial incentives to promote family planning is an innovative approach that has potential to make a contribution to efforts to better manage population growth. This report reviews eight studies that examined the effect of incentives on family planning. Method Published studies that tested the impact of incentives to promote some aspect of family planning and included an appropriate control or comparison condition were reviewed. Results Incentives have been used to promote attendance at contraceptive education sessions, adoption and continuation of contraceptive methods, sterilization, and to limit family size. All but one of the eight studies reviewed reported positive outcomes, but weaknesses in study design and execution limit the strength of the conclusions that can be drawn. Conclusion Review of this literature suggests that family planning behaviors, like other behaviors, are sensitive to incentives. Given the tremendous need for efficacious interventions in global efforts to manage population growth, further research on this topic using more rigorous experimental methods is warranted. PMID:22743293

  5. Programming gene expression with combinatorial promoters

    PubMed Central

    Cox, Robert Sidney; Surette, Michael G; Elowitz, Michael B

    2007-01-01

    Promoters control the expression of genes in response to one or more transcription factors (TFs). The architecture of a promoter is the arrangement and type of binding sites within it. To understand natural genetic circuits and to design promoters for synthetic biology, it is essential to understand the relationship between promoter function and architecture. We constructed a combinatorial library of random promoter architectures. We characterized 288 promoters in Escherichia coli, each containing up to three inputs from four different TFs. The library design allowed for multiple −10 and −35 boxes, and we observed varied promoter strength over five decades. To further analyze the functional repertoire, we defined a representation of promoter function in terms of regulatory range, logic type, and symmetry. Using these results, we identified heuristic rules for programming gene expression with combinatorial promoters. PMID:18004278

  6. Promotion to professor: a career development resource.

    PubMed

    Sanfey, Hilary

    2010-10-01

    By the time a faculty member is being considered for promotion to full professor, he/she will be about 10 years out of residency training and will almost certainly have prior experience with the academic promotion process. The preparation for promotion to full professor should begin soon after the promotion to associate professor. This is a time to reassess opportunities, resources, skills, and career goals. The timing of the promotion to full professor is usually less rigid than the timeframe for promotion at lower ranks, but schools vary in this regard.

  7. Natural selection promotes antigenic evolvability.

    PubMed

    Graves, Christopher J; Ros, Vera I D; Stevenson, Brian; Sniegowski, Paul D; Brisson, Dustin

    2013-01-01

    The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide an experimentally tractable system to test whether natural selection has favored mechanisms that increase evolvability. Many antigenic variation systems consist of paralogous unexpressed 'cassettes' that recombine into an expression site to rapidly alter the expressed protein. Importantly, the magnitude of antigenic change is a function of the genetic diversity among the unexpressed cassettes. Thus, evidence that selection favors among-cassette diversity is direct evidence that natural selection promotes antigenic evolvability. We used the Lyme disease bacterium, Borrelia burgdorferi, as a model to test the prediction that natural selection favors amino acid diversity among unexpressed vls cassettes and thereby promotes evolvability in a primary surface antigen, VlsE. The hypothesis that diversity among vls cassettes is favored by natural selection was supported in each B. burgdorferi strain analyzed using both classical (dN/dS ratios) and Bayesian population genetic analyses of genetic sequence data. This hypothesis was also supported by the conservation of highly mutable tandem-repeat structures across B. burgdorferi strains despite a near complete absence of sequence conservation. Diversification among vls cassettes due to natural selection and mutable repeat structures promotes long-term antigenic evolvability of VlsE. These findings provide a direct demonstration that molecular mechanisms that enhance evolvability of surface antigens are an evolutionary adaptation. The molecular evolutionary processes identified here can serve as a model for the evolution of antigenic evolvability in many pathogens which utilize similar strategies to establish chronic infections.

  8. Immune cell promotion of metastasis

    PubMed Central

    Kitamura, Takanori; Qian, Bin-Zhi; Pollard, Jeffrey W.

    2015-01-01

    Metastatic disease is the major cause of death from cancer, and immunotherapy and chemotherapy have had limited success in reversing its progression. Data from mouse models suggest that the recruitment of immunosuppressive cells to tumours protects metastatic cancer cells from surveillance by killer cells, which nullifies the effects of immunotherapy and thus establishes metastasis. Furthermore, in most cases, tumour-infiltrating immune cells differentiate into cells that promote each step of the metastatic cascade and thus are novel targets for therapy. In this Review, we describe how tumour-infiltrating immune cells contribute to the metastatic cascade and we discuss potential therapeutic strategies to target these cells. PMID:25614318

  9. 7 CFR 1209.17 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MUSHROOM PROMOTION, RESEARCH, AND CONSUMER INFORMATION ORDER Mushroom Promotion, Research, and Consumer Information Order Definitions § 1209... desirability of mushrooms, including paid advertising....

  10. 7 CFR 1209.17 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MUSHROOM PROMOTION, RESEARCH, AND CONSUMER INFORMATION ORDER Mushroom Promotion, Research, and Consumer Information Order Definitions § 1209... desirability of mushrooms, including paid advertising....

  11. 7 CFR 1209.17 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MUSHROOM PROMOTION, RESEARCH, AND CONSUMER INFORMATION ORDER Mushroom Promotion, Research, and Consumer Information Order Definitions § 1209... desirability of mushrooms, including paid advertising....

  12. 7 CFR 1209.17 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MUSHROOM PROMOTION, RESEARCH, AND CONSUMER INFORMATION ORDER Mushroom Promotion, Research, and Consumer Information Order Definitions § 1209... desirability of mushrooms, including paid advertising....

  13. 7 CFR 1212.20 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HONEY PACKERS AND IMPORTERS RESEARCH, PROMOTION, CONSUMER EDUCATION AND INDUSTRY INFORMATION ORDER Honey Packers and Importers Research, Promotion... action, including paid advertising and public relations that presents a favorable image for honey...

  14. 7 CFR 1208.20 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PROCESSED RASPBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Processed Raspberry Promotion, Research, and Information Order Definitions... raspberries to the general public and the food industry for the purpose of improving the competitive...

  15. 7 CFR 1208.20 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PROCESSED RASPBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Processed Raspberry Promotion, Research, and Information Order Definitions... raspberries to the general public and the food industry for the purpose of improving the competitive...

  16. 7 CFR 1212.20 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HONEY PACKERS AND IMPORTERS RESEARCH, PROMOTION, CONSUMER EDUCATION AND INDUSTRY INFORMATION ORDER Honey Packers and Importers Research, Promotion... action, including paid advertising and public relations that presents a favorable image for honey...

  17. 7 CFR 1212.20 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HONEY PACKERS AND IMPORTERS RESEARCH, PROMOTION, CONSUMER EDUCATION AND INDUSTRY INFORMATION ORDER Honey Packers and Importers Research, Promotion... action, including paid advertising and public relations that presents a favorable image for honey...

  18. 7 CFR 1212.20 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HONEY PACKERS AND IMPORTERS RESEARCH, PROMOTION, CONSUMER EDUCATION AND INDUSTRY INFORMATION ORDER Honey Packers and Importers Research, Promotion... action, including paid advertising and public relations that presents a favorable image for honey...

  19. Promotion of social change: a conceptual framework.

    PubMed

    Tseng, Vivian; Chesir-Teran, Daniel; Becker-Klein, Rachel; Chan, May L; Duran, Valkiria; Roberts, Ann; Bardoliwalla, Nenshad

    2002-06-01

    This paper argues for the need to advance promotion efforts and proposes a conceptual framework for promotion of social change. A brief review is presented of traditional frameworks for the prevention of mental and social disorders and the promotion of wellness and social competencies, with attention to the ways in which promotion of social change extends and departs from these frameworks. In a framework for promoting social change, we advocate for promoting dynamic processes within systems, rather than outcomes within individuals. Systems are viewed as flexible and capable of facilitating multiple adaptive pathways for individuals and groups. Promoting social change also involves careful attention to critical analysis, values, language, and contextual processes. Examples are discussed throughout to illustrate how these principles have been used in the past and can be implemented in future efforts to promote social change.

  20. 48 CFR 13.104 - Promoting competition.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false Promoting competition. 13... METHODS AND CONTRACT TYPES SIMPLIFIED ACQUISITION PROCEDURES Procedures 13.104 Promoting competition. The contracting officer must promote competition to the maximum extent practicable to obtain supplies and...

  1. 48 CFR 13.104 - Promoting competition.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Promoting competition. 13... METHODS AND CONTRACT TYPES SIMPLIFIED ACQUISITION PROCEDURES Procedures 13.104 Promoting competition. The contracting officer must promote competition to the maximum extent practicable to obtain supplies and...

  2. 7 CFR 1218.17 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1218.17 Section 1218.17 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.17...

  3. 7 CFR 1215.16 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1215.16 Section 1215.16 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POPCORN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Popcorn Promotion, Research, and Consumer Information Order Definitions §...

  4. 7 CFR 1221.23 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1221.23 Section 1221.23 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.23...

  5. 7 CFR 1209.17 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1209.17 Section 1209.17 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MUSHROOM PROMOTION, RESEARCH, AND CONSUMER INFORMATION ORDER Mushroom Promotion, Research, and Consumer Information Order Definitions §...

  6. 7 CFR 1230.22 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1230.22 Section 1230.22 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PORK PROMOTION, RESEARCH, AND CONSUMER INFORMATION Pork Promotion, Research, and Consumer Information Order Definitions §...

  7. 12 CFR 4.64 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 1 2010-01-01 2010-01-01 false Promotion. 4.64 Section 4.64 Banks and Banking...; Contracting for Goods and Services § 4.64 Promotion. (a) Scope. The OCC, under the direction of the Deputy Comptroller for Resource Management, engages in promotion and outreach activities designed to identify...

  8. 7 CFR 1219.22 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1219.22 Section 1219.22 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions §...

  9. 7 CFR 1220.121 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOYBEAN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Soybean Promotion and Research Order Definitions § 1220.121 Promotion. The term..., to enhance the image or desirability of soybeans or soybean products in domestic and foreign...

  10. 7 CFR 1220.121 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOYBEAN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Soybean Promotion and Research Order Definitions § 1220.121 Promotion. The term..., to enhance the image or desirability of soybeans or soybean products in domestic and foreign...

  11. 7 CFR 1220.121 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOYBEAN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Soybean Promotion and Research Order Definitions § 1220.121 Promotion. The term..., to enhance the image or desirability of soybeans or soybean products in domestic and foreign...

  12. 7 CFR 1220.121 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOYBEAN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Soybean Promotion and Research Order Definitions § 1220.121 Promotion. The term..., to enhance the image or desirability of soybeans or soybean products in domestic and foreign...

  13. 7 CFR 1220.121 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOYBEAN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Soybean Promotion and Research Order Definitions § 1220.121 Promotion. The term..., to enhance the image or desirability of soybeans or soybean products in domestic and foreign...

  14. Health Promotion: An Overview. Unit Technical Paper.

    ERIC Educational Resources Information Center

    Anderson, Robert

    Because health promotion is a relatively new concept in Europe, a study was undertaken to gather information on informal and organized health promoting behavior and programs in Europe. The study attempts, through a review of literature, interviews, and surveys, to clarify the meaning of health promotion in both theory and practice and to identify…

  15. 12 CFR 4.64 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Comptroller for Resource Management, engages in promotion and outreach activities designed to identify MWOBs... promotion events sponsored by other government agencies and attended by minorities, women and individuals... promotion events comprised of or attended by MWOBs and IDOBs to explain OCC contracting opportunities and...

  16. 48 CFR 13.104 - Promoting competition.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 1 2014-10-01 2014-10-01 false Promoting competition. 13... METHODS AND CONTRACT TYPES SIMPLIFIED ACQUISITION PROCEDURES Procedures 13.104 Promoting competition. The contracting officer must promote competition to the maximum extent practicable to obtain supplies and...

  17. 48 CFR 13.104 - Promoting competition.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 1 2012-10-01 2012-10-01 false Promoting competition. 13... METHODS AND CONTRACT TYPES SIMPLIFIED ACQUISITION PROCEDURES Procedures 13.104 Promoting competition. The contracting officer must promote competition to the maximum extent practicable to obtain supplies and...

  18. 48 CFR 13.104 - Promoting competition.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false Promoting competition. 13... METHODS AND CONTRACT TYPES SIMPLIFIED ACQUISITION PROCEDURES Procedures 13.104 Promoting competition. The contracting officer must promote competition to the maximum extent practicable to obtain supplies and...

  19. 17 CFR 33.8 - Promotional material.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 1 2010-04-01 2010-04-01 false Promotional material. 33.8... DOMESTIC EXCHANGE-TRADED COMMODITY OPTION TRANSACTIONS § 33.8 Promotional material. Each futures commission... promotional material it provides, directly or indirectly, to option customers as well as the true source...

  20. 17 CFR 33.8 - Promotional material.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 17 Commodity and Securities Exchanges 1 2012-04-01 2012-04-01 false Promotional material. 33.8... DOMESTIC EXCHANGE-TRADED COMMODITY OPTION TRANSACTIONS § 33.8 Promotional material. Each futures commission... promotional material it provides, directly or indirectly, to option customers as well as the true source...

  1. 21 CFR 314.550 - Promotional materials.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 5 2010-04-01 2010-04-01 false Promotional materials. 314.550 Section 314.550... Serious or Life-Threatening Illnesses § 314.550 Promotional materials. For drug products being considered... the agency for consideration during the preapproval review period copies of all promotional...

  2. Disease Prevention and Health Promotion

    PubMed Central

    Ali, Ather; Katz, David L.

    2015-01-01

    As a discipline, preventive medicine has traditionally been described to encompass primary, secondary, and tertiary prevention. The fields of preventive medicine and public health share the objectives of promoting general health, preventing disease, and applying epidemiologic techniques to these goals. This paper discusses a conceptual approach between the overlap and potential synergies of integrative medicine principles and practices with preventive medicine in the context of these levels of prevention, acknowledging the relative deficiency of research on the effectiveness of practice-based integrative care. One goal of integrative medicine is to make the widest array of appropriate options available to patients, ultimately blurring the boundaries between conventional and complementary medicine. Both disciplines should be subject to rigorous scientific inquiry so that interventions that are efficacious and effective are systematically distinguished from those that are not. Furthermore, principles of preventive medicine can be infused into prevalent practices in complementary and integrative medicine, promoting public health in the context of more-responsible practices. The case is made that an integrative preventive approach involves the responsible use of science with responsiveness to the needs of patients that persist when conclusive data are exhausted, providing a framework to make clinical decisions among integrative therapies. PMID:26477898

  3. Condom ads promote illicit sex.

    PubMed

    Kippley, J F

    1994-01-01

    Written in 1987, this opinion was republished in the wake of US President Bill Clinton's AIDS prevention media campaign promoting condom use which began January 1994, targeted at young adults aged 18-25. The author staunchly opposes condom use even though he admits that people do not consider abstinence from sex to be a serious option for the prevention of HIV/STD infection. He believes that there is no moral use of sex with a condom and that condoms have always been a sign of immorality, be it prostitution, adultery, fornication, or marital contraception. Likewise, the author laments the success enjoyed by Planned Parenthood in achieving the social acceptance of marital contraception and sex outside of marriage. The complete social acceptance of homosexual activity, however, remains to be achieved. Magazines, newspapers, and television receive income in exchange for publishing or airing advertisements. Finding offensive advertisements which promote the use of condoms against HIV infection, the author recommends writing letters of complaint to the responsible media sources. If the television stations or publications in question continue to advertise condoms to the public, stop watching them or end one's subscriptions to the particular printed media. Such action taken collectively among many individuals will reduce product sales and income, and potentially sway corporate policy against condom ads.

  4. Synthetic promoter design for new microbial chassis

    PubMed Central

    Gilman, James; Love, John

    2016-01-01

    The judicious choice of promoter to drive gene expression remains one of the most important considerations for synthetic biology applications. Constitutive promoter sequences isolated from nature are often used in laboratory settings or small-scale commercial production streams, but unconventional microbial chassis for new synthetic biology applications require well-characterized, robust and orthogonal promoters. This review provides an overview of the opportunities and challenges for synthetic promoter discovery and design, including molecular methodologies, such as saturation mutagenesis of flanking regions and mutagenesis by error-prone PCR, as well as the less familiar use of computational and statistical analyses for de novo promoter design. PMID:27284035

  5. Towards a relational health promotion.

    PubMed

    Veenstra, Gerry; Burnett, Patrick John

    2016-03-01

    The Ottawa Charter for Health Promotion exhibits a substantialist approach to the agency-structure dichotomy. From a substantialist point of view, both individual agency and social structure come preformed and subsequently relate to and influence one another, starkly positioning the choices made by individuals against the structured sets of opportunities and constraints in reference to which choices are made. From a relational perspective, however, relations between elements, not the elements themselves, are the primary ontological focus. We advocate for a relational approach to the structure-agency dichotomy, one that locates both agency and structure in social relations and thereby dissolves the stark distinction between them, suggesting that relational theories can provide useful insights into how and why people 'choose' to engage in health-related behaviours. Pierre Bourdieu's theory of practice, predicated upon the notions of field, capital and habitus, is exemplary in this regard.

  6. Promoting Teen Mothers' Mental Health.

    PubMed

    Freed, Patricia; SmithBattle, Lee

    2016-01-01

    In this second article in a two-part series, we call for the integration of strengths-based and trauma-informed care into services for teen mothers. Nurses working with teen mothers in health clinics, schools and home visiting programs can play a pivotal role in promoting their mental health. Many teen mothers have high levels of psychological distress and histories of adverse experiences that cannot be ignored, and cannot solely be addressed by referral to mental health services. Nurses must be prepared to assess for trauma and be open to listening to teen mothers' experiences. Principles of strengths-based and trauma-informed care are complementary and can be integrated in clinical services so that teen mothers' distress is addressed and their strengths and aspirations are supported. Potential screening tools, interviewing skills and basic strategies to alleviate teen mothers' distress are discussed.

  7. Promoter usage and alternative splicing.

    PubMed

    Kornblihtt, Alberto R

    2005-06-01

    Recent findings justify a renewed interest in alternative splicing (AS): the process is more a rule than an exception as it affects the expression of 60% of human genes; it explains how a vast mammalian proteomic complexity is achieved with a limited number of genes; and mutations in AS regulatory sequences are a widespread source of human disease. AS regulation not only depends on the interaction of splicing factors with their target sequences in the pre-mRNA but is coupled to transcription. A clearer picture is emerging of the mechanisms by which transcription affects AS through promoter identity and occupation. These mechanisms involve the recruitment of factors with dual functions in transcription and splicing (i.e. that contain both functional domains and hence link the two processes) and the control of RNA polymerase II elongation.

  8. Junking Science to Promote Tobacco

    PubMed Central

    Yach, Derek; Bialous, Stella Aguinaga

    2001-01-01

    Despite the tobacco industry's claims that it has changed its practices, the toll of tobacco-related disease and death continues to grow worldwide, and the industry continues to use a vast array of strategies to promote its products and increase profits. This commentary discusses the ways the tobacco industry has created controversy about risk assessment and about the scientific evidence of the health hazards of secondhand smoke. The authors recommend that policymakers be more vigilant and that they demand transparency about affiliations and linkages between allegedly independent scientists and tobacco companies. They also urge policymakers to be prepared for new and continuing challenges posed by the tobacco industry, because, despite the industry's claims, there is little evidence of fundamental change in its objectives. PMID:11684592

  9. Vessel Noise Promotes Hull Fouling.

    PubMed

    Stanley, Jenni A; Wilkens, Serena; McDonald, Justin I; Jeffs, Andrew G

    2016-01-01

    Fouling of submerged vessel hulls due to the rapid settlement of algae and invertebrates is a longstanding and costly problem. It is widely thought that the presence of extensive vacant surfaces on vessel hulls is responsible for the rapid attachment and growth of biofouling. We investigated whether noise from vessels in port could also be involved in promoting the settlement and growth of common biofouling organisms on vessel hulls. Three important biofouling species exhibited significantly faster development and settlement and better survival when exposed to vessel noise compared with control species. The extent of these responses appeared to vary in relation to the intensity of the vessel noise and may help to explain differences in biofouling observed on vessel hulls.

  10. NEDDylation promotes stress granule assembly

    PubMed Central

    Jayabalan, Aravinth Kumar; Sanchez, Anthony; Park, Ra Young; Yoon, Sang Pil; Kang, Gum-Yong; Baek, Je-Hyun; Anderson, Paul; Kee, Younghoon; Ohn, Takbum

    2016-01-01

    Stress granules (SGs) harbour translationally stalled messenger ribonucleoproteins and play important roles in regulating gene expression and cell fate. Here we show that neddylation promotes SG assembly in response to arsenite-induced oxidative stress. Inhibition or depletion of key components of the neddylation machinery concomitantly inhibits stress-induced polysome disassembly and SG assembly. Affinity purification and subsequent mass-spectrometric analysis of Nedd8-conjugated proteins from translationally stalled ribosomal fractions identified ribosomal proteins, translation factors and RNA-binding proteins (RBPs), including SRSF3, a previously known SG regulator. We show that SRSF3 is selectively neddylated at Lys85 in response to arsenite. A non-neddylatable SRSF3 (K85R) mutant do not prevent arsenite-induced polysome disassembly, but fails to support the SG assembly, suggesting that the neddylation pathway plays an important role in SG assembly. PMID:27381497

  11. Genes from the 20Q13 amplicon and their uses

    DOEpatents

    Gray, Joe; Collins, Colin; Hwang, Soo-in; Godfrey, Tony; Kowbel, David; Rommens, Johanna

    1999-01-01

    The present invention relates to cDNA sequences from a region of amplification on chromosome 20 associated with disease. The sequences can be used in hybridization methods for the identification of chromosomal abnormalities associated with various diseases. The sequences can also be used for treatment of diseases.

  12. Preconception care: promoting reproductive planning

    PubMed Central

    2014-01-01

    Introduction Preconception care recognizes that many adolescent girls and young women will be thrust into motherhood without the knowledge, skills or support they need. Sixty million adolescents give birth each year worldwide, even though pregnancy in adolescence has mortality rates at least twice as high as pregnancy in women aged 20-29 years. Reproductive planning and contraceptive use can prevent unintended pregnancies, unsafe abortions and sexually-transmitted infections in adolescent girls and women. Smaller families also mean better nutrition and development opportunities, yet 222 million couples continue to lack access to modern contraception. Method A systematic review and meta-analysis of the evidence was conducted to ascertain the possible impact of preconception care for adolescents, women and couples of reproductive age on MNCH outcomes. A comprehensive strategy was used to search electronic reference libraries, and both observational and clinical controlled trials were included. Cross-referencing and a separate search strategy for each preconception risk and intervention ensured wider study capture. Results Comprehensive interventions can prevent first pregnancy in adolescence by 15% and repeat adolescent pregnancy by 37%. Such interventions should address underlying social and community factors, include sexual and reproductive health services, contraceptive provision; personal development programs and emphasizes completion of education. Appropriate birth spacing (18-24 months from birth to next pregnancy compared to short intervals <6 months) can significantly lower maternal mortality, preterm births, stillbirths, low birth weight and early neonatal deaths. Conclusion Improving adolescent health and preventing adolescent pregnancy; and promotion of birth spacing through increasing correct and consistent use of effective contraception are fundamental to preconception care. Promoting reproductive planning on a wider scale is closely interlinked with the

  13. Does sleep promote false memories?

    PubMed

    Darsaud, Annabelle; Dehon, Hedwige; Lahl, Olaf; Sterpenich, Virginie; Boly, Mélanie; Dang-Vu, Thanh; Desseilles, Martin; Gais, Stephen; Matarazzo, Luca; Peters, Frédéric; Schabus, Manuel; Schmidt, Christina; Tinguely, Gilberte; Vandewalle, Gilles; Luxen, André; Maquet, Pierre; Collette, Fabienne

    2011-01-01

    Memory is constructive in nature so that it may sometimes lead to the retrieval of distorted or illusory information. Sleep facilitates accurate declarative memory consolidation but might also promote such memory distortions. We examined the influence of sleep and lack of sleep on the cerebral correlates of accurate and false recollections using fMRI. After encoding lists of semantically related word associates, half of the participants were allowed to sleep, whereas the others were totally sleep deprived on the first postencoding night. During a subsequent retest fMRI session taking place 3 days later, participants made recognition memory judgments about the previously studied associates, critical theme words (which had not been previously presented during encoding), and new words unrelated to the studied items. Sleep, relative to sleep deprivation, enhanced accurate and false recollections. No significant difference was observed in brain responses to false or illusory recollection between sleep and sleep deprivation conditions. However, after sleep but not after sleep deprivation (exclusive masking), accurate and illusory recollections were both associated with responses in the hippocampus and retrosplenial cortex. The data suggest that sleep does not selectively enhance illusory memories but rather tends to promote systems-level consolidation in hippocampo-neocortical circuits of memories subsequently associated with both accurate and illusory recollections. We further observed that during encoding, hippocampal responses were selectively larger for items subsequently accurately retrieved than for material leading to illusory memories. The data indicate that the early organization of memory during encoding is a major factor influencing subsequent production of accurate or false memories.

  14. Pmp27 promotes peroxisomal proliferation

    PubMed Central

    1995-01-01

    Peroxisomes perform many essential functions in eukaryotic cells. The weight of evidence indicates that these organelles divide by budding from preexisting peroxisomes. This process is not understood at the molecular level. Peroxisomal proliferation can be induced in Saccharomyces cerevisiae by oleate. This growth substrate is metabolized by peroxisomal enzymes. We have identified a protein, Pmp27, that promotes peroxisomal proliferation. This protein, previously termed Pmp24, was purified from peroxisomal membranes, and the corresponding gene, PMP27, was isolated and sequenced. Pmp27 shares sequence similarity with the Pmp30 family in Candida boidinii. Pmp27 is a hydrophobic peroxisomal membrane protein but it can be extracted by high pH, suggesting that it does not fully span the bilayer. Its expression is regulated by oleate. The function of Pmp27 was probed by observing the phenotype of strains in which the protein was eliminated by gene disruption or overproduced by expression from a multicopy plasmid. The strain containing the disruption (3B) was able to grow on all carbon sources tested, including oleate, although growth on oleate, glycerol, and acetate was slower than wild type. Strain 3B contained peroxisomes with all of the enzymes of beta-oxidation. However, in addition to the presence of a few modestly sized peroxisomes seen in a typical thin section of a cell growing on oleate-containing medium, cells of strain 3B also contained one or two very large peroxisomes. In contrast, cells in a strain in which Pmp27 was overexpressed contained an increased number of normal-sized peroxisomes. We suggest that Pmp27 promotes peroxisomal proliferation by participating in peroxisomal elongation or fission. PMID:7721939

  15. The Circadian Clock Mutation Promotes Intestinal Dysbiosis

    PubMed Central

    Voigt, Robin M.; Summa, Keith C.; Forsyth, Christopher B.; Green, Stefan J.; Engen, Phillip; Naqib, Ankur; Vitaterna, Martha H.; Turek, Fred W; Keshavarzian, Ali

    2016-01-01

    Background Circadian rhythm disruption is a prevalent feature of modern day society that is associated with an increase in pro-inflammatory diseases and there is a clear need for a better understanding of the mechanism(s) underlying this phenomenon. We have previously demonstrated that both environmental and genetic circadian rhythm disruption causes intestinal hyperpermeability and exacerbates alcohol-induced intestinal hyperpermeability and liver pathology. The intestinal microbiota can influence intestinal barrier integrity and impact immune system function; thus, in the current study, we sought to determine if genetic alteration of the core circadian clock gene, Clock, altered the intestinal microbiota community. Methods Male ClockΔ19 mutant mice (mice homozygous for a dominant-negative mutant allele) or littermate wild-type mice were fed one of three experimental diets: (1) a standard chow diet, (2) an alcohol-containing diet, or (3) an alcohol-control diet in which the alcohol calories were replaced with dextrose. Stool microbiota was assessed with 16S ribosomal RNA gene amplicon sequencing. Results The fecal microbial community of Clock mutant mice had lower taxonomic diversity, relative to wild type mice and the ClockΔ19 mutation was associated with intestinal dysbiosis when mice were fed either the alcohol-containing or the control diet. We found that alcohol consumption significantly altered the intestinal microbiota in both wild type and Clock mutant mice. Conclusion Our data support a model by which circadian rhythm disruption by the ClockΔ19 mutation perturbs normal intestinal microbial communities and this trend was exacerbated in the context of a secondary dietary intestinal stressor. PMID:26842252

  16. Rapid Fermentable Substance Modulates Interactions between Ruminal Commensals and Toll-Like Receptors in Promotion of Immune Tolerance of Goat Rumen

    PubMed Central

    Shen, Hong; Lu, Zhongyan; Chen, Zhan; Wu, Yufeng; Shen, Zanming

    2016-01-01

    Whether dietary non-fiber carbohydrate (NFC), a rapid fermentable substance, affects immune homeostasis of rumen through the modulation of interactions of ruminal microbiota and epithelial toll-like receptors (TLRs) remains unclear. A combination of 16S rRNA amplicon sequencing and quantitative PCRs was applied to study the synergetic responses of ruminal microbiota and epithelial TLRs to the dietary NFC switch from 15 to 31% in the goat model. The results showed that the 31% NFC diet caused the radical increases on the richness and diversity of rumen microbiota. The phylum Verrucomicrobia was most significantly expanded, whereas opportunistic pathogens, namely Rikenella, Anaeroplasma, and Olsenella, were significantly decreased. In rumen epithelium, the significantly increased expressions of TLR1, 6, 10 were associated with the significantly decreased expressions of pro-inflammatory cytokines interleukin-1beta (IL-1ß), IL-6, and anti-inflammatory cytokine IL-10. Constrained correlation analysis indicated that the increased abundance of commensal bacteria in Verrucomicrobia subdivision 5 contributed to the upregulation of TLR10 expression. Finally, the significantly increased concentrations of rumen short-chain fatty acids (SCFAs), coupled with the significantly upregulated expressions of epithelial genes related to SCFA absorption were observed in goats fed with 31% NFC diet. Thus, the NFC-induced expansion of rumen microbiota promoted epithelium tolerance by enhancement of the intensity of TLR10 signaling. The newly established equilibrium benefited to the transport of ruminal energy substances into the blood. PMID:27909428

  17. Single promoters as regulatory network motifs

    NASA Astrophysics Data System (ADS)

    Zopf, Christopher; Maheshri, Narendra

    2012-02-01

    At eukaryotic promoters, chromatin can influence the relationship between a gene's expression and transcription factor (TF) activity. This additional complexity might allow single promoters to exhibit dynamical behavior commonly attributed to regulatory motifs involving multiple genes. We investigate the role of promoter chromatin architecture in the kinetics of gene activation using a previously described set of promoter variants based on the phosphate-regulated PHO5 promoter in S. cerevisiae. Accurate quantitative measurement of transcription activation kinetics is facilitated by a controllable and observable TF input to a promoter of interest leading to an observable expression output in single cells. We find the particular architecture of these promoters can result in a significant delay in activation, filtering of noisy TF signals, and a memory of previous activation -- dynamical behaviors reminiscent of a feed-forward loop but only requiring a single promoter. We suggest this is a consequence of chromatin transactions at the promoter, likely passing through a long-lived ``primed'' state between its inactive and competent states. Finally, we show our experimental setup can be generalized as a ``gene oscilloscope'' to probe the kinetics of heterologous promoter architectures.

  18. Insights into the control of geminiviral promoters.

    PubMed

    Borah, B K; Zarreen, F; Baruah, G; Dasgupta, I

    2016-08-01

    Geminiviruses constitute one of the largest groups of plant viruses, having characteristic twinned geminate particles encapsidating small circular single-stranded DNA molecules. Geminiviral promoters are generally located within the intergenic region, although promoters have also been detected within the genes. Similarly, the geminivirus-associated betasatellite also harbours a promoter element for driving the expression of its only ORF. These regulatory elements of geminiviral and satellite origins have been subject of great interest to develop heterologous gene expression modules. Geminiviral promoter and regulatory elements show a complex regulation that is mediated by several host as well as viral proteins. Here, the structural and functional features of geminiviral and satellite promoters are discussed along with their regulation by plant and viral proteins. Although generalization in many cases is difficult and demands further studies, a pattern is seen to emerge on the regulation of the promoters.

  19. Hypercholesterolemia promotes an osteoporotic phenotype.

    PubMed

    Pelton, Kristine; Krieder, Jaclynn; Joiner, Danese; Freeman, Michael R; Goldstein, Steven A; Solomon, Keith R

    2012-09-01

    A role for hypercholesterolemia in the development of osteoporosis has been suggested in published reports. However, few studies contain direct evidence of a role for maintenance of cholesterol homeostasis in bone health. Using isocaloric high-fat/high-cholesterol and low-fat/no-cholesterol diets in a 4-month feeding study combined with micro computed tomography analysis, we demonstrated in two different mouse strains that mice with hypercholesterolemia lose cortical and trabecular bone in the femurs and vertebrae (bone mineral density was decreased on average by ≈90 mg/mL in the cortical vertebrae in one strain) and cortical bone in the calvariae (bone mineral density was decreased on average by ≈60 mg/mL in one strain). Mechanical testing of the femurs demonstrated that loss of bone in the mice with hypercholesterolemia caused changes in the mechanical properties of the bone including loss of failure load (failure load was decreased by ≈10 N in one strain) and energy to failure. Serologic and histomorphologic analyses suggested that hypercholesterolemia promotes osteoclastogenesis. These studies support a role for hypercholesterolemia in the development of osteoporosis and provide a model with which to test intervention strategies to reduce the effects of hypercholesterolemia on bone health.

  20. Promotional Guide for Navy Family Service Centers

    DTIC Science & Technology

    1985-10-01

    managers of FSCs with a guide to using effective marketing and to increase awareness of FSC products and services. Successful promotional strategies of...they are delivered. kiIl 14. SUBJECT TERMS 15. NUMBER OF PAGES Family Service Center, marketing , promotional approach, delivery system, evaluation 28...customers, the authors have prepared a guide that explains how to use various marketing and promotional strategies to achieve that end. A Marketing Approach

  1. Profit Negotiations and Promotion of Contractor Efficiency.

    DTIC Science & Technology

    1981-03-01

    AOA097 06 ARMY POCUREMNT RESARCH OFFICE FORT LEE VA F/B 5/I PROFIT NEGOTIATIONS AND PROMOTION OF CONTRACTOR EFFICIENCY.(U) MAR 81 R W NICK, 6 A...08 0.5 FINAL PROFIT NEGOTIATIONS MD PROMOTION OF CONTRACTOR EFFICIENCY MARCH 1981 Approved for Public Release; Distribution Unlimited APRO iU. ANIr...DRXMC-PRO 2 April 1981 SUBJECT: Army Procurement Research Office Report APRO 80-08, Profit Negotiations and Promotion of Contractor Efficiency SEE

  2. Promoter-Based Theranostics for Prostate Cancer

    DTIC Science & Technology

    2015-10-01

    AWARD NUMBER: W81XWH-14-1-0430 TITLE: Promoter -Based Theranostics for Prostate Cancer PRINCIPAL INVESTIGATOR: Martin Pomper CONTRACTING...ADDRESS. 1. REPORT DATE October 2015 2. REPORT TYPE Annual 3. DATES COVERED 15 Sep 2014 – 14 Sep 2015 4. TITLE AND SUBTITLE Promoter -Based...n/a 1. INTRODUCTION: This project leverages cancer-specific promoters for molecular- genetic endoradiotherapy and in the development of a universal

  3. An engineered strong promoter for streptomycetes.

    PubMed

    Wang, Weishan; Li, Xiao; Wang, Juan; Xiang, Sihai; Feng, Xiaozhou; Yang, Keqian

    2013-07-01

    Well-characterized promoters are essential tools for metabolic engineering and synthetic biology. In Streptomyces coelicolor, the native kasOp is a temporally expressed promoter strictly controlled by two regulators, ScbR and ScbR2. In this work, first, kasOp was engineered to remove a common binding site of ScbR and ScbR2 upstream of its core region, thus generating a stronger promoter, kasOp3. Second, another ScbR binding site internal to the kasOp3 core promoter region was abolished by random mutation and screening of the mutant library to obtain the strongest promoter, kasOp* (where the asterisk is used to distinguish the engineered promoter from the native promoter). The activities of kasOp* were compared with those of two known strong promoters, ermEp* and SF14p, in three Streptomyces species. kasOp* showed the highest activity at the transcription and protein levels in all three hosts. Furthermore, relative to ermEp* and SF14p, kasOp* was shown to confer the highest actinorhodin production level when used to drive the expression of actII-ORF4 in S. coelicolor. Therefore, kasOp* is a simple and well-defined strong promoter useful for gene overexpression in streptomycetes.

  4. An Engineered Strong Promoter for Streptomycetes

    PubMed Central

    Wang, Weishan; Li, Xiao; Wang, Juan; Xiang, Sihai; Feng, Xiaozhou

    2013-01-01

    Well-characterized promoters are essential tools for metabolic engineering and synthetic biology. In Streptomyces coelicolor, the native kasOp is a temporally expressed promoter strictly controlled by two regulators, ScbR and ScbR2. In this work, first, kasOp was engineered to remove a common binding site of ScbR and ScbR2 upstream of its core region, thus generating a stronger promoter, kasOp3. Second, another ScbR binding site internal to the kasOp3 core promoter region was abolished by random mutation and screening of the mutant library to obtain the strongest promoter, kasOp* (where the asterisk is used to distinguish the engineered promoter from the native promoter). The activities of kasOp* were compared with those of two known strong promoters, ermEp* and SF14p, in three Streptomyces species. kasOp* showed the highest activity at the transcription and protein levels in all three hosts. Furthermore, relative to ermEp* and SF14p, kasOp* was shown to confer the highest actinorhodin production level when used to drive the expression of actII-ORF4 in S. coelicolor. Therefore, kasOp* is a simple and well-defined strong promoter useful for gene overexpression in streptomycetes. PMID:23686264

  5. Career development resource: promotion to associate professor.

    PubMed

    Sanfey, Hilary; Hollands, Celeste

    2012-07-01

    This will most likely be the first time through the promotion and tenure process for the faculty member. The faculty member is responsible for meeting with the department chair and/or division chief on a regular basis to determine if she is on the right time line to successfully achieve promotion to associate professor. A physician will begin seriously preparing her portfolio for promotion to associate professor about 5 to 6 years out of training, at which time she will have some considerable experience running a practice and managing her time. However, the planning process for promotion should begin immediately upon starting the first academic position.

  6. WHO Health Promotion Glossary: new terms.

    PubMed

    Smith, Ben J; Tang, Kwok Cho; Nutbeam, Don

    2006-12-01

    The WHO Health Promotion Glossary was written to facilitate understanding, communication and cooperation among those engaged in health promotion at the local, regional, national and global levels. Two editions of the Glossary have been released, the first in 1986 and the second in 1998, and continued revision of the document is necessary to promote consensus regarding meanings and to take account of developments in thinking and practice. In this update 10 new terms that are to be included in the Glossary are presented. Criteria for the inclusion of terms in the Glossary are that they differentiate health promotion from other health concepts, or have a specific application or meaning when used in relation to health promotion. The terms defined here are: burden of disease; capacity building; evidence-based health promotion; global health; health impact assessment; needs assessment; self-efficacy; social marketing; sustainable health promotion strategies, and; wellness. WHO will continue to periodically update the Health Promotion Glossary to ensure its relevance to the international health promotion community.

  7. 76 FR 314 - Sorghum Promotion, Research, and Information Program: Referendum

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-04

    ... Agricultural Marketing Service Sorghum Promotion, Research, and Information Program: Referendum AGENCY: Agricultural Marketing Service, USDA. ACTION: Notice of Opportunity to Participate in the Sorghum Promotion... Promotion, Research, and Information Order (Order), as authorized under the Commodity Promotion,...

  8. Promoting Strong Written Communication Skills

    NASA Astrophysics Data System (ADS)

    Narayanan, M.

    2015-12-01

    The reason that an improvement in the quality of technical writing is still needed in the classroom is due to the fact that universities are facing challenging problems not only on the technological front but also on the socio-economic front. The universities are actively responding to the changes that are taking place in the global consumer marketplace. Obviously, there are numerous benefits of promoting strong written communication skills. They can be summarized into the following six categories. First, and perhaps the most important: The University achieves learner satisfaction. The learner has documented verbally, that the necessary knowledge has been successfully acquired. This results in learner loyalty that in turn will attract more qualified learners.Second, quality communication lowers the cost per pupil, consequently resulting in increased productivity backed by a stronger economic structure and forecast. Third, quality communications help to improve the cash flow and cash reserves of the university. Fourth, having high quality communication enables the university to justify the need for high costs of tuition and fees. Fifth, better quality in written communication skills result in attracting top-quality learners. This will lead to happier and satisfied learners, not to mention greater prosperity for the university as a whole. Sixth, quality written communication skills result in reduced complaints, thus meaning fewer hours spent on answering or correcting the situation. The University faculty and staff are thus able to devote more time on scholarly activities, meaningful research and productive community service. References Boyer, Ernest L. (1990). Scholarship reconsidered: Priorities of the Professorate.Princeton, NJ: Carnegie Foundation for the Advancement of Teaching. Hawkins, P., & Winter, J. (1997). Mastering change: Learning the lessons of the enterprise.London: Department for Education and Employment. Buzzel, Robert D., and Bradley T. Gale. (1987

  9. Mycogenic Mn(II) oxidation promotes remediation of acid mine drainage and other anthropogenically impacted environments

    NASA Astrophysics Data System (ADS)

    Santelli, C. M.; Chaput, D.; Hansel, C. M.; Burgos, W. D.

    2014-12-01

    Manganese is a pollutant in worldwide environments contaminated with metals and organics, such as acid mine drainage (AMD), freshwater ponds, and agricultural waste storage sites. Microorganisms contribute to the removal of dissolved Mn compounds in the environment by promoting Mn(II) oxidation reactions. The oxidation of Mn(II) results in the precipitation of sparingly soluble Mn(IV) oxide minerals, effectively removing the metal from the aqueous milieu (e.g., groundwater or wastewater streams). In recent years, our research has identified a diversity of Mn(II)-oxidizing fungi inhabiting these polluted environments, however their overall contribution to the remediation process in situ remains poorly understood. Here we present results of culture-based and Next Generation Sequencing (NGS) studies in AMD treatment systems actively remediating Mn and other metals where we profile the bacterial, fungal, algal and archaeal communities to determine the overall community diversity and to establish the relative abundance of known Mn(II) oxidizers. A variety of treatment systems with varying Mn-removal efficiencies were sampled to understand the relationship between remediation efficiency and microbial community composition and activity. Targeted-amplicon sequencing of DNA and RNA of the 16S rRNA genes (bacteria and archaea), 23S rRNA genes (algae) and ITS region (fungi) was performed using both 454 pyrosequencing and Illumina platforms. Results showed that only the fungal taxonomic profiles significantly differed between sites that removed the majority of influent Mn and those that did not. Specifically, Ascomycota (which include known Mn(II) oxidizers isolated from these treatment systems) dominated greater efficiency systems whereas less efficient systems were dominated by Basidiomycota. Furthermore, known Mn(II) oxidizers accounted for only a minor proportion of bacterial sequences but a far greater proportion of fungal sequences. These culture-independent studies lend

  10. Desert Farming Benefits from Microbial Potential in Arid Soils and Promotes Diversity and Plant Health

    PubMed Central

    Köberl, Martina; Müller, Henry; Ramadan, Elshahat M.; Berg, Gabriele

    2011-01-01

    Background To convert deserts into arable, green landscapes is a global vision, and desert farming is a strong growing area of agriculture world-wide. However, its effect on diversity of soil microbial communities, which are responsible for important ecosystem services like plant health, is still not known. Methodology/Principal Findings We studied the impact of long-term agriculture on desert soil in one of the most prominent examples for organic desert farming in Sekem (Egypt). Using a polyphasic methodological approach to analyse microbial communities in soil as well as associated with cultivated plants, drastic effects caused by 30 years of agriculture were detected. Analysing bacterial fingerprints, we found statistically significant differences between agricultural and native desert soil of about 60%. A pyrosequencing-based analysis of the 16S rRNA gene regions showed higher diversity in agricultural than in desert soil (Shannon diversity indices: 11.21/7.90), and displayed structural differences. The proportion of Firmicutes in field soil was significantly higher (37%) than in the desert (11%). Bacillus and Paenibacillus play the key role: they represented 96% of the antagonists towards phytopathogens, and identical 16S rRNA sequences in the amplicon library and for isolates were detected. The proportion of antagonistic strains was doubled in field in comparison to desert soil (21.6%/12.4%); disease-suppressive bacteria were especially enriched in plant roots. On the opposite, several extremophilic bacterial groups, e.g., Acidimicrobium, Rubellimicrobium and Deinococcus-Thermus, disappeared from soil after agricultural use. The N-fixing Herbaspirillum group only occurred in desert soil. Soil bacterial communities were strongly driven by the a-biotic factors water supply and pH. Conclusions/Significance After long-term farming, a drastic shift in the bacterial communities in desert soil was observed. Bacterial communities in agricultural soil showed a higher

  11. Scabies Mites Alter the Skin Microbiome and Promote Growth of Opportunistic Pathogens in a Porcine Model

    PubMed Central

    Swe, Pearl M.; Zakrzewski, Martha; Kelly, Andrew; Krause, Lutz; Fischer, Katja

    2014-01-01

    Background The resident skin microbiota plays an important role in restricting pathogenic bacteria, thereby protecting the host. Scabies mites (Sarcoptes scabiei) are thought to promote bacterial infections by breaching the skin barrier and excreting molecules that inhibit host innate immune responses. Epidemiological studies in humans confirm increased incidence of impetigo, generally caused by Staphylococcus aureus and Streptococcus pyogenes, secondary to the epidermal infestation with the parasitic mite. It is therefore possible that mite infestation could alter the healthy skin microbiota making way for the opportunistic pathogens. A longitudinal study to test this hypothesis in humans is near impossible due to ethical reasons. In a porcine model we generated scabies infestations closely resembling the disease manifestation in humans and investigated the scabies associated changes in the skin microbiota over the course of a mite infestation. Methodology/Principal Findings In a 21 week trial, skin scrapings were collected from pigs infected with S. scabies var. suis and scabies-free control animals. A total of 96 skin scrapings were collected before, during infection and after acaricide treatment, and analyzed by bacterial 16S rDNA tag-encoded FLX-titanium amplicon pyrosequencing. We found significant changes in the epidermal microbiota, in particular a dramatic increase in Staphylococcus correlating with the onset of mite infestation in animals challenged with scabies mites. This increase persisted beyond treatment from mite infection and healing of skin. Furthermore, the staphylococci population shifted from the commensal S. hominis on the healthy skin prior to scabies mite challenge to S. chromogenes, which is increasingly recognized as being pathogenic, coinciding with scabies infection in pigs. In contrast, all animals in the scabies-free cohort remained relatively free of Staphylococcus throughout the trial. Conclusions/Significance This is the first

  12. Marketing and Promotion of Library Services

    NASA Astrophysics Data System (ADS)

    Nicholas, Julie

    As librarians we should be actively marketing and promoting our library services. This paper aims to demystify marketing for librarians. Practical solutions are provided on how to implement a marketing strategy, with particular emphasis on the value of using electronic information resources. It also shows the link between promoting library services and raising the profile of the library.

  13. Promoting Relationships and Eliminating Violence in Canada

    ERIC Educational Resources Information Center

    Pepler, Debra; Craig, Wendy

    2011-01-01

    The Promoting Relationships and Eliminating Violence Network (PREVNet) involves Canadian researchers and national organizations working to promote healthy relationships and prevent bullying. In this paper, we provide the rationale for establishing PREVNet, a description of the work of the network, and an assessment of the success of PREVNet.…

  14. Linking Core Promoter Classes to Circadian Transcription

    PubMed Central

    Westermark, Pål O.

    2016-01-01

    Circadian rhythms in transcription are generated by rhythmic abundances and DNA binding activities of transcription factors. Propagation of rhythms to transcriptional initiation involves the core promoter, its chromatin state, and the basal transcription machinery. Here, I characterize core promoters and chromatin states of genes transcribed in a circadian manner in mouse liver and in Drosophila. It is shown that the core promoter is a critical determinant of circadian mRNA expression in both species. A distinct core promoter class, strong circadian promoters (SCPs), is identified in mouse liver but not Drosophila. SCPs are defined by specific core promoter features, and are shown to drive circadian transcriptional activities with both high averages and high amplitudes. Data analysis and mathematical modeling further provided evidence for rhythmic regulation of both polymerase II recruitment and pause release at SCPs. The analysis provides a comprehensive and systematic view of core promoters and their link to circadian mRNA expression in mouse and Drosophila, and thus reveals a crucial role for the core promoter in regulated, dynamic transcription. PMID:27504829

  15. Cloning the human SUMO1 promoter.

    PubMed

    Nanos-Webb, Angela; Deyrieux, Adeline; Bian, Xue-lin; Rosas-Acosta, Germán; Wilson, Van G

    2010-03-01

    Regulation of the sumoylation system at the level of gene expression has not yet been explored. To begin to define transcriptional regulatory features, the promoter region for the SUMO1 gene was cloned from human genomic DNA and characterized. Initially, a 532 base pair fragment upstream of and including the predicted SUMO1 transcription start site (TSS) was cloned and shown to possess promoter activity. Subsequent deletion analysis showed that a smaller fragment containing 158 bp upstream of the TSS region exhibited basal promoter activity in both human and rodent cell lines. Within this basal promoter fragment, there were predicted binding sites for numerous transcription factors, including the nude mouse gene product, Whn (FoxN1). Electrophoretic mobility shift assays showed that Whn could bind to an ACGC motif adjacent to the TSR, and in transfection studies Whn stimulated a 3-fold increase in transcription from this cloned promoter in keratinocytes (HaCaT cells). Mutation of the ACGC motif abrogated both Whn binding and transcriptional activation, indicating that the Whn effect is likely due to direct interaction with this promoter element. Consistent with these observations on the cloned promoter region, Whn also modestly stimulated transcription from the endogenous, genomic SUMO1 promoter in HaCaT cells, consistent with Whn potentially playing a regulatory role for SUMO1 transcription in keratinocytes.

  16. Promoting Transformative Learning through Reading Fiction

    ERIC Educational Resources Information Center

    Hoggan, Chad; Cranton, Patricia

    2015-01-01

    This article is a report on research into the role of fiction in promoting transformative learning in higher education settings. Participants were 131 undergraduate and graduate students from two universities in the United States. To determine the type of learning promoted by reading fiction, we performed qualitative analyses on participants'…

  17. Media Characteristics in Promoting Management Education Courses

    ERIC Educational Resources Information Center

    Berry, Dick

    1978-01-01

    The effectiveness of direct media employed for promoting marketing, service and sales management conferences and institutes was analyzed. Direct mail was the preferred media for program promotion for business clientele. Three enrollment tables illustrate the effectiveness of different media by program area and course type. (EM)

  18. Promoting people's health: challenges and opportunities.

    PubMed

    Heitkamp, P

    1998-01-01

    Promoting health underlines the right of each individual to the highest attainable standard of health. It stresses the importance of the participation of people and recognizes different sociocultural values and beliefs that are prevalent throughout the world. Working on health development has a sustainable effect only when done comprehensively: personal development, community development, organizational development, and political development. The international conferences that have marked the way of health promotion have been goal posts of an energetic movement to strengthen health worldwide. The Ottawa Charter on Health Promotion has been a worldwide source of guidance for health promotion through its five strategies: building health policy, creating supportive elements, strengthening community action, developing personal skills, and reorienting health services. Moreover, the Jakarta Declaration on "Leading Health Promotion into the 21st Century" identifies five priorities in the next millennium: 1) promote social responsibility for health; 2) increase investments for health development; 3) consolidate and expand partnerships for health; 4) increase community capacity and empower the individual in matters of health; and 5) secure an infrastructure for health promotion. Increasing the investment in health development calls for the need to find new mechanisms for funding as well as reorienting existing resources towards health promotion and health education.

  19. How should vasectomy be promoted in Guatemala?

    PubMed

    Piotrow, P T; Kincaid, D L

    1988-01-01

    In the article "Evaluation of a Communications Program to Increase Adoption of Vasectomy in Guatemala" by J.T. Bertrand et al (Stud Fam Plann 1987 Nov/Dec), the authors conclude that the use of a male promoter alone was 4 times more cost-effective in increasing the number of vasectomies than the use of radio alone because the costs of the radio program were 4 times higher. This conclusion is questionable for several reasons. 1) The district where the promoter was used alone was twice as large as the radio-only district. 2) In one of the promoter-only districts the same promoter worked throughout the program, but in the other, 3 different promoters had to be recruited and trained, due to high personnel turnover. 3) The initial costs of a radio program may be higher, but 1 program can be broadcast in all districts with little or no extra cost, whereas the costs of a promoter would have to be multiplied by the number of districts. 4) Although the promoter and the radio program produced approximately equal numbers of vasectomies, the radio messages reached over 70% of the people surveyed. Thus, on a national basis, radio broadcasts would be far more cost-effective than the use of salaried promoters in each district.

  20. The Literature on Social Promotion versus Retention.

    ERIC Educational Resources Information Center

    Southwest Educational Development Lab., Austin, TX.

    This general review of the relative merits of social promotion and retention examines research on the benefits of each, describes current strategies for resolving the policy dilemma involved, and considers issues raised by abolishing social promotion and establishing remedial programs. A summary of the history of the widespread adoption of the…

  1. The Grade Retention/Social Promotion Debate.

    ERIC Educational Resources Information Center

    Lindelow, John

    1985-01-01

    This publication focuses on the retention/promotion debate regarding failing and low-achieving students. An introductory essay describes the inherent limitation in the research done on this issue--the impossibility of obtaining an appropriate control group--and suggests that the retention/promotion quandary can best be resolved by accommodating…

  2. Health-Promoting Behaviours in Conservatoire Students

    ERIC Educational Resources Information Center

    Kreutz, Gunter; Ginsborg, Jane; Williamon, Aaron

    2009-01-01

    This study focuses on health-promoting behaviours in students from two conservatoires, the Royal Northern College of Music (RNCM, Manchester, UK; n =199) and the Royal College of Music (RCM, London, UK; n = 74). The research questions concern (a) the levels and types of health-promoting behaviours among performance students and (b) the association…

  3. Effective Promotion in a Greater Metropolitan Area.

    ERIC Educational Resources Information Center

    Kelly, J. Terence

    Community colleges offering or contemplating offering television-based courses will be successful in promotional efforts if the fundamental, philosophical purpose is built upon a need for expanding current educational services to a population thus far unaffected by on-campus programs. Efforts in promoting television courses should be made so that…

  4. Identifying Opinion Leaders to Promote Behavior Change

    ERIC Educational Resources Information Center

    Valente, Thomas W.; Pumpuang, Patchareeya

    2007-01-01

    This article reviews 10 techniques used to identify opinion leaders to promote behavior change. Opinion leaders can act as gatekeepers for interventions, help change social norms, and accelerate behavior change. Few studies document the manner in which opinion leaders are identified, recruited, and trained to promote health. The authors categorize…

  5. [Concept of tumor promoters in hepatocarcinogenesis].

    PubMed

    Frayssinet, C; Lafarge-Frayssinet, C

    1990-01-01

    Tumor promotion is defined as 1 step of carcinogenesis. Its intervention occurred when cell DNA was already damaged by a carcinogen and is the support of quiescent mutations. This leads to a complete malignant transformation of the cells by a non genotoxic mechanism. The presence of tumor promoters in our environment, although difficult to estimate, must be taken into account. This requires further research.

  6. 27 CFR 6.96 - Consumer promotions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Consumer promotions. 6.96... OF THE TREASURY LIQUORS âTIED-HOUSEâ Exceptions § 6.96 Consumer promotions. (a) Coupons. The act by an industry member of furnishing to consumers coupons which are redeemable at a retail...

  7. 17 CFR 200.70 - Business promotions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 2 2010-04-01 2010-04-01 false Business promotions. 200.70 Section 200.70 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION ORGANIZATION; CONDUCT AND ETHICS; AND INFORMATION AND REQUESTS Canons of Ethics § 200.70 Business promotions. A member...

  8. 27 CFR 6.96 - Consumer promotions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Consumer promotions. 6.96 Section 6.96 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS âTIED-HOUSEâ Exceptions § 6.96 Consumer promotions. (a) Coupons. The act...

  9. Promotion in Call Centres: Opportunities and Determinants

    ERIC Educational Resources Information Center

    Gorjup, Maria Tatiana; Valverde, Mireia; Ryan, Gerard

    2008-01-01

    Purpose: The purpose of this paper is to examine the quality of jobs in call centres by focusing on the opportunities for promotion in this sector. More specifically, the research questions focus on discovering whether promotion is common practise in the call centre sector and on identifying the factors that affect this.…

  10. Activities for Engaging Schools in Health Promotion

    ERIC Educational Resources Information Center

    Bardi, Mohammad; Burbank, Andrea; Choi, Wayne; Chow, Lawrence; Jang, Wesley; Roccamatisi, Dawn; Timberley-Berg, Tonia; Sanghera, Mandeep; Zhang, Margaret; Macnab, Andrew J.

    2014-01-01

    Purpose: The purpose of this paper is to describe activities used to initiate health promotion in the school setting. Design/Methodology/Approach: Description of successful pilot Health Promoting School (HPS) initiatives in Canada and Uganda and the validated measures central to each program. Evaluation methodologies: quantitative data from the…

  11. 17 CFR 200.70 - Business promotions.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 17 Commodity and Securities Exchanges 3 2014-04-01 2014-04-01 false Business promotions. 200.70... AND ETHICS; AND INFORMATION AND REQUESTS Canons of Ethics § 200.70 Business promotions. A member must not engage in any other business, employment or vocation while in office, nor may he ever use...

  12. 17 CFR 200.70 - Business promotions.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 17 Commodity and Securities Exchanges 2 2013-04-01 2013-04-01 false Business promotions. 200.70... AND ETHICS; AND INFORMATION AND REQUESTS Canons of Ethics § 200.70 Business promotions. A member must not engage in any other business, employment or vocation while in office, nor may he ever use...

  13. 17 CFR 200.70 - Business promotions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 17 Commodity and Securities Exchanges 2 2011-04-01 2011-04-01 false Business promotions. 200.70... AND ETHICS; AND INFORMATION AND REQUESTS Canons of Ethics § 200.70 Business promotions. A member must not engage in any other business, employment or vocation while in office, nor may he ever use...

  14. 17 CFR 200.70 - Business promotions.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 17 Commodity and Securities Exchanges 2 2012-04-01 2012-04-01 false Business promotions. 200.70... AND ETHICS; AND INFORMATION AND REQUESTS Canons of Ethics § 200.70 Business promotions. A member must not engage in any other business, employment or vocation while in office, nor may he ever use...

  15. Composing a Tumor Specific Bacterial Promoter

    PubMed Central

    Deyneko, Igor V.; Kasnitz, Nadine; Leschner, Sara; Weiss, Siegfried

    2016-01-01

    Systemically applied Salmonella enterica spp. have been shown to invade and colonize neoplastic tissues where it retards the growth of many tumors. This offers the possibility to use the bacteria as a vehicle for the tumor specific delivery of therapeutic molecules. Specificity of such delivery is solely depending on promoter sequences that control the production of a target molecule. We have established the functional structure of bacterial promoters that are transcriptionally active exclusively in tumor tissues after systemic application. We observed that the specific transcriptional activation is accomplished by a combination of a weak basal promoter and a strong FNR binding site. This represents a minimal set of control elements required for such activation. In natural promoters, additional DNA remodeling elements are found that alter the level of transcription quantitatively. Inefficiency of the basal promoter ensures the absence of transcription outside tumors. As a proof of concept, we compiled an artificial promoter sequence from individual motifs representing FNR and basal promoter and showed specific activation in a tumor microenvironment. Our results open possibilities for the generation of promoters with an adjusted level of expression of target proteins in particular for applications in bacterial tumor therapy. PMID:27171245

  16. [Promoting "well-treatment" in medical imaging].

    PubMed

    Renouf, Nicole; Llop, Marc

    2012-12-01

    A project to promote "well-treatment" has been initiated in the medical imaging department of a Parisian hospital. With the aim of promoting the well-being of the patient and developing shared values of empathy and respect, the members of this medico-technical team have undertaken to build a culture of "well-treatment" which respects the patient's dignity and rights.

  17. The double jeopardy of sales promotions.

    PubMed

    Jones, J P

    1990-01-01

    The maturing of most consumer markets in the United States has put great pressure on manufacturers in their search for growth. They have concentrated on building sales and expanding share proportions in the stagnant markets with devices like niche products, product extensions, mergers, and international ventures. They have shifted emphasis to sales promotions at the expense of advertising. But promotions, when you come right down to it, mean price reductions. Trade promotions are almost always rebates, and consumer promotions are usually temporary price reductions or coupons. The cost in reduced profit, demonstrated mathematically through calculations of price elasticity, is severe. Besides, when the promotion is over, the manufacturer has not moved forward an inch in shoring up the brand franchise. Promotions bring volatile demand, whereas the producer seeks stable demand. By sustaining a brand image and building customer loyalty, on the other hand, theme advertising can stabilize demand. Moreover, this type of advertising is less likely than promotion is to invite destructive competitive retaliation. Calculation of the advertising elasticity of a brand indicates that sometimes even modest sales increases can produce healthy profit improvement. In a well-planned marketing campaign, there is often good reason to include trade or consumer promotion--to counter a leading competitor's moves, for example. But there is no point in carrying out wild swings at rivals in a struggle for market share. Mathematical techniques can aid the efficiency of marketing planning and put on a more rational basis the decision on where to put the dollars.

  18. Promoting Tech Schools within Your Community

    ERIC Educational Resources Information Center

    Nikirk, Martin

    2010-01-01

    This article describes a number of promotional events and practices that the author's school engages in. The author hopes that his school's experiences will serve as an inspiration and useful model for other "Tech Directions" readers. There are many methods for successfully promoting one's tech program. Two key strategies are: (1) Connect in…

  19. Promoting School Connectedness through Whole School Approaches

    ERIC Educational Resources Information Center

    Rowe, Fiona; Stewart, Donald; Patterson, Carla

    2007-01-01

    Purpose: The purpose of this paper is to develop a framework to demonstrate the contribution of whole school approaches embodied by the health-promoting school approach, to the promotion of school connectedness, defined as the cohesiveness between diverse groups in the school community, including students, families, school staff and the wider…

  20. 7 CFR 1214.18 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE CHRISTMAS TREE PROMOTION, RESEARCH, AND INFORMATION ORDER Christmas Tree Promotion, Research, and Information Order Definitions § 1214.18... favorable image of Christmas trees to the general public with the intent of improving the perception...

  1. 7 CFR 1214.18 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE CHRISTMAS TREE PROMOTION, RESEARCH, AND INFORMATION ORDER Christmas Tree Promotion, Research, and Information Order Definitions § 1214.18... favorable image of Christmas trees to the general public with the intent of improving the perception...

  2. 7 CFR 1214.18 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE CHRISTMAS TREE PROMOTION, RESEARCH, AND INFORMATION ORDER Christmas Tree Promotion, Research, and Information Order Definitions § 1214.18... favorable image of Christmas trees to the general public with the intent of improving the perception...

  3. 77 FR 56552 - Holiday Mobile Shopping Promotion

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-13

    ...] [Pages 56552-56554] [FR Doc No: 2012-22507] POSTAL SERVICE 39 CFR Part 111 Holiday Mobile Shopping... Holiday Mobile Shopping Promotion (``Promotion''), which will take place between November 7, 2012 and.../ Cyber Monday holiday shopping timeframe. In this final rule, the Postal Service provides a...

  4. Transcription Factor Binding Site Positioning in Yeast: Proximal Promoter Motifs Characterize TATA-Less Promoters

    PubMed Central

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of ‘proximal promoter motifs’ (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters. PMID:21931670

  5. Transcription factor binding site positioning in yeast: proximal promoter motifs characterize TATA-less promoters.

    PubMed

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of 6 'proximal promoter motifs' (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters.

  6. The Dual Lives of Bidirectional Promoters

    PubMed Central

    Wakano, Clay; Byun, Jung S.; Di, Li-Jun; Gardner, Kevin

    2012-01-01

    The sequencing of the human genome led to many insights into gene organization and structure. One interesting observation was the high frequency of bidirectional promoters characterized by two protein encoding genes whose promoters are arranged in a divergent or “head-to-head” configuration with less than 2000 base pairs of intervening sequence. Computational estimates published by various groups indicate that nearly 10% of the coding gene promoters are arranged in such a manner and the extent of this bias is a unique feature of mammalian genomes. Moreover, as a class, head-to-head promoters appear to be enriched in specific categories of gene function. Here we review the structure, composition, genomic properties and functional classifications of genes controlled by bidirectional promoters and explore the biological implication of these features. PMID:22366276

  7. The Nordic Health Promotion Research Network (NHPRN).

    PubMed

    Ringsberg, Karin C

    2015-08-01

    The Nordic Health Promotion Research Network (NHPRN) was established in 2007 at the Nordic School of Public Health (NHV). This article aims to describe the foundation of the NHPRN, the development and the present status of the work of NHPRN. The NHPRN consists of about 50 senior and junior researchers from all Nordic countries. It is a working network that aims to develop the theoretical understanding of health promotion, to create research cooperation in health promotion from a Nordic perspective and to extend the scope of health promotion through education. Network members meet biannually to discuss and further develop research within the field and are also responsible for the Nordic conference on Health Promotion, organized every 3 years. The NHV hosted the network between 2007 and 2014; and the World Health Organisation (WHO) will assume this role in 2015.

  8. Plant growth-promoting bacterial endophytes.

    PubMed

    Santoyo, Gustavo; Moreno-Hagelsieb, Gabriel; Orozco-Mosqueda, Ma del Carmen; Glick, Bernard R

    2016-02-01

    Bacterial endophytes ubiquitously colonize the internal tissues of plants, being found in nearly every plant worldwide. Some endophytes are able to promote the growth of plants. For those strains the mechanisms of plant growth-promotion known to be employed by bacterial endophytes are similar to the mechanisms used by rhizospheric bacteria, e.g., the acquisition of resources needed for plant growth and modulation of plant growth and development. Similar to rhizospheric plant growth-promoting bacteria, endophytic plant growth-promoting bacteria can act to facilitate plant growth in agriculture, horticulture and silviculture as well as in strategies for environmental cleanup (i.e., phytoremediation). Genome comparisons between bacterial endophytes and the genomes of rhizospheric plant growth-promoting bacteria are starting to unveil potential genetic factors involved in an endophytic lifestyle, which should facilitate a better understanding of the functioning of bacterial endophytes.

  9. Interventions for promoting physical activity

    PubMed Central

    Foster, Charles; Hillsdon, Melvyn; Thorogood, Margaret; Kaur, Asha; Wedatilake, Thamindu

    2014-01-01

    Background Little is known about the effectiveness of strategies to enable people to achieve and maintain recommended levels of physical activity. Objectives To assess the effectiveness of interventions designed to promote physical activity in adults aged 16 years and older, not living in an institution. Search methods We searched The Cochrane Library (issue 1 2005), MEDLINE, EMBASE, CINAHL, PsycLIT, BIDS ISI, SPORTDISCUS, SIGLE, SCISEARCH (from earliest dates available to December 2004). Reference lists of relevant articles were checked. No language restrictions were applied. Selection criteria Randomised controlled trials that compared different interventions to encourage sedentary adults not living in an institution to become physically active. Studies required a minimum of six months follow up from the start of the intervention to the collection of final data and either used an intention-to-treat analysis or, failing that, had no more than 20% loss to follow up. Data collection and analysis At least two reviewers independently assessed each study quality and extracted data. Study authors were contacted for additional information where necessary. Standardised mean differences and 95% confidence intervals were calculated for continuous measures of self-reported physical activity and cardio-respiratory fitness. For studies with dichotomous outcomes, odds ratios and 95% confidence intervals were calculated. Main results The effect of interventions on self-reported physical activity (19 studies; 7598 participants) was positive and moderate (pooled SMD random effects model 0.28 95% CI 0.15 to 0.41) as was the effect of interventions (11 studies; 2195 participants) on cardio-respiratory fitness (pooled SMD random effects model 0.52 95% CI 0.14 to 0.90). There was significant heterogeneity in the reported effects as well as heterogeneity in characteristics of the interventions. The heterogeneity in reported effects was reduced in higher quality studies, when physical

  10. Health promotion in Canada: 1986 to 2006.

    PubMed

    Jackson, Suzanne F; Riley, Barbara L

    2007-01-01

    The evolution of health promotion in Canada between 1986 and 2006 is characterized by three major eras: Health Promotion in the Limelight, 1986-1992, Health Promotion Behind the Scenes, 1993-2003, and Health Promotion Restaged, 2003-2006. These eras are illustrated using the Canadian Heart Health Initiative as an example. The first era, backed by strong federal government leadership and support, was a progressive time of developing concepts, collaborations and infrastructure for health promotion across the country. Despite significant progress, by the end of this era, health promotion was neither sufficiently developed nor funded to make it a cornerstone of the health system. In addition, the emphasis was heavily biased towards changing individual behaviour. In the second era, health promotion continued to develop in pockets across the country and debates within the field intensified. However, these events went largely unnoticed and massive overall cuts at federal and provincial levels of government made acute care a much higher priority than health promotion. The third era, mostly shaped by fears linked to public health threats, saw a restaging of health promotion through efforts to strengthen public health infrastructure. Nevertheless, at the end of this era, the necessary intersectoral partnerships (such as in health, housing, education, food, income) remained scarce, and little progress was made to decrease health inequalities. The Canadian Heart Health Initiative was implemented over the same time period as the three eras. Its legacy includes collegial relationships across various levels of government and with non-government organizations, a culture that values pan-Canadian initiatives, and support for integration of research, evaluation, surveillance, policy and practice. It remains to be seen how quickly it will be possible to advance the vision of health promotion conceived during the Limelight Era in Canada.

  11. Synthetic promoter libraries for Corynebacterium glutamicum.

    PubMed

    Rytter, Jakob Vang; Helmark, Søren; Chen, Jun; Lezyk, Mateusz Jakub; Solem, Christian; Jensen, Peter Ruhdal

    2014-03-01

    The ability to modulate gene expression is an important genetic tool in systems biology and biotechnology. Here, we demonstrate that a previously published easy and fast PCR-based method for modulating gene expression in lactic acid bacteria is also applicable to Corynebacterium glutamicum. We constructed constitutive promoter libraries based on various combinations of a previously reported C. glutamicum -10 consensus sequence (gngnTA(c/t)aaTgg) and the Escherichia coli -35 consensus, either with or without an AT-rich region upstream. A promoter library based on consensus sequences frequently found in low-GC Gram-positive microorganisms was also included. The strongest promoters were found in the library with a -35 region and a C. glutamicum -10 consensus, and this library also represents the largest activity span. Using the alternative -10 consensus TATAAT, which can be found in many other prokaryotes, resulted in a weaker but still useful promoter library. The upstream AT-rich region did not appear to affect promoter strength in C. glutamicum. In addition to the constitutive promoters, a synthetic inducible promoter library, based on the E. coli lac-promoter, was constructed by randomizing the 17-bp spacer between -35 and -10 consensus sequences and the sequences surrounding these. The inducible promoter library was shown to result in β-galactosidase activities ranging from 284 to 1,665 Miller units when induced by IPTG, and the induction fold ranged from 7-59. We find that the synthetic promoter library (SPL) technology is convenient for modulating gene expression in C. glutamicum and should have many future applications, within basic research as well as for optimizing industrial production organisms.

  12. Promoting Health in Early Childhood Environments: A Health-Promotion Approach

    ERIC Educational Resources Information Center

    Minniss, Fiona Rowe; Wardrope, Cheryl; Johnston, Donni; Kendall, Elizabeth

    2013-01-01

    This paper investigates the mechanisms by which a health-promotion intervention might influence the health-promoting behaviours of staff members working in early childhood centres. The intervention was an ecological health-promotion initiative that was implemented within four early childhood centres in South-East Queensland, Australia. In-depth,…

  13. Recurrent patterns of DNA methylation in the ZNF154, CASP8, and VHL promoters across a wide spectrum of human solid epithelial tumors and cancer cell lines

    PubMed Central

    Sánchez-Vega, Francisco; Gotea, Valer; Petrykowska, Hanna M; Margolin, Gennady; Krivak, Thomas C; DeLoia, Julie A; Bell, Daphne W; Elnitski, Laura

    2013-01-01

    The study of aberrant DNA methylation in cancer holds the key to the discovery of novel biological markers for diagnostics and can help to delineate important mechanisms of disease. We have identified 12 loci that are differentially methylated in serous ovarian cancers and endometrioid ovarian and endometrial cancers with respect to normal control samples. The strongest signal showed hypermethylation in tumors at a CpG island within the ZNF154 promoter. We show that hypermethylation of this locus is recurrent across solid human epithelial tumor samples for 15 of 16 distinct cancer types from TCGA. Furthermore, ZNF154 hypermethylation is strikingly present across a diverse panel of ENCODE cell lines, but only in those derived from tumor cells. By extending our analysis from the Illumina 27K Infinium platform to the 450K platform, to sequencing of PCR amplicons from bisulfite treated DNA, we demonstrate that hypermethylation extends across the breadth of the ZNF154 CpG island. We have also identified recurrent hypomethylation in two genomic regions associated with CASP8 and VHL. These three genes exhibit significant negative correlation between methylation and gene expression across many cancer types, as well as patterns of DNaseI hypersensitivity and histone marks that reflect different chromatin accessibility in cancer vs. normal cell lines. Our findings emphasize hypermethylation of ZNF154 as a biological marker of relevance for tumor identification. Epigenetic modifications affecting the promoters of ZNF154, CASP8, and VHL are shared across a vast array of tumor types and may therefore be important for understanding the genomic landscape of cancer. PMID:24149212

  14. Data describing the effect of DRD4 promoter polymorphisms on promoter activity.

    PubMed

    Tei, Shoin; Mitsuhashi, Hiroaki; Ishiura, Shoichi

    2016-06-01

    This data article tested whether polymorphisms within the dopamine D4 receptor (DRD4) gene promoter can lead to differences in the promoter activity. The variants, a 120-bp variable number tandem repeat (VNTR), -906 T/C, -809 G/A, -616G/C, and -521C/T, were introduced into the DRD4 promoter and the promoter activity was measured in a neural cell line using the luciferase assay. However, no differences were detected among the haplotypes investigated, and the in vitro data obtained from our protocol could not support the involvement of DRD4 promoter polymorphisms in heritable human traits.

  15. Overview of tumor promotion in animals

    PubMed Central

    Slaga, T. J.

    1983-01-01

    Our present understanding of two-stage carcinogenesis encompasses almost four decades of research. Evidence for chemical promotion or cocarcinogenesis was first provided by Berenblum, who reported that a regimen of croton oil (weak or noncarcinogenic) applied alternately with small doses of benzo(a)pyrene (BP) to mouse skin induced a larger number of tumors than BP alone. Subsequently, Moltram found that a single subcarcinogenic dose of BP followed by multiple applications of croton oil could induce a large number of skin tumors. These investigations as well as a number of others, such as Boutwell, Van Duuren and Hecker, were responsible in defining many important aspects of the initiation and promotion of two-stage carcinogenesis. The initiation stage in mouse skin requires only a single application of either a direct-acting carcinogen or a procarcinogen and is essentially an irreversible step which as data suggests probably involves a somatic cell mutation. The promotion stage in mouse skin can be accomplished by a wide variety of weak or noncarcinogenic agents and is initially reversible later becoming irreversible. Current information suggests that skin tumor promoters are not mutagenic but bring about a number of important epigenetic changes, such as epidermal hyperplasia, and an increase in polyamines, prostaglandins and dark basal keratinocytes as well as other embryonic conditions. Recently, tumor promotion in mouse skin was shown to consist of at least two stages, in which each stage can be accomplished by either a known promoter or a weak or nonpromoting agent. Some of the important characteristics of the first stage of promotion are: (1) only one application of a first-stage promoter, such as phorbol ester tumor promoters, calcium ionophore A23187, hydrogen peroxide and wounding is needed; (2) the action is partially irreversible; (3) an increase in dark basal keratinocytes and prostaglandins is important; and (4) such an increase can be inhibited by

  16. New health promotion movement: a critical examination.

    PubMed

    Robertson, A; Minkler, M

    1994-01-01

    In the last decade, a revolution has been occurring in the field of health promotion. Guided to a large extent by position papers disseminated by the World Health Organization (WHO) Europe Health Promotion Office, and furthered by the Ottawa Charter, the Epp Report in Canada, the Healthy Cities project, as well as by other efforts, this new health promotion movement has introduced new ideas, new language, and new concepts about what constitutes health and how health promotion efforts should be configured to achieve health. Punctuated by the terms like empowerment and community participation, this movement has generated a whole new discourse about the theory and practice of health promotion. This paper explores the multiple meanings that surround these terms, and the implications for practice, by addressing questions like: What does health mean in this new context? What is empowerment? What does participation look like? Has the tyranny of the professional been replaced by the tyranny of the community? Has anything changed about the practice of health promotion other than the language? Finally, it is argued that an acknowledgment of the multidimensionality of these concepts may facilitate their translation from rhetoric into health promotion practice.

  17. Corynebacterium glutamicum promoters: a practical approach

    PubMed Central

    Pátek, Miroslav; Holátko, Jiří; Busche, Tobias; Kalinowski, Jörn; Nešvera, Jan

    2013-01-01

    Summary Transcription initiation is the key step in gene expression in bacteria, and it is therefore studied for both theoretical and practical reasons. Promoters, the traffic lights of transcription initiation, are used as construction elements in biotechnological efforts to coordinate ‘green waves’ in the metabolic pathways leading to the desired metabolites. Detailed analyses of Corynebacterium glutamicum promoters have already provided large amounts of data on their structures, regulatory mechanisms and practical capabilities in metabolic engineering. In this minireview the main aspects of promoter studies, the methods developed for their analysis and their practical use in C. glutamicum are discussed. These include definitions of the consensus sequences of the distinct promoter classes, promoter localization and characterization, activity measurements, the functions of transcriptional regulators and examples of practical uses of constitutive, inducible and modified promoters in biotechnology. The implications of the introduction of novel techniques, such as in vitro transcription and RNA sequencing, to C. glutamicum promoter studies are outlined. PMID:23305350

  18. Vector promoters used in Klebsiella pneumoniae.

    PubMed

    Jiang, Xiao; Zhu, Chengqian; Lin, Jie; Li, Jingkang; Fu, Shuilin; Gong, Heng

    2016-09-01

    Much effort has been devoted to the metabolic engineering of Klebsiella pneumoniae; however, our knowledge of the actual expression level of promoters used in K. pneumoniae is limited. In this study, the expression levels of three promoters were compared systematically by using the lacZ reporter gene with different carbon sources in K. pneumoniae. The results showed that, although promoters PT5 and Ptac designed for Escherichia coli were functional, PT5 appeared more efficient and the induction/repression ratio of Ptac was decreased extremely in K. pneumoniae. The basal level of Ptac for lacZ expression reached 396.5 U/mg, which was 9.5-fold higher compared with PT5 in LB medium, indicating Ptac can be used as an efficient "constitutive" promoter as well as an efficient induced promoter in K. pneumoniae. In different carbon sources medium, a newly constructed endogenous constitutive Pbud proved to be a stable and weak promoter. On the basis of our data, a set of Pbud and Ptac promoters could meet the broad range (about 1,000 orders of magnitude) of gene expression needed for engineered K. pneumoniae in glycerol-based medium.

  19. [Prediction of Promoter Motifs in Virophages].

    PubMed

    Gong, Chaowen; Zhou, Xuewen; Pan, Yingjie; Wang, Yongjie

    2015-07-01

    Virophages have crucial roles in ecosystems and are the transport vectors of genetic materials. To shed light on regulation and control mechanisms in virophage--host systems as well as evolution between virophages and their hosts, the promoter motifs of virophages were predicted on the upstream regions of start codons using an analytical tool for prediction of promoter motifs: Multiple EM for Motif Elicitation. Seventeen potential promoter motifs were identified based on the E-value, location, number and length of promoters in genomes. Sputnik and zamilon motif 2 with AT-rich regions were distributed widely on genomes, suggesting that these motifs may be associated with regulation of the expression of various genes. Motifs containing the TCTA box were predicted to be late promoter motif in mavirus; motifs containing the ATCT box were the potential late promoter motif in the Ace Lake mavirus . AT-rich regions were identified on motif 2 in the Organic Lake virophage, motif 3 in Yellowstone Lake virophage (YSLV)1 and 2, motif 1 in YSLV3, and motif 1 and 2 in YSLV4, respectively. AT-rich regions were distributed widely on the genomes of virophages. All of these motifs may be promoter motifs of virophages. Our results provide insights into further exploration of temporal expression of genes in virophages as well as associations between virophages and giant viruses.

  20. Development of an efficient bi-directional promoter with tripartite enhancer employing three viral promoters.

    PubMed

    Patro, Sunita; Maiti, Indu B; Dey, Nrisingha

    2013-02-10

    We have developed a novel bi-directional promoter (FsFfCBD) by placing two heterogeneous core-promoters from the Figwort mosaic virus sub-genomic transcript promoter (FsCP, -69 to +31) and Cauliflower mosaic virus 35S promoter (CCP, -89 to +1) respectively on upstream (5') and downstream (3') ends of a tri-hybrid enhancer (FsEFfECE), in reverse orientation. The FsEFfECE domain encompasses three heterologous enhancer fragments from Figwort mosaic virus sub-genomic transcript promoter (FsE, 101 bp, -70 to -170), Figwort mosaic virus full-length transcript promoter (FfE, 196 bp, -249 to -54) and Cauliflower mosaic virus 35S promoter (CE, 254 bp, -343 to -90). The bi-directional nature of the FsFfCBD promoter (coupled to GFP and GUS) was established both in transient systems (onion epidermal cells and tobacco protoplasts) and transgenic plant (Nicotiana tabacum samsun NN) by monitoring the simultaneous expression of GFP and GUS employing fluorescence (for GFP) and biochemical (for GUS) based assays. In transgenic plants, the FsFfCBD promoter was found to be 6.8 and 2.5 times stronger than two parent promoters; Fs and FfC respectively. The bi-directional compound promoter FsFfCBD, composed of three heterologous enhancers with enhanced activity could become a valuable additional tool for efficient plant metabolic engineering and molecular pharming.

  1. The impact of the distance-dependent promotional effect on the promotion cost sharing decision

    NASA Astrophysics Data System (ADS)

    Sheen, Gwo-Ji; Wang, Shih-Yen; Yeh, Yingchieh

    2016-02-01

    This paper considers the promotion cost sharing decision between a supplier and a retailer. The customer demand is affected by both national and local promotional effects while the local promotional effect on a customer is dependent on the distance between the retailer and this customer. We propose a continuous approximation approach to modelling the sum of the customer demand in the whole market area served by the retailer. A model is provided to help managers decide on the retail price, the local advertising expenditure, the national advertising expenditure, and the supplier participation rate, with consideration of the influence of distance on the promotional effect. We also find that the supplier's promotion cost sharing rate increases as the market size increases or the influence of distance on the promotional effect decreases. A numerical example is given to show that the nature of distance-dependent promotional effect has a significant impact on the decisions and profits.

  2. NCDs, health promotion and public health.

    PubMed

    McQueen, David V

    2013-12-01

    Though not necessarily using the same terminology historically, people concerned with the public's health have long been addressing the social context of non-communicable diseases (NCDs) and the actions of promoting health. This commentary places the current interest in NCDs within that history and discusses the challenges that continue to face institutions in dealing with NCDs. It makes a particular plea for the role of health promotion as the area of public health that takes actions to address the global burden of NCDs. Without a health promotion focus, we will just continue to describe the NCD burden rather than reduce it.

  3. Characterization of human TCR Vbeta gene promoter. Role of the dodecamer motif in promoter activity.

    PubMed

    Deng, X; Sun, G R; Zheng, Q; Li, Y

    1998-09-11

    During T-lymphocyte development, the T-cell antigen receptor (TCR) gene expression is controlled by its promoter and enhancer elements and regulated in tissue- and development stage-specific manner. To uncover the promoter function and to define positive and negative regulatory elements in TCR gene promoters, the promoter activities from 13 human TCR Vbeta genes were determined by the transient transfection system and luciferase reporter assay. Although most of the TCR Vbeta gene promoters that we tested are inactive by themselves, some promoters were found to be constitutively strong. Among them, Vbeta6.7 is the strongest. 5'-Deletion and fragmentation experiments have narrowed the full promoter activity of Vbeta6.7 to a fragment of 147 base pairs immediately 5' to the transcription initiation site. A decanucleotide motif with the consensus sequence AGTGAYRTCA has been found to be conserved in most TCR Vbeta gene promoters. There are three such decamer motifs in the promoter region of Vbeta6.7, and the contribution of each such motif to the promoter activity has been examined. Further site-directed mutagenesis analyses showed that: 1) when two Ts in the decamer were mutated, the promoter activity was totally abolished; 2) when two additional nucleotides 3' to the end of decamer were mutated, the promoter activity was decreased to two-thirds of the full level; and 3) when the element with the sequence AGTGATGTCACT was inserted into other promoters, the original weak promoters become very strong. Taken together, our data suggest that the positive regulatory element in Vbeta6.7 should be considered a dodecamer rather than a decamer and that it confers strong basal transcriptional activity on TCR Vbeta genes.

  4. Promoted Combustion Test Propagation Rate Data

    NASA Technical Reports Server (NTRS)

    Borstorff, J.; Jones, P.; Lowery, F.

    2002-01-01

    Combustion propagation rate data were examined for potential use in benchmarking a thermal model of the Promoted Combustion Test (PCT), and also for potential use in measuring the repeatability of PCT results.

  5. Responsible Assertive Behavior Promotes Effective Interpersonal Communication.

    ERIC Educational Resources Information Center

    Hulbert, Jack E.

    1983-01-01

    Assertive behavior promotes the development of mutually satisfying relationships, with none of the disadvantages of passive or aggressive behavior. Because of the interpersonal and organizational benefits, managers should encourage assertive behavior throughout their organizations. (SK)

  6. 21 CFR 314.640 - Promotional materials.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Efficacy Studies Are Not Ethical or Feasible § 314.640 Promotional materials. For drug products being... publication within 120 days following marketing approval. After 120 days following marketing approval,...

  7. Health promotion: challenges revealed in successful practices

    PubMed Central

    Silva, Kênia Lara; de Sena, Roseni Rosângela; Belga, Stephanie Marques Moura Franco; Silva, Paloma Morais; Rodrigues, Andreza Trevenzoli

    2014-01-01

    OBJECTIVE To examine successful practices of health promotion in health, education, culture, welfare and sport, leisure, identifying the elements of success and challenges in the field. METHODS A qualitative study with data obtained from in-depth analysis that included participant observation, interviews with managers, coordinators, professionals and participants from 29 practices reported as successful for promoting health in six municipalities of the metropolitan region of Belo Horizonte, MG, Southeastern Brazil, in 2011. The variables of the study were concept, dimension, dissemination and ease of access, identified in practices guided by content analysis. RESULTS The results indicate a conceptual and methodological uncertainty about health promotion as evidenced by conflicting objects and contradictory purposes. The practices differ in size, coverage and ease of access, determined by inter-sector coordination and political and financial investment. CONCLUSIONS We identified challenges to health promotion focusing on vulnerable populations, limits to financing and intersectoral partnerships. PMID:24789640

  8. Promoting Community Health Resources: Preferred Communication Strategies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Community health promotion efforts involve communicating resource information to priority populations. Which communication strategies are most effective is largely unknown for specific populations. Objective: A random-dialed telephone survey was conducted to assess health resource comm...

  9. What Promotes Racial and Ethnic Tolerance?

    ERIC Educational Resources Information Center

    Holloway, John H.

    2003-01-01

    Reviews selected research on efforts to promote racial and ethnic tolerance through the adoption of certain school and classroom practices and improved preservice and inservice teacher-training activities. (PKP)

  10. A decision theoretical approach for diffusion promotion

    NASA Astrophysics Data System (ADS)

    Ding, Fei; Liu, Yun

    2009-09-01

    In order to maximize cost efficiency from scarce marketing resources, marketers are facing the problem of which group of consumers to target for promotions. We propose to use a decision theoretical approach to model this strategic situation. According to one promotion model that we develop, marketers balance between probabilities of successful persuasion and the expected profits on a diffusion scale, before making their decisions. In the other promotion model, the cost for identifying influence information is considered, and marketers are allowed to ignore individual heterogeneity. We apply the proposed approach to two threshold influence models, evaluate the utility of each promotion action, and provide discussions about the best strategy. Our results show that efforts for targeting influentials or easily influenced people might be redundant under some conditions.

  11. The Causes and Costs of Promotion Trauma.

    ERIC Educational Resources Information Center

    Darling, Lu Ann W.; McGrath, Loraine G.

    1983-01-01

    Minimizing the stress that nurses experience after a promotion requires an understanding of the factors associated with moving up the managerial ladder. Knowing these factors, nursing administrators can plan their programs to support their managers in transition. (SSH)

  12. Promoting consumer empowerment through entrepreneurship: a proposal.

    PubMed

    Prince, Jonathan D

    2007-01-01

    In order to promote life control, this proposal suggests strategies to help people who use community mental health services to access small loans or "microcredit" for entrepreneurship in self-help groups.

  13. The health promotion internship: a sponsor's perspective.

    PubMed

    Smoczyk, C M

    1987-01-01

    Health promotion internships are an essential link between the university classroom and the corporate or public service environment. A well structured internship experience will provide the future health promotion professional with an opportunity to transfer learned theory into practical application within a controlled and supervised environment. The success of the internship experience is contingent upon the mutual effort of the student, academic institution, and sponsoring organization working together to develop a model that will reflect the goals and objectives of each group with primary long-term benefit accruing to the future health promotion professional. The purpose of this article is to explore the components of a successful health promotion internship model developed from the perspective of a sponsoring organization.

  14. Promoting Business Education through Student Organization Activities.

    ERIC Educational Resources Information Center

    Yelverton, Sandra

    1983-01-01

    Discusses the promotion of business education through the activities of student organizations. Describes specific programs, projects, and leadership development activities and their effectiveness in publicizing business education programs. (JOW)

  15. Worksite health promotion programs in college settings

    PubMed Central

    Hill-Mey, Patricia E.; Kumpfer, Karol L.; Merrill, Ray M.; Reel, Justine; Hyatt-Neville, Beverly; Richardson, Glenn E.

    2015-01-01

    The purpose of this paper is to describe the multifaceted nature and benefits of worksite health promotion programs (WHPPs), with emphasis on the college setting. An assessment of the peer-reviewed literature was conducted of articles published since 2000. Several search engines were accessed and selected key words were used. Most studies examining WHPPs have focused on return on investment and productivity. Research that targets the softer side-benefits of health promotion programs in the workplace is less available. Although the college setting offers some advantages for implementing health promotion programs. They may also have unique challenges due to their large and diverse employee population. There is little research to show the effectiveness and unique challenges of college-based health promotion programs. PMID:25861657

  16. Perceived effectiveness of messages promoting hand hygiene.

    PubMed

    Taylor, Ronald E

    2017-03-01

    Eighty-six infection control specialists evaluated 24 messages promoting hand hygiene (HH). Brief, simple messages using appeals to social situations and to ego (self-efficacy) were rated as most likely to increase HH compliance.

  17. Reviewing health promotion in nursing education.

    PubMed

    Whitehead, Dean

    2007-04-01

    The [World Health Organisation, 2000. Nurse and Midwives for Health: A WHO European Strategy for Nursing and Midwifery Education. WHO Regional Office for Europe, Copenhagen] European Strategy for Nursing and Midwifery Education calls for the explicit inclusion and application of health promotion in all nursing curricula. Prior to this strategy, and subsequently, studies have explored the nature and extent of health promotion in nursing education. This article extensively reviews this body of literature. Overall, the literature, both included in this review and its supporting discussion, presents a picture suggesting that the call for effective inclusion of health promotion has in many cases gone unheeded. The literature also identifies that the educational delivery of broader elements of health promotion is muted in comparison to the 'traditional' constructs of health education. Considerations for wider reform, born out of the literature, are presented.

  18. Cultural aspects of ageing and health promotion.

    PubMed

    Mariño, R J

    2015-03-01

    The emphasis of Australian Government policy is on the promotion of good health in later life and positive experiences with ageing. Conceptually, a new gerontology framework has replaced the study of disease, decline, loss and disability. Within this framework, health promotion offers a mechanism by which individuals can be assisted to create environments that offer better opportunities for continued participation in society and improved quality of health and self-care. Oral health is instrumental to older people's health, life satisfaction, quality of life and perception of self. Australia is culturally diverse, composed of numerous ethno-cultural groups coexisting within a larger, predominant culture, creating a multicultural and multiracial society. However, despite this cultural diversity, the well documented ageing profile of the Australian population and repeated calls for comprehensive geriatric assessment, the oral health of older adults remains a challenge for oral health providers and for society. A major challenge will be to translate existing knowledge and experience of disease prevention and health promotion into appropriate programmes for older adults. Health promotion is the key to improving oral health in later life as it encourages older adults to be proactive in regard to their health. Therefore, increased efforts should be directed towards identifying opportunities for health promotion activities and the development of community based models that encourage older people to improve and maintain their oral health. Ignoring opportunities for health promotion may increase inequalities in oral health and may lead to even greater demands for curative and oral rehabilitative services from these groups This article firstly provides a brief rationale for oral health promotion. Its second part explores the influence of culture on health beliefs, behaviours and outcomes in older adults and how oral health can relate to cultural background. The last section

  19. Characterizing yeast promoters used in Kluyveromyces marxianus.

    PubMed

    Yang, Chun; Hu, Shenglin; Zhu, Songli; Wang, Dongmei; Gao, Xiaolian; Hong, Jiong

    2015-10-01

    Fermentation at higher temperatures can potentially reduce the cooling cost in large-scale fermentation and reduce the contamination risk. Thus, the thermotolerant yeast, Kluyveromyces marxianus, which can grow and ferment at elevated temperatures, is a promising biotechnological tool for future applications. However, the promoters used in K. marxianus are not well characterized, especially at elevated temperatures, which is important in efficient metabolic pathway construction. In this study, six constitutive promoters (P(TDH3), P(PGK), and P(ADH1) from both Saccharomyces cerevisiae and K. marxianus) were evaluated in K. marxianus through the heterologous expression of the KlLAC4, GUSA, and SH BLE genes at various temperatures, with various carbon sources and oxygen conditions. The expression was evaluated at the transcription and protein level using real-time PCR and protein activity determination to eliminate the effect of heterologous protein stability. While the transcription of all the promoters decreased at higher temperatures, the order of their promoting strength at various temperatures with glucose as the carbon source was P(KmPGK) > P(KmTDH3) > P(ScPGK) > P(ScTDH3) > P(KmADH1) > P(ScADH1). When glycerol or xylose was supplied as the carbon source at 42 °C, the order of promoter strength was P(KmPGK) > P(ScPGK) > P(KmADH1) > P(ScADH1) > P(ScTDH3) > P(KmTDH3). The promoter activity of P TDH3 decreased significantly, while the promoter activity of both of the P(ADH1) promoters increased. Oxygen conditions had non-significant effect. The results of this study provide important information for fine-tuned pathway construction for the metabolic engineering of K. marxianus.

  20. Heated Promoted Combustion-Initial Test Results

    NASA Technical Reports Server (NTRS)

    Engel, Carl D.; Herald, Stephen; Davis, S. Eddie

    2005-01-01

    The purpose of the STD 6001 test 17 is to determine the flammability of materials in GOX at ambient temperature and at use pressure. The purpose of the new Heated Promoted combustion test is to determine the flammability of material in GOX at use temperature and pressure. The objective is to present the new heated promoted combustion method and show initial data and trends for three representative metals.

  1. Evidence for promotion of nontenured faculty.

    PubMed

    Sorrell, Jeanne M; Cangelosi, Pamela R; Black, Patricia L; Bunty, Kathy; Filak, Carol; Hobbs, Deborah L; Sookratree, Monruedee

    2008-01-01

    An increasing number of faculty members are choosing the nontenured path in academia. These faculty members bring expertise in teaching that must be recognized and valued. With the shortage of nurse educators, procedures to attract and retain these faculty members are needed. To address this problem, guidelines for promotion of nontenured faculty were developed. The authors describe the development process and the final criteria for promotion.

  2. Kinetic analysis of T7 RNA polymerase-promoter interactions with small synthetic promoters.

    PubMed

    Martin, C T; Coleman, J E

    1987-05-19

    Specific interactions between T7 RNA polymerase and its promoter have been studied by a simple steady-state kinetic assay using synthetic oligonucleotide promoters that produce a short five-base message. A series of promoters with upstream lengths extending to promoter positions -19, -17, -14, and -12 show that promoters extending to -19 and -17 produce very specific transcripts with initiation rate constant Kcat = 50 min-1 and a Michaelis constant Km = 0.02 microM, indicating that the consensus sequence to position -17 is sufficient for maximum promoter usage. Shortening the upstream region of the promoter to -14 substantially increases Km (0.3 microM) but does not significantly reduce the maximum velocity (kcat = 30 min-1). Finally, truncation of the promoter at position -12 results in extremely low levels of specific transcription. The coding and noncoding strands appear to make different contributions to promoter recognition. Although the double-stranded promoter of upstream length -12 is very poor as a transcription template, extension of only the noncoding strand to -17 very significantly improves both Kcat and Km. In contrast, extension of only the coding strand results in no significant improvement. Substitution of an AT base pair at position -10 by CG (as found in T3 RNA polymerase promoters) produces a 10-fold increase in Km, with little effect on Kcat. Comparison of two promoters containing a base pair mismatch at this site (AG or CT) demonstrates that promoter recognition is very sensitive to the nature of the base on the noncoding strand and is only slightly affected by the presence of a mismatch created by a wrong base in the coding strands.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Implementation of Mexico's Health Promotion Operational Model.

    PubMed

    Santos-Burgoa, Carlos; Rodríguez-Cabrera, Lucero; Rivero, Lilia; Ochoa, Jorge; Stanford, Adriana; Latinovic, Ljubica; Rueda, Gretel

    2009-01-01

    Mexico is undergoing profound health reform, extending health insurance to previously uninsured populations and changing the way health care services are delivered. Legislation enacted in 2003 and implemented in 2004 mandated funding and infrastructure that will allow 52% of Mexico's population to access medical care at no cost by 2010. This ambitious social reform has not been without challenges, particularly financial sustainability. Health promotion, because of its potential to prevent or delay chronic diseases and injuries and their associated costs, is a key component of health care reform. In 2006, the Ministry of Health's General Directorate of Health Promotion developed the Health Promotion Operational Model. Based on Ottawa Charter functions, the model integrates health promotion activities within the overall health care system. The main goal of this model is to build strong human capital and to improve organizational capacity for health promotion starting at the local level by training health care personnel to implement health promotion activities. Organizational development workshops started in 2006, and implementation plans in all 32 Mexican states were in place by end of 2008.

  4. Promoter Motifs in NCLDVs: An Evolutionary Perspective.

    PubMed

    Oliveira, Graziele Pereira; Andrade, Ana Cláudia Dos Santos Pereira; Rodrigues, Rodrigo Araújo Lima; Arantes, Thalita Souza; Boratto, Paulo Victor Miranda; Silva, Ludmila Karen Dos Santos; Dornas, Fábio Pio; Trindade, Giliane de Souza; Drumond, Betânia Paiva; La Scola, Bernard; Kroon, Erna Geessien; Abrahão, Jônatas Santos

    2017-01-20

    For many years, gene expression in the three cellular domains has been studied in an attempt to discover sequences associated with the regulation of the transcription process. Some specific transcriptional features were described in viruses, although few studies have been devoted to understanding the evolutionary aspects related to the spread of promoter motifs through related viral families. The discovery of giant viruses and the proposition of the new viral order Megavirales that comprise a monophyletic group, named nucleo-cytoplasmic large DNA viruses (NCLDV), raised new questions in the field. Some putative promoter sequences have already been described for some NCLDV members, bringing new insights into the evolutionary history of these complex microorganisms. In this review, we summarize the main aspects of the transcription regulation process in the three domains of life, followed by a systematic description of what is currently known about promoter regions in several NCLDVs. We also discuss how the analysis of the promoter sequences could bring new ideas about the giant viruses' evolution. Finally, considering a possible common ancestor for the NCLDV group, we discussed possible promoters' evolutionary scenarios and propose the term "MEGA-box" to designate an ancestor promoter motif ('TATATAAAATTGA') that could be evolved gradually by nucleotides' gain and loss and point mutations.

  5. Firefly luciferase gene contains a cryptic promoter

    PubMed Central

    Vopálenský, Václav; Mašek, Tomáš; Horváth, Ondřej; Vicenová, Blanka; Mokrejš, Martin; Pospíšek, Martin

    2008-01-01

    A firefly luciferase (FLuc) counts among the most popular reporters of present-day molecular and cellular biology. In this study, we report a cryptic promoter activity in the luc+ gene, which is the most frequently used version of the firefly luciferase. The FLuc coding region displays cryptic promoter activity both in mammalian and yeast cells. In human CCL13 and Huh7 cells, cryptic transcription from the luc+ gene is 10–16 times weaker in comparison to the strong immediate-early cytomegalovirus promoter. Additionally, we discuss a possible impact of the FLuc gene cryptic promoter on experimental results especially in some fields of the RNA-oriented research, for example, in analysis of translation initiation or analysis of miRNA/siRNA function. Specifically, we propose how this newly described cryptic promoter activity within the FLuc gene might contribute to the previous determination of the strength of the cryptic promoter found in the cDNA corresponding to the hepatitis C virus internal ribosome entry site. Our findings should appeal to the researchers to be more careful when designing firefly luciferase-based assays as well as open the possibility of performing some experiments with the hepatitis C virus internal ribosome entry site, which could not be considered until now. PMID:18697919

  6. 7 CFR 1207.335 - Research and promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POTATO RESEARCH AND PROMOTION PLAN Potato Research and Promotion Plan Research and Promotion § 1207.335 Research and promotion. The... administration of appropriate programs or projects for the advertising and promotion of potatoes and...

  7. 7 CFR 1207.335 - Research and promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POTATO RESEARCH AND PROMOTION PLAN Potato Research and Promotion Plan Research and Promotion § 1207.335 Research and promotion. The... administration of appropriate programs or projects for the advertising and promotion of potatoes and...

  8. 7 CFR 1207.335 - Research and promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POTATO RESEARCH AND PROMOTION PLAN Potato Research and Promotion Plan Research and Promotion § 1207.335 Research and promotion. The... administration of appropriate programs or projects for the advertising and promotion of potatoes and...

  9. 7 CFR 1207.335 - Research and promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POTATO RESEARCH AND PROMOTION PLAN Potato Research and Promotion Plan Research and Promotion § 1207.335 Research and promotion. The... administration of appropriate programs or projects for the advertising and promotion of potatoes and...

  10. 7 CFR 1230.60 - Promotion, research, and consumer information.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Promotion, research, and consumer information. 1230... PORK PROMOTION, RESEARCH, AND CONSUMER INFORMATION Pork Promotion, Research, and Consumer Information Order Promotion, Research, and Consumer Information § 1230.60 Promotion, research, and...

  11. Nurses and Teachers: Partnerships for Green Health Promotion

    ERIC Educational Resources Information Center

    Sendall, Marguerite C.; Lidstone, John; Fleming, MaryLou; Domocol, Michelle

    2013-01-01

    Background: The term "green health promotion" is given to health promotion underpinned by the principles of ecological health and sustainability. Green health promotion is supported philosophically by global health promotion documents such as the 1986 Ottawa Charter for Health Promotion and the ecological public health movement. Green…

  12. 7 CFR 1230.60 - Promotion, research, and consumer information.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion, research, and consumer information. 1230... PORK PROMOTION, RESEARCH, AND CONSUMER INFORMATION Pork Promotion, Research, and Consumer Information Order Promotion, Research, and Consumer Information § 1230.60 Promotion, research, and...

  13. 5 CFR 335.103 - Agency promotion programs.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Agency promotion programs. 335.103... PROMOTION AND INTERNAL PLACEMENT General Provisions § 335.103 Agency promotion programs. (a) Merit promotion plans. Except as otherwise specifically authorized by OPM, an agency may make promotions under §...

  14. 7 CFR 1250.341 - Research, education, and promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Research, education, and promotion. 1250.341 Section... RESEARCH AND PROMOTION Egg Research and Promotion Order Research, Education, and Promotion § 1250.341 Research, education, and promotion. The Board shall develop and submit to the Secretary for approval...

  15. 7 CFR 1205.333 - Research and promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Research and promotion. 1205.333 Section 1205.333... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE COTTON RESEARCH AND PROMOTION Cotton Research and Promotion Order Research and Promotion § 1205.333 Research and promotion. The...

  16. 7 CFR 1207.335 - Research and promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Research and promotion. 1207.335 Section 1207.335... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POTATO RESEARCH AND PROMOTION PLAN Potato Research and Promotion Plan Research and Promotion § 1207.335 Research and promotion....

  17. 7 CFR 1250.341 - Research, education, and promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Research, education, and promotion. 1250.341 Section... RESEARCH AND PROMOTION Egg Research and Promotion Order Research, Education, and Promotion § 1250.341 Research, education, and promotion. The Board shall develop and submit to the Secretary for approval...

  18. 7 CFR 1250.341 - Research, education, and promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 10 2012-01-01 2012-01-01 false Research, education, and promotion. 1250.341 Section... RESEARCH AND PROMOTION Egg Research and Promotion Order Research, Education, and Promotion § 1250.341 Research, education, and promotion. The Board shall develop and submit to the Secretary for approval...

  19. 7 CFR 1250.341 - Research, education, and promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 10 2013-01-01 2013-01-01 false Research, education, and promotion. 1250.341 Section... RESEARCH AND PROMOTION Egg Research and Promotion Order Research, Education, and Promotion § 1250.341 Research, education, and promotion. The Board shall develop and submit to the Secretary for approval...

  20. 7 CFR 1205.333 - Research and promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 10 2013-01-01 2013-01-01 false Research and promotion. 1205.333 Section 1205.333... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE COTTON RESEARCH AND PROMOTION Cotton Research and Promotion Order Research and Promotion § 1205.333 Research and promotion. The...

  1. Development of a promoter shutoff system in Aspergillus oryzae using a sorbitol-sensitive promoter.

    PubMed

    Oda, Ken; Terado, Shiho; Toyoura, Rieko; Fukuda, Hisashi; Kawauchi, Moriyuki; Iwashita, Kazuhiro

    2016-09-01

    Promoter shutoff is a general method for analyzing essential genes, but in the fungus Aspergillus oryzae, no tightly repressed promoters have been reported. To overcome the current limitations of conditional promoters, we examined sorbitol- and galactose-responsive genes using microarrays to identify regulatable genes with only minor physiological and genetic effects. We identified two sorbitol-induced genes (designated as sorA and sorB), cloned their promoters, and built a regulated egfp and brlA expression system. Growth medium-dependent enhanced green fluorescence protein (EGFP) fluorescence and conidiation were confirmed for egfp and brlA under the control of their respective promoters. We also used this shutoff system to regulate the essential rhoA, which demonstrated the expected growth inhibition under repressed growth conditions. Our new sorbitol promoter shutoff system developed can serve as a valuable new tool for essential gene analyses of filamentous fungi.

  2. Assessing environmental assets for health promotion program planning: a practical framework for health promotion practitioners

    PubMed Central

    Springer, Andrew E.; Evans, Alexandra E.

    2016-01-01

    Conducting a health needs assessment is an important if not essential first step for health promotion planning. This paper explores how health needs assessments may be further strengthened for health promotion planning via an assessment of environmental assets rooted in the multiple environments (policy, information, social and physical environments) that shape health and behavior. Guided by a behavioral-ecological perspective- one that seeks to identify environmental assets that can influence health behavior, and an implementation science perspective- one that seeks to interweave health promotion strategies into existing environmental assets, we present a basic framework for assessing environmental assets and review examples from the literature to illustrate the incorporation of environmental assets into health program design. Health promotion practitioners and researchers implicitly identify and apply environmental assets in the design and implementation of health promotion interventions;this paper provides foundation for greater intentionality in assessing environmental assets for health promotion planning. PMID:27579254

  3. Assessing environmental assets for health promotion program planning: a practical framework for health promotion practitioners.

    PubMed

    Springer, Andrew E; Evans, Alexandra E

    2016-01-01

    Conducting a health needs assessment is an important if not essential first step for health promotion planning. This paper explores how health needs assessments may be further strengthened for health promotion planning via an assessment of environmental assets rooted in the multiple environments (policy, information, social and physical environments) that shape health and behavior. Guided by a behavioral-ecological perspective- one that seeks to identify environmental assets that can influence health behavior, and an implementation science perspective- one that seeks to interweave health promotion strategies into existing environmental assets, we present a basic framework for assessing environmental assets and review examples from the literature to illustrate the incorporation of environmental assets into health program design. Health promotion practitioners and researchers implicitly identify and apply environmental assets in the design and implementation of health promotion interventions;this paper provides foundation for greater intentionality in assessing environmental assets for health promotion planning.

  4. Structure and Regulation of the Versican Promoter

    PubMed Central

    Domenzain-Reyna, Clelia; Hernández, Daniel; Miquel-Serra, Laia; Docampo, María José; Badenas, Celia; Fabra, Angels; Bassols, Anna

    2009-01-01

    Versican is a large chondroitin sulfate proteoglycan of the extracellular matrix that is involved in a variety of cellular processes. We showed previously that versican, which is overexpressed in cutaneous melanomas as well as in premalignant lesions, contributes to melanoma progression, favoring the detachment of cells and the metastatic dissemination. Here, we investigated the transcriptional regulation of the versican promoter in melanoma cell lines with different levels of biological aggressiveness and stages of differentiation. We show that versican promoter up-regulation accounts for the differential expression levels of mRNA and protein detected in the invasive SK-mel-131 human melanoma cells. The activity of the versican promoter increased 5-fold in these cells in comparison with that measured in non-invasive MeWo melanoma cells. Several transcriptional regulatory elements were identified in the proximal promoter, including AP-1, Sp1, AP-2, and two TCF-4 sites. We show that promoter activation is mediated by the ERK/MAPK and JNK signaling pathways acting on the AP-1 site, suggesting that BRAF mutation present in SK-mel-131 cells impinge upon the up-regulation of the versican gene through signaling elicited by the ERK/MAPK pathway. This is the first time the AP-1 transcription factor family has been shown to be related to the regulation of versican expression. Furthermore, deletion of the TCF-4 binding sites caused a 60% decrease in the promoter activity in SK-mel-131 cells. These results showing that AP-1 and TCF-4 binding sites are the main regulatory regions directing versican production provide new insights into versican promoter regulation during melanoma progression. PMID:19269971

  5. TERT promoter mutations in melanoma survival.

    PubMed

    Nagore, Eduardo; Heidenreich, Barbara; Rachakonda, Sívaramakrishna; Garcia-Casado, Zaida; Requena, Celia; Soriano, Virtudes; Frank, Christoph; Traves, Victor; Quecedo, Esther; Sanjuan-Gimenez, Josefa; Hemminki, Kari; Landi, Maria Teresa; Kumar, Rajiv

    2016-07-01

    Despite advances in targeted therapies, the treatment of advanced melanoma remains an exercise in disease management, hence a need for biomarkers for identification of at-risk primary melanoma patients. In this study, we aimed to assess the prognostic value of TERT promoter mutations in primary melanomas. Tumors from 300 patients with stage I/II melanoma were sequenced for TERT promoter and BRAF/NRAS mutations. Cumulative curves were drawn for patients with and without mutations with progression-free and melanoma-specific survival as outcomes. Cox proportional hazard regression models were used to determine the effect of the mutations on survivals. Individually, presence of TERT promoter and BRAF/NRAS mutations associated with poor disease-free and melanoma-specific survival with modification of the effect by the rs2853669 polymorphism within the TERT promoter. Hazard ratio (HR) for simultaneous occurrence of TERT promoter and BRAF/NRAS mutations for disease-free survival was 2.3 (95% CI 1.2-4.4) and for melanoma-specific survival 5.8 (95% CI 1.9-18.3). The effect of the mutations on melanoma-specific survival in noncarriers of variant allele of the polymorphism was significant (HR 4.5, 95% CI 1.4-15.2) but could not be calculated for the carriers due to low number of events. The variant allele per se showed association with increased survival (HR 0.3, 95% CI 0.1-0.9). The data in this study provide preliminary evidence that TERT promoter mutations in combination with BRAF/NRAS mutations can be used to identify patients at risk of aggressive disease and the possibility of refinement of the classification with inclusion of the rs2853669 polymorphism within TERT promoter.

  6. Reduced capacity of alternative σs to melt promoters ensures stringent promoter recognition

    PubMed Central

    Koo, Byoung-Mo; Rhodius, Virgil A.; Nonaka, Gen; deHaseth, Pieter L.; Gross, Carol A.

    2009-01-01

    In bacteria, multiple σs direct RNA polymerase to distinct sets of promoters. Housekeeping σs direct transcription from thousands of promoters, whereas most alternative σs are more selective, recognizing more highly conserved promoter motifs. For σ32 and σ28, two Escherichia coli Group 3 σs, altering a few residues in Region 2.3, the portion of σ implicated in promoter melting, to those universally conserved in housekeeping σs relaxed their stringent promoter requirements and significantly enhanced melting of suboptimal promoters. All Group 3 σs and the more divergent Group 4 σs have nonconserved amino acids at these positions and rarely transcribe >100 promoters. We suggest that the balance of “melting” and “recognition” functions of σs is critical to setting the stringency of promoter recognition. Divergent σs may generally use a nonoptimal Region 2.3 to increase promoter stringency, enabling them to mount a focused response to altered conditions. PMID:19833768

  7. Database construction for PromoterCAD: synthetic promoter design for mammals and plants.

    PubMed

    Nishikata, Koro; Cox, Robert Sidney; Shimoyama, Sayoko; Yoshida, Yuko; Matsui, Minami; Makita, Yuko; Toyoda, Tetsuro

    2014-03-21

    Synthetic promoters can control a gene's timing, location, and expression level. The PromoterCAD web server ( http://promotercad.org ) allows the design of synthetic promoters to control plant gene expression, by novel arrangement of cis-regulatory elements. Recently, we have expanded PromoterCAD's scope with additional plant and animal data: (1) PLACE (Plant Cis-acting Regulatory DNA Elements), including various sized sequence motifs; (2) PEDB (Mammalian Promoter/Enhancer Database), including gene expression data for mammalian tissues. The plant PromoterCAD data now contains 22 000 Arabidopsis thaliana genes, 2 200 000 microarray measurements in 20 growth conditions and 79 tissue organs and developmental stages, while the new mammalian PromoterCAD data contains 679 Mus musculus genes and 65 000 microarray measurements in 96 tissue organs and cell types ( http://promotercad.org/mammal/ ). This work presents step-by-step instructions for adding both regulatory motif and gene expression data to PromoterCAD, to illustrate how users can expand PromoterCAD functionality for their own applications and organisms.

  8. Efficient chimeric plant promoters derived from plant infecting viral promoter sequences.

    PubMed

    Acharya, Sefali; Ranjan, Rajiv; Pattanaik, Sitakanta; Maiti, Indu B; Dey, Nrisingha

    2014-02-01

    In the present study, we developed a set of three chimeric/hybrid promoters namely FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt incorporating different important domains of Figwort Mosaic Virus sub-genomic transcript promoter (FSgt, -270 to -60), Mirabilis Mosaic Virus sub-genomic transcript promoter (MSgt, -306 to -125) and Peanut Chlorotic Streak Caulimovirus full-length transcript promoter (PFlt-, -353 to +24 and PFlt-UAS, -353 to -49). We demonstrated that these chimeric/hybrid promoters can drive the expression of reporter genes in different plant species including tobacco, Arabidopsis, petunia, tomato and spinach. FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt promoters showed 4.2, 1.5 and 1.2 times stronger GUS activities compared to the activity of the CaMV35S promoter, respectively, in tobacco protoplasts. Protoplast-derived recombinant promoter driven GFP showed enhanced accumulation compared to that obtained under the CaMV35S promoter. FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt promoters showed 3.0, 1.3 and 1.0 times stronger activities than the activity of the CaMV35S² (a modified version of the CaMV35S promoter with double enhancer domain) promoter, respectively, in tobacco (Nicotiana tabacum, var. Samsun NN). Alongside, we observed a fair correlation between recombinant promoter-driven GUS accumulation with the corresponding uidA-mRNA level in transgenic tobacco. Histochemical (X-gluc) staining of whole transgenic seedlings and fluorescence images of ImaGene Green™ treated floral parts expressing the GUS under the control of recombinant promoters also support above findings. Furthermore, we confirmed that these chimeric promoters are inducible in the presence of 150 μM salicylic acid (SA) and abscisic acid (ABA). Taken altogether, we propose that SA/ABA inducible chimeric/recombinant promoters could be used for strong expression of gene(s) of interest in crop plants.

  9. 65 FR 10599 - Honey Research, Promotion, and Consumer Information Order

    Federal Register 2010, 2011, 2012, 2013, 2014

    2000-02-28

    ... Agriculture Agricultural Marketing Service 7 CFR Part 1240 Honey Research, Promotion, and Consumer Information... Honey Research, Promotion, and Consumer Information Order AGENCY: Agricultural Marketing Service, USDA... comments regarding proposed amendments to the Honey Research, Promotion, and Consumer Information...

  10. Expression of Arabidopsis LINEs from two promoters.

    PubMed

    Ohta, Yoshizu; Noma, Kenichi; Tsuchimoto, Suguru; Ohtsubo, Eiichi; Ohtsubo, Hisako

    2002-12-01

    Most Arabidopsis long interspersed elements (LINEs, called ATLNs) have two open reading frames, orf1 and orf2. In the 5' untranslated regions (UTRs) located upstream of orf1, the most proximal segments of tens of base pairs long are not homologous even in two ATLN members with almost identical sequences. In this study, we first show that RT-PCR products from ATLN39, a member of ATLN, can be detected only in total RNA from the hypomethylation mutant ddm1 or from suspension-cultured cells treated with a DNA methylation inhibitor 5-azacytidine, indicating that the expression of ATLN39 is negatively regulated by DNA methylation. We then show that orf1 fused in frame with the luciferase (luc) gene is expressed in suspension-cultured cells of A. thaliana when the 5' UTR is present in the region upstream of orf1. Analysis of deletion in the 5' UTR revealed that the 5' UTR has two promoters, designated here as P1 and P2. Analysis of transcripts by 5' RACE showed that their 5' ends were located at sites immediately upstream of the P1 region or at sites downstream of the P2 region. This observation and the fact that the P1 region contains no TATA sequence indicate that P1 is an internal promoter that initiates transcription from sites upstream of the promoter. A sequence containing GGCGA with a CpG methylatable site is conserved in the P1 regions in members closely related to ATLN39. The P2 region, however, contains the TATA sequence as well as another sequence with a CpG site. The TATA sequence is conserved in members closely related to ATLN39 but not in the other ATLN members, suggesting that P2 is the promoter uniquely present in the ATLN39-related members. Transcripts from promoter P1 can be used as templates to give new copies proficient in retroposition, but those from promoter P2 cannot because of the lack of the proximal half region of the 5' UTR sequence. Transcripts from promoter P2, as well as those from promoter P1 can, however, be used for the production of a

  11. Localization and regulation of bacteriophage Mu promoters

    SciTech Connect

    Stoddard, S.F. ); Howe, M.M. )

    1989-06-01

    Mu promoters active during the lytic cycle were located by isolating RNA at various times after induction of Mu prophages, radiolabeling it by capping in vitro, and hybridizing it to Mu DNA fragments on Southern blots. Signals were detected from four new promoters in addition to the previously characterized P{sub e} (early), P{sub cM} (repressor), and P{sub mom} (late) promoters. A major signal upstream of C was first observed at 12 min and intensified thereafter with RNA from cts and C amber but not replication-defective prophages; these characteristics indicate that this signal arises from a middle promoter, which we designate P{sub m}. With 20- and 40-min RNA, four additional major signals originated in the C-lys, F-G-I, N-P, and com-mom regions. These signals were missing with RNA from C amber and replication-defective prophages and therefore reflected the activity of late promoters, one of which we presume was P{sub mom}. Uninduced lysogens showed weak signals from five regions, one from the early regulatory region, three between genes B and lys, and one near the late genes K, L, and M. The first of these probably resulted from P{sub cM} activity; the others remain to be identified.

  12. 'Settings' based health promotion: a review.

    PubMed

    Whitelaw, S; Baxendale, A; Bryce, C; MacHardy, L; Young, I; Witney, E

    2001-12-01

    Over the past 10 years, 'settings' based health promotion has become a central feature of efforts to promote health that recognize the significance of context. Emerging in part from a perception of an over-reliance on individualistic methods, the approach was built on a profound belief in its value and deployed a range of novel theoretical resources, mainly from organizational sociology and psychology. This initial enthusiasm has been maintained within policy directives, in the published literature and, from our own experience, amongst health promotion practitioners. At the same time, with the maturing of the approach, has come a healthy element of critical review. Drawing upon the literature and based upon our experiences within the Health Education Board for Scotland, this paper seeks to bring together a range of perspectives, casting a critical yet constructive eye on current settings theory and practice. The paper first reviews the nature of settings based work, highlighting the varied bases and expectations that underpin it. Similarly, the many factors that influence the ability of health promoters to deliver such activities are considered. In relation to the construction and delivery of such activity, the paper suggests that there needs to be an explicit and detailed assessment of the nature of the setting, the skills of the health promoter and the associated expectations.

  13. Promoter Motifs in NCLDVs: An Evolutionary Perspective

    PubMed Central

    Oliveira, Graziele Pereira; Andrade, Ana Cláudia dos Santos Pereira; Rodrigues, Rodrigo Araújo Lima; Arantes, Thalita Souza; Boratto, Paulo Victor Miranda; Silva, Ludmila Karen dos Santos; Dornas, Fábio Pio; Trindade, Giliane de Souza; Drumond, Betânia Paiva; La Scola, Bernard; Kroon, Erna Geessien; Abrahão, Jônatas Santos

    2017-01-01

    For many years, gene expression in the three cellular domains has been studied in an attempt to discover sequences associated with the regulation of the transcription process. Some specific transcriptional features were described in viruses, although few studies have been devoted to understanding the evolutionary aspects related to the spread of promoter motifs through related viral families. The discovery of giant viruses and the proposition of the new viral order Megavirales that comprise a monophyletic group, named nucleo-cytoplasmic large DNA viruses (NCLDV), raised new questions in the field. Some putative promoter sequences have already been described for some NCLDV members, bringing new insights into the evolutionary history of these complex microorganisms. In this review, we summarize the main aspects of the transcription regulation process in the three domains of life, followed by a systematic description of what is currently known about promoter regions in several NCLDVs. We also discuss how the analysis of the promoter sequences could bring new ideas about the giant viruses’ evolution. Finally, considering a possible common ancestor for the NCLDV group, we discussed possible promoters’ evolutionary scenarios and propose the term “MEGA-box” to designate an ancestor promoter motif (‘TATATAAAATTGA’) that could be evolved gradually by nucleotides’ gain and loss and point mutations. PMID:28117683

  14. Roles of Rad51 paralogs for promoting homologous recombination in Leishmania infantum

    PubMed Central

    Genois, Marie-Michelle; Plourde, Marie; Éthier, Chantal; Roy, Gaétan; Poirier, Guy G.; Ouellette, Marc; Masson, Jean-Yves

    2015-01-01

    To achieve drug resistance Leishmania parasite alters gene copy number by using its repeated sequences widely distributed through the genome. Even though homologous recombination (HR) is ascribed to maintain genome stability, this eukaryote exploits this potent mechanism driven by the Rad51 recombinase to form beneficial extrachromosomal circular amplicons. Here, we provide insights on the formation of these circular amplicons by analyzing the functions of the Rad51 paralogs. We purified three Leishmania infantum Rad51 paralogs homologs (LiRad51-3, LiRad51-4 and LiRad51-6) all of which directly interact with LiRad51. LiRad51-3, LiRad51-4 and LiRad51-6 show differences in DNA binding and annealing capacities. Moreover, it is also noteworthy that LiRad51-3 and LiRad51-4 are able to stimulate Rad51-mediated D-loop formation. In addition, we succeed to inactivate the LiRad51-4 gene and report a decrease of circular amplicons in this mutant. The LiRad51-3 gene was found to be essential for cell viability. Thus, we propose that the LiRad51 paralogs play crucial functions in extrachromosomal circular DNA amplification to circumvent drug actions and preserve survival. PMID:25712090

  15. Promoting healthy people 2010 through small grants.

    PubMed

    Hartwig, Kari A; Dunville, Richard Louis; Kim, Michael H; Levy, Becca; Zaharek, Margot M; Njike, Valentine Y; Katz, David L

    2009-01-01

    The Department of Health and Human Services initiated a pilot "microgrant" or small grants program in 2001 to promote Healthy People 2010 (HP 2010) implemented by the Yale-Griffin Prevention Research Center. This article describes the 103 agencies funded under this initiative and 67 control group agencies. It evaluates the HP 2010 focus areas targeted and the effectiveness of promoting HP 2010 objectives through microgrants. Forty-four percent of the grant recipients and 79% of the control group agencies indicated low levels of familiarity with HP 2010 goals. Changes in knowledge of HP 2010 goals for the microgrant group increased significantly from 5.24 +/- 3.67 to 7.83 +/- 1.86 (p < .05). The results suggest that microgrants can be a useful mechanism to plant the seeds for developing community and organizational capacity to define local health priorities, practice and test new initiatives or expand existing programs and promote knowledge about HP 2010.

  16. Universal light-switchable gene promoter system

    DOEpatents

    Quail, Peter H.; Huq, Enamul; Tepperman, James; Sato, Sae

    2005-02-22

    An artificial promoter system that can be fused upstream of any desired gene enabling reversible induction or repression of the expression of the gene at will in any suitable host cell or organisms by light is described. The design of the system is such that a molecule of the plant photoreceptor phytochrome is targeted to the specific DNA binding site in the promoter by a protein domain that is fused to the phytochrome and that specifically recognizes this binding site. This bound phytochrome, upon activation by light, recruits a second fusion protein consisting of a protein that binds to phytochrome only upon light activation and a transcriptional activation domain that activates expression of the gene downstream of the promoter.

  17. [Health promotion in the Pankararu indigenous community].

    PubMed

    Oliveira, Jonas Welton Barros; Aquino, Jael Maria; Monteiro, Estela Maria Leite Meirelles

    2012-01-01

    The objective of the study was to know how the Pankararu indigenous perceive their health situation and identify actions they prioritize as necessary to promote health in their community. Qualitative research, in which the declarations collected were subjected to the technique of analyzing the Collective Subject Discourse. It was identified that in the indigenous perception, as the health status of their community, there is a lack of general assistance, and a lack of professionals to assist them meeting their needs. In relation to actions that the Indigenous prioritize as necessary to promote the health of their community, it was highlighted provision of health unit with trained professionals and access to health education actions. It was, thus, proposed an overhaul of the organizations and establishments of the subsystems in promoting indigenous health.

  18. Initiatives and resources to promote antimicrobial stewardship.

    PubMed

    Paño-Pardo, José Ramón; Campos, José; Natera Kindelán, Clara; Ramos, Antonio

    2013-09-01

    The development of an antimicrobial stewardship program (ASP) requires institutional support. However, obtaining sufficient institutional support is often a complex task that requires convincing the hospital's managers of the benefits of these programs. Additionally, in the design and implementation of an ASP, antimicrobial stewardship (AS) leaders need tools for diverse purposes, such as measuring antimicrobial consumption, education and training and designing protocols. In this review we provide useful information for AS promoters to facilitate the task of designing and implementing an ASP. First, we summarize information about various institutions that promote AS and include evidence that supports the need for and benefits of these programs. Then, several campaigns promoting AS are described. Finally, online resources for professionals dealing with AS are briefly summarized.

  19. Efficient promotion strategies in hierarchical organizations

    NASA Astrophysics Data System (ADS)

    Pluchino, Alessandro; Rapisarda, Andrea; Garofalo, Cesare

    2011-10-01

    The Peter principle has recently been investigated by means of an agent-based simulation, and its validity has been numerically corroborated. It has been confirmed that, within certain conditions, it can really influence in a negative way the efficiency of a pyramidal organization adopting meritocratic promotions. It was also found that, in order to bypass these effects, alternative promotion strategies should be adopted, as for example a random selection choice. In this paper, within the same line of research, we study promotion strategies in a more realistic hierarchical and modular organization, and we show the robustness of our previous results, extending their validity to a more general context. We also discuss why the adoption of these strategies could be useful for real organizations.

  20. Promotion of PDT efficacy by bile acids

    NASA Astrophysics Data System (ADS)

    Castelli, Michelle; Reiners, John, Jr.; Kessel, David

    2003-06-01

    We had previously described the use of relatively hydrophobic bile acids, notably UDCA (ursodeoxycholate) for the promotion of the apoptotic response to photodynamic therapy. Further study revealed that this effect occurred only when the target for photodamage was the anti-apoptotic protein Bcl-2. The efficacy of lysosomal photodamage, leading to a cleavage of the protein Bid, was not influenced by UDCA. Moreover, the apoptotic cell death resulting from treatment of cells with the non-peptidic Bcl-2 inhibitor HA 14-1 was also promoted by UDCA. These results are consistent with the proposal that the pro-apoptotic effects of UDCA are directed against Bcl-2, promoting inactivation by HA 14-1 or photodamage.