Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

PubMed

Marine, Rachel; McCarren, Coleen; Vorrasane, Vansay; Nasko, Dan; Crowgey, Erin; Polson, Shawn W; Wommack, K Eric

2014-01-30

Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested. Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes. MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.

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

PubMed Central

Mellerup, Anders; Ståhl, Marie

2015-01-01

The aim of this article was to define the sampling level and method combination that captures antibiotic resistance at pig herd level utilizing qPCR antibiotic resistance gene quantification and culture-based quantification of antibiotic resistant coliform indicator bacteria. Fourteen qPCR assays for commonly detected antibiotic resistance genes were developed, and used to quantify antibiotic resistance genes in total DNA from swine fecal samples that were obtained using different sampling and pooling methods. In parallel, the number of antibiotic resistant coliform indicator bacteria was determined in the same swine fecal samples. The results showed that the qPCR assays were capable of detecting differences in antibiotic resistance levels in individual animals that the coliform bacteria colony forming units (CFU) could not. Also, the qPCR assays more accurately quantified antibiotic resistance genes when comparing individual sampling and pooling methods. qPCR on pooled samples was found to be a good representative for the general resistance level in a pig herd compared to the coliform CFU counts. It had significantly reduced relative standard deviations compared to coliform CFU counts in the same samples, and therefore differences in antibiotic resistance levels between samples were more readily detected. To our knowledge, this is the first study to describe sampling and pooling methods for qPCR quantification of antibiotic resistance genes in total DNA extracted from swine feces. PMID:26114765

Physical mapping of complex genomes

DOEpatents

Evans, G.A.

1993-06-15

A method for the simultaneous identification of overlapping cosmid clones among multiple cosmid clones and the use of the method for mapping complex genomes are provided. A library of cosmid clones that contains the DNA to be mapped is constructed and arranged in a manner such that individual clones can be identified and replicas of the arranged clones prepared. In preferred embodiments, the clones are arranged in a two dimensional matrix. In such embodiments, the cosmid clones in a row are pooled, mixed probes complementary to the ends of the DNA inserts in the pooled clones are synthesized, hybridized to a first replica of the library. Hybridizing clones, which include the pooled row, are identified. A second portion of clones is prepared by pooling cosmid clones that correspond to a column in the matrix. The second pool thereby includes one clone from the first portion pooled clones. This common clone is located on the replica at the intersection of the column and row. Mixed probes complementary to the ends of the DNA inserts in the second pooled portion of clones are prepared and hybridized to a second replica of the library. The hybridization pattern on the first and second replicas of the library are compared and cross-hybridizing clones, other than the clones in the pooled column and row, that hybridize to identical clones in the first and second replicas are identified. These clones necessarily include DNA inserts that overlap with the DNA insert in the common clone located at the intersection of the pooled row and pooled column. The DNA in the entire library may be mapped by pooling the clones in each of the rows and columns of the matrix, preparing mixed end-specific probes and hybridizing the probes from each row or column to a replica of the library. Since all clones in the library are located at the intersection of a column and a row, the overlapping clones for all clones in the library may be identified and a physical map constructed.

Quantitative analysis of low-density SNP data for parentage assignment and estimation of family contributions to pooled samples.

PubMed

Henshall, John M; Dierens, Leanne; Sellars, Melony J

2014-09-02

While much attention has focused on the development of high-density single nucleotide polymorphism (SNP) assays, the costs of developing and running low-density assays have fallen dramatically. This makes it feasible to develop and apply SNP assays for agricultural species beyond the major livestock species. Although low-cost low-density assays may not have the accuracy of the high-density assays widely used in human and livestock species, we show that when combined with statistical analysis approaches that use quantitative instead of discrete genotypes, their utility may be improved. The data used in this study are from a 63-SNP marker Sequenom® iPLEX Platinum panel for the Black Tiger shrimp, for which high-density SNP assays are not currently available. For quantitative genotypes that could be estimated, in 5% of cases the most likely genotype for an individual at a SNP had a probability of less than 0.99. Matrix formulations of maximum likelihood equations for parentage assignment were developed for the quantitative genotypes and also for discrete genotypes perturbed by an assumed error term. Assignment rates that were based on maximum likelihood with quantitative genotypes were similar to those based on maximum likelihood with perturbed genotypes but, for more than 50% of cases, the two methods resulted in individuals being assigned to different families. Treating genotypes as quantitative values allows the same analysis framework to be used for pooled samples of DNA from multiple individuals. Resulting correlations between allele frequency estimates from pooled DNA and individual samples were consistently greater than 0.90, and as high as 0.97 for some pools. Estimates of family contributions to the pools based on quantitative genotypes in pooled DNA had a correlation of 0.85 with estimates of contributions from DNA-derived pedigree. Even with low numbers of SNPs of variable quality, parentage testing and family assignment from pooled samples are sufficiently accurate to provide useful information for a breeding program. Treating genotypes as quantitative values is an alternative to perturbing genotypes using an assumed error distribution, but can produce very different results. An understanding of the distribution of the error is required for SNP genotyping platforms.

Pooled Genome-Wide Analysis to Identify Novel Risk Loci for Pediatric Allergic Asthma

PubMed Central

Ricci, Giampaolo; Astolfi, Annalisa; Remondini, Daniel; Cipriani, Francesca; Formica, Serena; Dondi, Arianna; Pession, Andrea

2011-01-01

Background Genome-wide association studies of pooled DNA samples were shown to be a valuable tool to identify candidate SNPs associated to a phenotype. No such study was up to now applied to childhood allergic asthma, even if the very high complexity of asthma genetics is an appropriate field to explore the potential of pooled GWAS approach. Methodology/Principal Findings We performed a pooled GWAS and individual genotyping in 269 children with allergic respiratory diseases comparing allergic children with and without asthma. We used a modular approach to identify the most significant loci associated with asthma by combining silhouette statistics and physical distance method with cluster-adapted thresholding. We found 97% concordance between pooled GWAS and individual genotyping, with 36 out of 37 top-scoring SNPs significant at individual genotyping level. The most significant SNP is located inside the coding sequence of C5, an already identified asthma susceptibility gene, while the other loci regulate functions that are relevant to bronchial physiopathology, as immune- or inflammation-mediated mechanisms and airway smooth muscle contraction. Integration with gene expression data showed that almost half of the putative susceptibility genes are differentially expressed in experimental asthma mouse models. Conclusion/Significance Combined silhouette statistics and cluster-adapted physical distance threshold analysis of pooled GWAS data is an efficient method to identify candidate SNP associated to asthma development in an allergic pediatric population. PMID:21359210

A High-throughput AFLP-based Method for Constructing Integrated Genetic and Physical Maps: Progress Toward a Sorghum Genome Map

PubMed Central

Klein, Patricia E.; Klein, Robert R.; Cartinhour, Samuel W.; Ulanch, Paul E.; Dong, Jianmin; Obert, Jacque A.; Morishige, Daryl T.; Schlueter, Shannon D.; Childs, Kevin L.; Ale, Melissa; Mullet, John E.

2000-01-01

Sorghum is an important target for plant genomic mapping because of its adaptation to harsh environments, diverse germplasm collection, and value for comparing the genomes of grass species such as corn and rice. The construction of an integrated genetic and physical map of the sorghum genome (750 Mbp) is a primary goal of our sorghum genome project. To help accomplish this task, we have developed a new high-throughput PCR-based method for building BAC contigs and locating BAC clones on the sorghum genetic map. This task involved pooling 24,576 sorghum BAC clones (∼4× genome equivalents) in six different matrices to create 184 pools of BAC DNA. DNA fragments from each pool were amplified using amplified fragment length polymorphism (AFLP) technology, resolved on a LI-COR dual-dye DNA sequencing system, and analyzed using Bionumerics software. On average, each set of AFLP primers amplified 28 single-copy DNA markers that were useful for identifying overlapping BAC clones. Data from 32 different AFLP primer combinations identified ∼2400 BACs and ordered ∼700 BAC contigs. Analysis of a sorghum RIL mapping population using the same primer pairs located ∼200 of the BAC contigs on the sorghum genetic map. Restriction endonuclease fingerprinting of the entire collection of sorghum BAC clones was applied to test and extend the contigs constructed using this PCR-based methodology. Analysis of the fingerprint data allowed for the identification of 3366 contigs each containing an average of 5 BACs. BACs in ∼65% of the contigs aligned by AFLP analysis had sufficient overlap to be confirmed by DNA fingerprint analysis. In addition, 30% of the overlapping BACs aligned by AFLP analysis provided information for merging contigs and singletons that could not be joined using fingerprint data alone. Thus, the combination of fingerprinting and AFLP-based contig assembly and mapping provides a reliable, high-throughput method for building an integrated genetic and physical map of the sorghum genome. [The sequence data described in this paper have been submitted to the GenBank data library under accession no. AF218263.] PMID:10854411

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

NASA Astrophysics Data System (ADS)

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

1996-04-01

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

An accurate algorithm for the detection of DNA fragments from dilution pool sequencing experiments.

PubMed

Bansal, Vikas

2018-01-01

The short read lengths of current high-throughput sequencing technologies limit the ability to recover long-range haplotype information. Dilution pool methods for preparing DNA sequencing libraries from high molecular weight DNA fragments enable the recovery of long DNA fragments from short sequence reads. These approaches require computational methods for identifying the DNA fragments using aligned sequence reads and assembling the fragments into long haplotypes. Although a number of computational methods have been developed for haplotype assembly, the problem of identifying DNA fragments from dilution pool sequence data has not received much attention. We formulate the problem of detecting DNA fragments from dilution pool sequencing experiments as a genome segmentation problem and develop an algorithm that uses dynamic programming to optimize a likelihood function derived from a generative model for the sequence reads. This algorithm uses an iterative approach to automatically infer the mean background read depth and the number of fragments in each pool. Using simulated data, we demonstrate that our method, FragmentCut, has 25-30% greater sensitivity compared with an HMM based method for fragment detection and can also detect overlapping fragments. On a whole-genome human fosmid pool dataset, the haplotypes assembled using the fragments identified by FragmentCut had greater N50 length, 16.2% lower switch error rate and 35.8% lower mismatch error rate compared with two existing methods. We further demonstrate the greater accuracy of our method using two additional dilution pool datasets. FragmentCut is available from https://bansal-lab.github.io/software/FragmentCut. vibansal@ucsd.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

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

PubMed Central

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

2015-01-01

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

Time- and Cost-Efficient Identification of T-DNA Insertion Sites through Targeted Genomic Sequencing

PubMed Central

Lepage, Étienne; Zampini, Éric; Boyle, Brian; Brisson, Normand

2013-01-01

Forward genetic screens enable the unbiased identification of genes involved in biological processes. In Arabidopsis, several mutant collections are publicly available, which greatly facilitates such practice. Most of these collections were generated by agrotransformation of a T-DNA at random sites in the plant genome. However, precise mapping of T-DNA insertion sites in mutants isolated from such screens is a laborious and time-consuming task. Here we report a simple, low-cost and time efficient approach to precisely map T-DNA insertions simultaneously in many different mutants. By combining sequence capture, next-generation sequencing and 2D-PCR pooling, we developed a new method that allowed the rapid localization of T-DNA insertion sites in 55 out of 64 mutant plants isolated in a screen for gyrase inhibition hypersensitivity. PMID:23951038

Phylogeographic Differentiation of Mitochondrial DNA in Han Chinese

PubMed Central

Yao, Yong-Gang; Kong, Qing-Peng; Bandelt, Hans-Jürgen; Kivisild, Toomas; Zhang, Ya-Ping

2002-01-01

To characterize the mitochondrial DNA (mtDNA) variation in Han Chinese from several provinces of China, we have sequenced the two hypervariable segments of the control region and the segment spanning nucleotide positions 10171–10659 of the coding region, and we have identified a number of specific coding-region mutations by direct sequencing or restriction-fragment–length–polymorphism tests. This allows us to define new haplogroups (clades of the mtDNA phylogeny) and to dissect the Han mtDNA pool on a phylogenetic basis, which is a prerequisite for any fine-grained phylogeographic analysis, the interpretation of ancient mtDNA, or future complete mtDNA sequencing efforts. Some of the haplogroups under study differ considerably in frequencies across different provinces. The southernmost provinces show more pronounced contrasts in their regional Han mtDNA pools than the central and northern provinces. These and other features of the geographical distribution of the mtDNA haplogroups observed in the Han Chinese make an initial Paleolithic colonization from south to north plausible but would suggest subsequent migration events in China that mainly proceeded from north to south and east to west. Lumping together all regional Han mtDNA pools into one fictive general mtDNA pool or choosing one or two regional Han populations to represent all Han Chinese is inappropriate for prehistoric considerations as well as for forensic purposes or medical disease studies. PMID:11836649

Quantitative cytochemistry of nuclear and cytoplasmic proteins using the Naphthol Yellow S and dinitrofluorobenzene staining methods.

PubMed

Tas, J; James, J

1981-09-01

The 'total protein staining' of biological specimens with the electrostatically binding Naphthol Yellow S or the covalently binding dinitrofluorobenzene must be interpreted as methods which yield data on the specific amino acid pool of the proteins concerned. Both dyes bind to certain free amino-acid side-chains, giving different dye--protein ratios for various proteins. In the presence of DNA, dinitrofluorobenzene stains all proteins present in cell nuclei, whereas Naphthol Yellow S only stains the majority of the non-histone proteins. When protein staining methods are combined with the Feulgen--Pararosanile (SO2) procedure for DNA, decreased Feulgen--DNA contents were measured in dinitrofluorobenzene-stained isolated nuclei and lymphocytes.

Physical mapping of complex genomes

DOEpatents

Evans, Glen A.

1993-01-01

Method for simultaneous identification of overlapping cosmid clones among multiple cosmid clones and the use of the method for mapping complex genomes are provided. A library of cosmid clones that contains the DNA to be mapped is constructed and arranged in a manner such that individual clones can be identified and replicas of the arranged clones prepared. In preferred embodiments, the clones are arranged in a two dimensional matrix. In such embodiments, the cosmid clones in a row are pooled, mixed probes complementary to the ends of the DNA inserts int he pooled clones are synthesized, hybridized to a first replica of the library. Hybridizing clones, which include the pooled row, are identified. A second portion of clones is prepared by pooling cosmid clones that correspond to a column in the matrix. The second pool thereby includes one clone from the first portion pooled clones. This common clone is located on the replica at the intersection of the column and row. Mixed probes complementary to the ends of the DNA inserts in the second pooled portion of clones are prepared and hybridized to a second replica of the library. The hybridization pattern on the first and second replicas of the library are compared and cross-hybridizing clones, other than the clones in the pooled column and row, that hybridize to identical clones in the first and second replicas are identified. These clones necessarily include DNA inserts that overlap with the DNA insert int he common clone located at the intersection of the pooled row and pooled column. The DNA in the entire library may be mapped by pooling the clones in each of the rows and columns of the matrix, preparing mixed end-specific probes and hybridizing the probes from each row or column to a replica of the library. Since all clones in the library are located at the intersection of a column and a row, the overlapping clones for all clones in the library may be identified and a physical map constructed. In other preferred embodiments, the cosmid clones are arranged in a three dimensional matrix, pooled and compared in threes according to intersecting planes of the three dimensional matrix. Arrangements corresponding to geometries of higher dimensions may also be prepared and used to simultaneously identify overlapping clones in highly complex libraries with relatively few hybridization reactions.

DNA precursor pool: a significant target for N-methyl-N-nitrosourea in C3H/10T1/2 clone 8 cells.

PubMed Central

Topal, M D; Baker, M S

1982-01-01

Synchronized C3H/10T1/2 clone 8 cells were treated in vitro with a nontoxic dose of N-methyl-N-nitrosourea during their S phase. Chromatographic isolation of the deoxyribonucleotide DNA precursor pool and measurement of the precursor content per cell showed that a nucleic acid residue in the precursor pool is 190-13,000 times more susceptible to methylation than a residue in the DNA duplex, depending on the site of methylation. This conclusion comes from measurements indicating that, for example, the N-1 position of adenine in dATP is 6.3 times more methylated than the same position in the DNA, even though the adenine content of the pool is only a fraction (0.0005) of the adenine content of the DNA helix. The comparative susceptibility between pool and DNA was found to vary with the site of methylation in the order the N-1 position of adenine greater than phosphate greater than the N-3 position of adenine greater than the O6 position of guanine greater than the N-7 position of guanine. The significance of these results for chemical mutagenesis and carcinogenesis is discussed. PMID:6954535

Investigation of Bartonella infection in ixodid ticks from California.

PubMed

Chang, Chao-chin; Hayashidani, Hideki; Pusterla, Nicola; Kasten, Rickie W; Madigan, John E; Chomel, Bruno B

2002-07-01

A total of 1253 ixodid ticks (254 tick pools) collected between the end of 1995 and the spring of 1997 from six California counties (El Dorado, Los Angeles, Orange, Santa Cruz, Shasta and Sonoma) were examined for the presence of Bartonella DNA by PCR of the citrate synthase gene. Of 1,119 adult Ixodes pacificus ticks tested, 26 (11.6%) of 224 pools, each containing five ticks, were positive (minimum percentage of ticks harboring detectable Bartonella DNA, 2.3%). Bartonella PCR-positive ticks were identified in five counties but none of the ticks from Los Angeles County was positive. Among 47 nymphal I. pacificus ticks collected in Sonoma County, one (10%) positive pool out of 10 pools was identified (minimum percentage of ticks harboring detectable Bartonella DNA, 2.1%). Among the 54 Dermacentor occidentalis grouped in 12 pools from Orange County, one pool (8.3%) was PCR positive for Bartonella and similarly one pool (14.3%) was positive among the 30 Dermacentor variabilis ticks grouped in seven pools. None of the three D. occidentalis from El Dorado County were positive. None of the nine tick pools positive for Ehrlichia phagocytophila were positive for Bartonella. Following our previous findings of Bartonella PCR-positive adult I. pacificus ticks in central coastal California, this is the first preliminary report of the presence of Bartonella DNA in I. pacificus nymphs and in Dermacentor sp. ticks. Distribution of Bartonella among ixodid ticks appears widespread in California.

Rapid gene identification in sugar beet using deep sequencing of DNA from phenotypic pools selected from breeding panels.

PubMed

Ries, David; Holtgräwe, Daniela; Viehöver, Prisca; Weisshaar, Bernd

2016-03-15

The combination of bulk segregant analysis (BSA) and next generation sequencing (NGS), also known as mapping by sequencing (MBS), has been shown to significantly accelerate the identification of causal mutations for species with a reference genome sequence. The usual approach is to cross homozygous parents that differ for the monogenic trait to address, to perform deep sequencing of DNA from F2 plants pooled according to their phenotype, and subsequently to analyze the allele frequency distribution based on a marker table for the parents studied. The method has been successfully applied for EMS induced mutations as well as natural variation. Here, we show that pooling genetically diverse breeding lines according to a contrasting phenotype also allows high resolution mapping of the causal gene in a crop species. The test case was the monogenic locus causing red vs. green hypocotyl color in Beta vulgaris (R locus). We determined the allele frequencies of polymorphic sequences using sequence data from two diverging phenotypic pools of 180 B. vulgaris accessions each. A single interval of about 31 kbp among the nine chromosomes was identified which indeed contained the causative mutation. By applying a variation of the mapping by sequencing approach, we demonstrated that phenotype-based pooling of diverse accessions from breeding panels and subsequent direct determination of the allele frequency distribution can be successfully applied for gene identification in a crop species. Our approach made it possible to identify a small interval around the causative gene. Sequencing of parents or individual lines was not necessary. Whenever the appropriate plant material is available, the approach described saves time compared to the generation of an F2 population. In addition, we provide clues for planning similar experiments with regard to pool size and the sequencing depth required.

H3-THYMIDINE DERIVATIVE POOLS IN RELATION TO MACRONUCLEAR DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS

PubMed Central

Stone, G. E.; Miller, O. L.; Prescott, D. M.

1965-01-01

The formation of a soluble H3-thymidine derivative pool has been examined in Tetrahymena pyriformis as a function of macronuclear DNA synthesis during the cell life cycle. An autoradiographic technique which allows the detection of water-soluble materials within a cell has shown that these cells do not take up and retain exogenous H3-thymidine during G1 or G2. Uptake of H3-thymidine is restricted to the S period of the cell cycle. Additional autoradiographic experiments show, however, that a soluble pool of H3-thymidine derivatives persists from the end of one DNA synthesis period to the beginning of the next synthesis period in the subsequent cell cycle. Since this persisting pool cannot be labeled with H3-thymidine, the pool does not turn over during non-S periods. PMID:19866660

Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

PubMed

Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

2017-01-01

Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

Pneumocystis jirovecii multilocus genotyping in pooled DNA samples: a new approach for clinical and epidemiological studies.

PubMed

Esteves, F; Gaspar, J; de Sousa, B; Antunes, F; Mansinho, K; Matos, O

2012-06-01

Specific single-nucleotide polymorphisms (SNPs) are recognized as important DNA sequence variations influencing the pathogenesis of Pneumocystis jirovecii and the clinical outcome of Pneumocystis pneumonia, which is a major worldwide cause of illness among immunocompromised patients. Genotyping platforms for pooled DNA samples are promising methodologies for genetic characterization of infectious organisms. We have developed a new typing strategy for P. jirovecii, which consisted of DNA pools prepared according to clinical data (HIV diagnosis, microscopic and molecular detection of P. jirovecii, parasite burden, clinical diagnosis and follow-up of infection) from individual samples using quantitative real-time PCR followed by multiplex-PCR/single base extension (MPCR/SBE). The frequencies of multiple P. jirovecii SNPs (DHFR312, mt85, SOD215 and SOD110) encoded at three distinct loci, the dihydrofolate reductase (DHFR), the mitochondrial large-subunit rRNA (mtLSU rRNA) and the superoxide dismutase (SOD) loci, were estimated in seven DNA pooled samples, representing a total of 100 individual samples. The studied SNPs were confirmed to be associated with distinct clinical parameters of infection such as parasite burden and follow-up. The MPCR/SBE-DNA pooling methodology, described in the present study, was demonstrated to be a useful high-throughput procedure for large-scale P. jirovecii SNPs screening and a powerful tool for evaluation of clinically relevant SNPs potentially related to parasite burden, clinical diagnosis and follow-up of P. jirovecii infection. In further studies, the candidate SNPs mt85, SOD215 and SOD110 may be used as molecular markers in association with MPCR/SBE-DNA pooling to generate useful information for understanding the patterns and causes of Pneumocystis pneumonia. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

Blow flies as urban wildlife sensors.

PubMed

Hoffmann, Constanze; Merkel, Kevin; Sachse, Andreas; Rodríguez, Pablo; Leendertz, Fabian H; Calvignac-Spencer, Sébastien

2018-05-01

Wildlife detection in urban areas is very challenging. Conventional monitoring techniques such as direct observation are faced with the limitation that urban wildlife is extremely elusive. It was recently shown that invertebrate-derived DNA (iDNA) can be used to assess wildlife diversity in tropical rainforests. Flies, which are ubiquitous and very abundant in most cities, may also be used to detect wildlife in urban areas. In urban ecosystems, however, overwhelming quantities of domestic mammal DNA could completely mask the presence of wild mammal DNA. To test whether urban wild mammals can be detected using fly iDNA, we performed DNA metabarcoding of pools of flies captured in Berlin, Germany, using three combinations of blocking primers. Our results show that domestic animal sequences are, as expected, very dominant in urban environments. Nevertheless, wild mammal sequences can often be retrieved, although they usually only represent a minor fraction of the sequence reads. Fly iDNA metabarcoding is therefore a viable approach for quick scans of urban wildlife diversity. Interestingly, our study also shows that blocking primers can interact with each other in ways that affect the outcome of metabarcoding. We conclude that the use of complex combinations of blocking primers, although potentially powerful, should be carefully planned when designing experiments. © 2018 John Wiley & Sons Ltd.

Polymorphism in the PER3 promoter associates with diurnal preference and delayed sleep phase disorder.

PubMed

Archer, Simon N; Carpen, Jayshan D; Gibson, Mark; Lim, Gim Hui; Johnston, Jonathan D; Skene, Debra J; von Schantz, Malcolm

2010-05-01

To screen the PER3 promoter for polymorphisms and investigate the phenotypic associations of these polymorphisms with diurnal preference, delayed sleep phase disorder/syndrome (DSPD/DSPS), and their effects on reporter gene expression. Interspecific comparison was used to define the approximate extent of the PER3 promoter as the region between the transcriptional start site and nucleotide position -874. This region was screened in DNA pools using PCR and direct sequencing, which was also used to screen DNA from individual participants. The different promoter alleles were cloned into a luciferase expression vector and a deletion library created. Promoter activation was measured by chemiluminescence. N/A. DNA samples were obtained from volunteers with defined diurnal preference (3 x 80, selected from a pool of 1,590), and DSPD patients (n=23). N/A. We verified three single nucleotide polymorphisms (G -320T, C -319A, G -294A), and found a novel variable number tandem repeat (VNTR) polymorphism (-318 1/2 VNTR). The -320T and -319A alleles occurred more frequently in DSPD compared to morning (P = 0.042 for each) or evening types (P = 0.006 and 0.033). The allele combination TA2G was more prevalent in DSPD compared to morning (P 0.033) or evening types (P = 0.002). Luciferase expression driven by the TA2G combination was greater than for the more common GC2A (P < 0.05) and the rarer TA1G (P < 0.001) combinations. Deletion reporter constructs identified two enhancer regions (-703 to -605, and -283 to -80). Polymorphisms in the PER3 promoter could affect its expression, leading to potential differences in the observed functions of PER3.

Rational development of two flowthrough purification strategies for adenovirus type 5 and retro virus-like particles.

PubMed

Nestola, Piergiuseppe; Peixoto, Cristina; Villain, Louis; Alves, Paula M; Carrondo, Manuel J T; Mota, José P B

2015-12-24

We report on the rational design and implementation of flowthrough (FT) platforms for purification of virus vectors (VVs) and virus-like particles (VLPs), combining anion-exchange polyallylamine membranes (Sartobind STIC) and core-shell octylamine resins (CaptoCore 700). In one configuration, the VV bulk is concentrated and conditioned with appropriate buffer in a ultra/diafiltration (UF/DF) unit prior to injection into the STIC chromatography membrane. The FT pool and an intermediate cut of the elution pool of the STIC membrane are admixed and directed to a second UF/DF. Finally, the retentate is injected into a CC700 packed bed adsorber where the purified VVs are collected in the FT pool, whereas the residual amount of DNA and host cell protein (HCP) are discarded in the eluate. The experimental recovery achieved with this downstream processing (DSP) platform is close to 100%, the DNA clearance is roughly a 4-log reduction, and the HCP level is reduced by 5 logs. The platform developed for VLP purification is simpler than the previous one, as the STIC membrane adsorber and CC700 bed are connected in series with no UF/DF unit in between. Experimentally, the FT scheme for VLP purification gave a recovery yield of 45% in the chromatography train; the experimental log reduction of DNA and HCP were 2.0 and 3.5, respectively. These results are in line with other purification strategies in the specific field of enveloped VLPs. Both DSP platforms were successfully developed from an initial design space of the binding of the major contaminant (DNA) to the two ligands, determined by surface plasmon resonance, which was subsequently scaled up and confirmed experimentally. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular Detection of Leishmania DNA in Wild-Caught Phlebotomine Sand Flies (Diptera: Psychodidae) From a Cave in the State of Minas Gerais, Brazil.

PubMed

Carvalho, G M L; Brazil, R P; Rêgo, F D; Ramos, M C N F; Zenóbio, A P L A; Andrade Filho, J D

2017-01-01

Leishmania spp. are distributed throughout the world, and different species are associated with varying degrees of disease severity. In Brazil, Leishmania transmission involves several species of phlebotomine sand flies that are closely associated with different parasites and reservoirs, and thereby giving rise to different transmission cycles. Infection occurs during the bloodmeals of sand flies obtained from a variety of wild and domestic animals, and sometimes from humans. The present study focused on detection of Leishmania DNA in phlebotomine sand flies from a cave in the state of Minas Gerais. Detection of Leishmania in female sand flies was performed with ITS1 PCR-RFLP (internal transcribed spacer 1) using HaeIII enzyme and genetic sequencing for SSUrRNA target. The survey of Leishmania DNA was carried out on 232 pools and the parasite DNA was detected in four: one pool of Lutzomyia cavernicola (Costa Lima, 1932), infected with Le. infantum (ITS1 PCR-RFLP), two pools of Evandromyia sallesi (Galvão & Coutinho, 1939), both infected with Leishmania braziliensis complex (SSUrRNA genetic sequencing analysis), and one pool of Sciopemyia sordellii (Shannon & Del Ponte, 1927), infected with subgenus Leishmania (SSUrRNA genetic sequencing analysis). The present study identified the species for Leishmania DNA detected in four pools of sand flies, all of which were captured inside the cave. These results represent the first molecular detection of Lu cavernicola with Le infantum DNA, Sc sordellii with subgenus Leishmania DNA, and Ev sallesi with Leishmania braziliensis complex DNA. The infection rate in females captured for this study was 0.17%. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Novel quantitative real-time LCR for the sensitive detection of SNP frequencies in pooled DNA: method development, evaluation and application.

PubMed

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-19

Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food.

Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

PubMed Central

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-01

Background Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. Methods The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. Conclusions The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. Significance The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food. PMID:21283808

DNA pooling strategies for categorical (ordinal) traits

USDA-ARS?s Scientific Manuscript database

Despite reduced genotyping costs in recent years, obtaining genotypes for all individuals in a population may still not be feasible when sample size is large. DNA pooling provides a useful alternative to determining genotype effects. Clustering algorithms allow for grouping of individuals (observati...

Highly multiplexed targeted DNA sequencing from single nuclei.

PubMed

Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

2016-02-01

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

Effort versus Reward: Preparing Samples for Fungal Community Characterization in High-Throughput Sequencing Surveys of Soils

PubMed Central

Song, Zewei; Schlatter, Dan; Kennedy, Peter; Kinkel, Linda L.; Kistler, H. Corby; Nguyen, Nhu; Bates, Scott T.

2015-01-01

Next generation fungal amplicon sequencing is being used with increasing frequency to study fungal diversity in various ecosystems; however, the influence of sample preparation on the characterization of fungal community is poorly understood. We investigated the effects of four procedural modifications to library preparation for high-throughput sequencing (HTS). The following treatments were considered: 1) the amount of soil used in DNA extraction, 2) the inclusion of additional steps (freeze/thaw cycles, sonication, or hot water bath incubation) in the extraction procedure, 3) the amount of DNA template used in PCR, and 4) the effect of sample pooling, either physically or computationally. Soils from two different ecosystems in Minnesota, USA, one prairie and one forest site, were used to assess the generality of our results. The first three treatments did not significantly influence observed fungal OTU richness or community structure at either site. Physical pooling captured more OTU richness compared to individual samples, but total OTU richness at each site was highest when individual samples were computationally combined. We conclude that standard extraction kit protocols are well optimized for fungal HTS surveys, but because sample pooling can significantly influence OTU richness estimates, it is important to carefully consider the study aims when planning sampling procedures. PMID:25974078

Investigation of rare and low-frequency variants using high-throughput sequencing with pooled DNA samples

PubMed Central

Wang, Jingwen; Skoog, Tiina; Einarsdottir, Elisabet; Kaartokallio, Tea; Laivuori, Hannele; Grauers, Anna; Gerdhem, Paul; Hytönen, Marjo; Lohi, Hannes; Kere, Juha; Jiao, Hong

2016-01-01

High-throughput sequencing using pooled DNA samples can facilitate genome-wide studies on rare and low-frequency variants in a large population. Some major questions concerning the pooling sequencing strategy are whether rare and low-frequency variants can be detected reliably, and whether estimated minor allele frequencies (MAFs) can represent the actual values obtained from individually genotyped samples. In this study, we evaluated MAF estimates using three variant detection tools with two sets of pooled whole exome sequencing (WES) and one set of pooled whole genome sequencing (WGS) data. Both GATK and Freebayes displayed high sensitivity, specificity and accuracy when detecting rare or low-frequency variants. For the WGS study, 56% of the low-frequency variants in Illumina array have identical MAFs and 26% have one allele difference between sequencing and individual genotyping data. The MAF estimates from WGS correlated well (r = 0.94) with those from Illumina arrays. The MAFs from the pooled WES data also showed high concordance (r = 0.88) with those from the individual genotyping data. In conclusion, the MAFs estimated from pooled DNA sequencing data reflect the MAFs in individually genotyped samples well. The pooling strategy can thus be a rapid and cost-effective approach for the initial screening in large-scale association studies. PMID:27633116

Genomewide association study of liver abscess in beef cattle.

PubMed

Keele, J W; Kuehn, L A; McDaneld, T G; Tait, R G; Jones, S A; Keel, B N; Snelling, W M

2016-02-01

Fourteen percent of U.S. cattle slaughtered in 2011 had liver abscesses, resulting in reduced carcass weight, quality, and value. Liver abscesses can result from a common bacterial cause, , which inhabits rumen lesions caused by acidosis and subsequently escapes into the blood stream, is filtered by the liver, and causes abscesses in the liver. Our aim was to identify SNP associated with liver abscesses in beef cattle. We used lung samples as a DNA source because they have low economic value, they have abundant DNA, and we had unrestricted access to sample them. We collected 2,304 lung samples from a beef processing plant: 1,152 from animals with liver abscess and 1,152 from animals without liver abscess. Lung tissue from pairs of animals, 1 with abscesses and another without, were collected from near one another on the viscera table to ensure that pairs of phenotypically extreme animals came from the same lot. Within each phenotype (abscess or no abscess), cattle were pooled by slaughter sequence into 12 pools of 96 cattle for each phenotype for a total of 24 pools. The pools were constructed by equal volume of frozen lung tissue from each animal. The DNA needed to allelotype each pool was then extracted from pooled lung tissue and the BovineHD Bead Array (777,962 SNP) was run on all 24 pools. Total intensity (TI), an indicator of copy number variants, was the sum of intensities from red and green dyes. Pooling allele frequency (PAF) was red dye intensity divided TI. Total intensity and PAF were weighted by the inverse of their respective genomic covariance matrices computed over all SNP across the genome. A false discovery rate ≤ 5% was achieved for 15 SNP for PAF and 20 SNP for TI. Genes within 50 kbp from significant SNP were in diverse pathways including maintenance of pH homeostasis in the gastrointestinal tract, maintain immune defenses in the liver, migration of leukocytes from the blood into infected tissues, transport of glutamine into the kidney in response to acidosis to facilitate production of bicarbonate to increase pH, aggregate platelets to liver injury to facilitate liver repair, and facilitate axon guidance. Evidence from the 35 detected SNP associations combined with evidence of polygenic variation indicate that there is adequate genetic variation in incidence rate of liver abscesses, which could be exploited to select sires for reduced susceptibility to subacute acidosis and associated liver abscess.

A new approach to epigenome-wide discovery of non-invasive methylation biomarkers for colorectal cancer screening in circulating cell-free DNA using pooled samples.

PubMed

Gallardo-Gómez, María; Moran, Sebastian; Páez de la Cadena, María; Martínez-Zorzano, Vicenta Soledad; Rodríguez-Berrocal, Francisco Javier; Rodríguez-Girondo, Mar; Esteller, Manel; Cubiella, Joaquín; Bujanda, Luis; Castells, Antoni; Balaguer, Francesc; Jover, Rodrigo; De Chiara, Loretta

2018-01-01

Colorectal cancer is the fourth cause of cancer-related deaths worldwide, though detection at early stages associates with good prognosis. Thus, there is a clear demand for novel non-invasive tests for the early detection of colorectal cancer and premalignant advanced adenomas, to be used in population-wide screening programs. Aberrant DNA methylation detected in liquid biopsies, such as serum circulating cell-free DNA (cfDNA), is a promising source of non-invasive biomarkers. This study aimed to assess the feasibility of using cfDNA pooled samples to identify potential serum methylation biomarkers for the detection of advanced colorectal neoplasia (colorectal cancer or advanced adenomas) using microarray-based technology. cfDNA was extracted from serum samples from 20 individuals with no colorectal findings, 20 patients with advanced adenomas, and 20 patients with colorectal cancer (stages I and II). Two pooled samples were prepared for each pathological group using equal amounts of cfDNA from 10 individuals, sex-, age-, and recruitment hospital-matched. We measured the methylation levels of 866,836 CpG positions across the genome using the MethylationEPIC array. Pooled serum cfDNA methylation data meets the quality requirements. The proportion of detected CpG in all pools (> 99% with detection p value < 0.01) exceeded Illumina Infinium methylation data quality metrics of the number of sites detected. The differential methylation analysis revealed 1384 CpG sites (5% false discovery rate) with at least 10% difference in the methylation level between no colorectal findings controls and advanced neoplasia, the majority of which were hypomethylated. Unsupervised clustering showed that cfDNA methylation patterns can distinguish advanced neoplasia from healthy controls, as well as separate tumor tissue from healthy mucosa in an independent dataset. We also observed that advanced adenomas and stage I/II colorectal cancer methylation profiles, grouped as advanced neoplasia, are largely homogenous and clustered close together. This preliminary study shows the viability of microarray-based methylation biomarker discovery using pooled serum cfDNA samples as an alternative approach to tissue specimens. Our strategy sets an open door for deciphering new non-invasive biomarkers not only for colorectal cancer detection, but also for other types of cancers.

Parvovirus B19V DNA contamination in Chinese plasma and plasma derivatives

PubMed Central

2012-01-01

Background To ensure the safety of plasma derivatives, screening for human parvovirus B19V genomic DNA in donated plasma using a pooling strategy is performed in some countries. We investigated the prevalence of B19V DNA and anti-B19V antibodies in Chinese plasma pools, plasma derivatives and plasma donations to evaluate the risk posed by B19V. Methods Using a Q-PCR assay developed in-house, we tested for B19V genomic DNA in 142 plasma pools collected between January 2009 and June 2011 from two Chinese blood products manufacturers. Plasma derivatives collected between 1993–1995 (10 batches of albumin, 155 batches of intravenous immunoglobulin, IVIG) and 2009–2011 (50 batches of albumin, 54 batches of IVIG, 35 batches of factor VIII, 7 batches of fibrinogen, and 17 batches of prothrombin complex concentrate, PCC) were also tested for B19V contamination. In addition, B19V genome prevalence in minipools(including 90 individual donations) of 49680 individual plasma samples collected between August 2011 and March 2012 by a single Chinese manufacturer was investigated. IgM/IgG was also investigated in plasma pools/derivatives and in minipools with B19V-DNA titers above 1x104 and 1x106 geq/mL using B19 ELISA IgM/IgG assay(Virion-Serion, Würzburg, Germany), respectively. Results B19V-DNA was detected in 54.2% of plasma pools from two Chinese blood product manufacturers; among recently produced blood products, B19V was detected in 21/54 IVIG samples, 19/35 factor VIII samples, 6/7 fibrinogen samples, and 12/17 PCC samples, but not in albumin samples. The levels of B19V-DNA in these samples varied from 102-107 geq/mL. In samples with >104 geq/mL genome DNA, B19V-specific IgG was also found in all corresponding plasma pools and IVIG, whereas none was detected in the majority of other plasma derivatives. Screening of plasma donations indicated that most minipools were contaminated with B19V-DNA (102-108 geq/mL) and one donation had 1.09 × 1010 geq/mL B19V genomic DNA along with a non-classical IgG/IgM profile. Conclusions Despite the implementation of some inactivation/removal methods designed to prevent viral contamination, B19V DNA was detectable in Chinese plasma pools and plasma derivatives. Thus, the introduction of B19V screening and discard donation with high viramic concentration for Chinese plasma donors would be desirable. PMID:22978673

Oxidation in the nucleotide pool, the DNA damage response and cellular senescence: Defective bricks build a defective house.

PubMed

Rai, Priyamvada

2010-11-28

Activation of persistent DNA damage response (DDR) signaling is associated with the induction of a permanent proliferative arrest known as cellular senescence, a phenomenon intrinsically linked to both tissue aging as well as tumor suppression. The DNA damage observed in senescent cells has been attributed to elevated levels of reactive oxygen species (ROS), failing DNA damage repair processes, and/or oncogenic activation. It is not clear how labile molecules such as ROS are able to damage chromatin-bound DNA to a sufficient extent to invoke persistent DNA damage and DDR signaling. Recent evidence suggests that the nucleotide pool is a significant target for oxidants and that oxidized nucleotides, once incorporated into genomic DNA, can lead to the induction of a DNA strand break-associated DDR that triggers senescence in normal cells and in cells sustaining oncogene activation. Evasion of this DDR and resulting senescence is a key step in tumor progression. This review will explore the role of oxidation in the nucleotide pool as a major effector of oxidative stress-induced genotoxic damage and DDR in the context of cellular senescence and tumorigenic transformation. 2010 Elsevier B.V. All rights reserved.

Impact and quantification of the sources of error in DNA pooling designs.

PubMed

Jawaid, A; Sham, P

2009-01-01

The analysis of genome wide variation offers the possibility of unravelling the genes involved in the pathogenesis of disease. Genome wide association studies are also particularly useful for identifying and validating targets for therapeutic intervention as well as for detecting markers for drug efficacy and side effects. The cost of such large-scale genetic association studies may be reduced substantially by the analysis of pooled DNA from multiple individuals. However, experimental errors inherent in pooling studies lead to a potential increase in the false positive rate and a loss in power compared to individual genotyping. Here we quantify various sources of experimental error using empirical data from typical pooling experiments and corresponding individual genotyping counts using two statistical methods. We provide analytical formulas for calculating these different errors in the absence of complete information, such as replicate pool formation, and for adjusting for the errors in the statistical analysis. We demonstrate that DNA pooling has the potential of estimating allele frequencies accurately, and adjusting the pooled allele frequency estimates for differential allelic amplification considerably improves accuracy. Estimates of the components of error show that differential allelic amplification is the most important contributor to the error variance in absolute allele frequency estimation, followed by allele frequency measurement and pool formation errors. Our results emphasise the importance of minimising experimental errors and obtaining correct error estimates in genetic association studies.

Genomic analyses of tropical beef cattle fertility based on genotyping pools of Brahman cows with unknown pedigree.

PubMed

Reverter, A; Porto-Neto, L R; Fortes, M R S; McCulloch, R; Lyons, R E; Moore, S; Nicol, D; Henshall, J; Lehnert, S A

2016-10-01

We introduce an innovative approach to lowering the overall cost of obtaining genomic EBV (GEBV) and encourage their use in commercial extensive herds of Brahman beef cattle. In our approach, the DNA genotyping of cow herds from 2 independent properties was performed using a high-density bovine SNP chip on DNA from pooled blood samples, grouped according to the result of a pregnancy test following their first and second joining opportunities. For the DNA pooling strategy, 15 to 28 blood samples from the same phenotype and contemporary group were allocated to pools. Across the 2 properties, a total of 183 pools were created representing 4,164 cows. In addition, blood samples from 309 bulls from the same properties were also taken. After genotyping and quality control, 74,584 remaining SNP were used for analyses. Pools and individual DNA samples were related by means of a "hybrid" genomic relationship matrix. The pooled genotyping analysis of 2 large and independent commercial populations of tropical beef cattle was able to recover significant and plausible associations between SNP and pregnancy test outcome. We discuss 24 SNP with significant association ( < 1.0 × 10) and mapped within 40 kb of an annotated gene. We have established a method to estimate the GEBV in young herd bulls for a trait that is currently unable to be predicted at all. In summary, our novel approach allowed us to conduct genomic analyses of fertility in 2 large commercial Brahman herds managed under extensive pastoral conditions.

High-resolution chromosome mapping of BACs using multi-colour FISH and pooled-BAC FISH as a backbone for sequencing tomato chromosome 6.

PubMed

Szinay, Dóra; Chang, Song-Bin; Khrustaleva, Ludmila; Peters, Sander; Schijlen, Elio; Bai, Yuling; Stiekema, Willem J; van Ham, Roeland C H J; de Jong, Hans; Klein Lankhorst, René M

2008-11-01

Within the framework of the International Solanaceae Genome Project, the genome of tomato (Solanum lycopersicum) is currently being sequenced. We follow a 'BAC-by-BAC' approach that aims to deliver high-quality sequences of the euchromatin part of the tomato genome. BACs are selected from various libraries of the tomato genome on the basis of markers from the F2.2000 linkage map. Prior to sequencing, we validated the precise physical location of the selected BACs on the chromosomes by five-colour high-resolution fluorescent in situ hybridization (FISH) mapping. This paper describes the strategies and results of cytogenetic mapping for chromosome 6 using 75 seed BACs for FISH on pachytene complements. The cytogenetic map obtained showed discrepancies between the actual chromosomal positions of these BACs and their markers on the linkage group. These discrepancies were most notable in the pericentromere heterochromatin, thus confirming previously described suppression of cross-over recombination in that region. In a so called pooled-BAC FISH, we hybridized all seed BACs simultaneously and found a few large gaps in the euchromatin parts of the long arm that are still devoid of seed BACs and are too large for coverage by expanding BAC contigs. Combining FISH with pooled BACs and newly recruited seed BACs will thus aid in efficient targeting of novel seed BACs into these areas. Finally, we established the occurrence of repetitive DNA in heterochromatin/euchromatin borders by combining BAC FISH with hybridization of a labelled repetitive DNA fraction (Cot-100). This strategy provides an excellent means to establish the borders between euchromatin and heterochromatin in this chromosome.

Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus

PubMed Central

Cao, Xiaoxiao; Li, Shaohua; Chen, Liucun; Ding, Hongmei; Xu, Hua; Huang, Yanping; Li, Jie; Liu, Nongle; Cao, Weihong; Zhu, Yanjun; Shen, Beifen; Shao, Ningsheng

2009-01-01

In this article, a panel of ssDNA aptamers specific to Staphylococcus aureus was obtained by a whole bacterium-based SELEX procedure and applied to probing S. aureus. After several rounds of selection with S. aureus as the target and Streptococcus and S. epidermidis as counter targets, the highly enriched oligonucleic acid pool was sequenced and then grouped under different families on the basis of the homology of the primary sequence and the similarity of the secondary structure. Eleven sequences from different families were selected for further characterization by confocal imaging and flow cytometry analysis. Results showed that five aptamers demonstrated high specificity and affinity to S. aureus individually. The five aptamers recognize different molecular targets by competitive experiment. Combining these five aptamers had a much better effect than the individual aptamer in the recognition of different S. aureus strains. In addition, the combined aptamers can probe single S. aureus in pyogenic fluids. Our work demonstrates that a set of aptamers specific to one bacterium can be used in combination for the identification of the bacterium instead of a single aptamer. PMID:19498077

Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus.

PubMed

Cao, Xiaoxiao; Li, Shaohua; Chen, Liucun; Ding, Hongmei; Xu, Hua; Huang, Yanping; Li, Jie; Liu, Nongle; Cao, Weihong; Zhu, Yanjun; Shen, Beifen; Shao, Ningsheng

2009-08-01

In this article, a panel of ssDNA aptamers specific to Staphylococcus aureus was obtained by a whole bacterium-based SELEX procedure and applied to probing S. aureus. After several rounds of selection with S. aureus as the target and Streptococcus and S. epidermidis as counter targets, the highly enriched oligonucleic acid pool was sequenced and then grouped under different families on the basis of the homology of the primary sequence and the similarity of the secondary structure. Eleven sequences from different families were selected for further characterization by confocal imaging and flow cytometry analysis. Results showed that five aptamers demonstrated high specificity and affinity to S. aureus individually. The five aptamers recognize different molecular targets by competitive experiment. Combining these five aptamers had a much better effect than the individual aptamer in the recognition of different S. aureus strains. In addition, the combined aptamers can probe single S. aureus in pyogenic fluids. Our work demonstrates that a set of aptamers specific to one bacterium can be used in combination for the identification of the bacterium instead of a single aptamer.

The Deoxynucleoside Triphosphate Triphosphohydrolase Activity of SAMHD1 Protein Contributes to the Mitochondrial DNA Depletion Associated with Genetic Deficiency of Deoxyguanosine Kinase*

PubMed Central

Franzolin, Elisa; Salata, Cristiano; Bianchi, Vera; Rampazzo, Chiara

2015-01-01

The dNTP triphosphohydrolase SAMHD1 is a nuclear antiviral host restriction factor limiting HIV-1 infection in macrophages and a major regulator of dNTP concentrations in human cells. In normal human fibroblasts its expression increases during quiescence, contributing to the small dNTP pool sizes of these cells. Down-regulation of SAMHD1 by siRNA expands all four dNTP pools, with dGTP undergoing the largest relative increase. The deoxyguanosine released by SAMHD1 from dGTP can be phosphorylated inside mitochondria by deoxyguanosine kinase (dGK) or degraded in the cytosol by purine nucleoside phosphorylase. Genetic mutations of dGK cause mitochondrial (mt) DNA depletion in noncycling cells and hepato-cerebral mtDNA depletion syndrome in humans. We studied if SAMHD1 and dGK interact in the regulation of the dGTP pool during quiescence employing dGK-mutated skin fibroblasts derived from three unrelated patients. In the presence of SAMHD1 quiescent mutant fibroblasts manifested mt dNTP pool imbalance and mtDNA depletion. When SAMHD1 was silenced by siRNA transfection the composition of the mt dNTP pool approached that of the controls, and mtDNA copy number increased, compensating the depletion to various degrees in the different mutant fibroblasts. Chemical inhibition of purine nucleoside phosphorylase did not improve deoxyguanosine recycling by dGK in WT cells. We conclude that the activity of SAMHD1 contributes to the pathological phenotype of dGK deficiency. Our results prove the importance of SAMHD1 in the regulation of all dNTP pools and suggest that dGK inside mitochondria has the function of recycling the deoxyguanosine derived from endogenous dGTP degraded by SAMHD1 in the nucleus. PMID:26342080

Oligonucleotide Based Magnetic Bead Capture of Onchocerca volvulus DNA for PCR Pool Screening of Vector Black Flies

PubMed Central

Gopal, Hemavathi; Hassan, Hassan K.; Rodríguez-Pérez, Mario A.; Toé, Laurent D.; Lustigman, Sara; Unnasch, Thomas R.

2012-01-01

Background Entomological surveys of Simulium vectors are an important component in the criteria used to determine if Onchocerca volvulus transmission has been interrupted and if focal elimination of the parasite has been achieved. However, because infection in the vector population is quite rare in areas where control has succeeded, large numbers of flies need to be examined to certify transmission interruption. Currently, this is accomplished through PCR pool screening of large numbers of flies. The efficiency of this process is limited by the size of the pools that may be screened, which is in turn determined by the constraints imposed by the biochemistry of the assay. The current method of DNA purification from pools of vector black flies relies upon silica adsorption. This method can be applied to screen pools containing a maximum of 50 individuals (from the Latin American vectors) or 100 individuals (from the African vectors). Methodology/Principal Findings We have evaluated an alternative method of DNA purification for pool screening of black flies which relies upon oligonucleotide capture of Onchocerca volvulus genomic DNA from homogenates prepared from pools of Latin American and African vectors. The oligonucleotide capture assay was shown to reliably detect one O. volvulus infective larva in pools containing 200 African or Latin American flies, representing a two-four fold improvement over the conventional assay. The capture assay requires an equivalent amount of technical time to conduct as the conventional assay, resulting in a two-four fold reduction in labor costs per insect assayed and reduces reagent costs to $3.81 per pool of 200 flies, or less than $0.02 per insect assayed. Conclusions/Significance The oligonucleotide capture assay represents a substantial improvement in the procedure used to detect parasite prevalence in the vector population, a major metric employed in the process of certifying the elimination of onchocerciasis. PMID:22724041

Pooled-DNA Sequencing for Elucidating New Genomic Risk Factors, Rare Variants Underlying Alzheimer's Disease.

PubMed

Jin, Sheng Chih; Benitez, Bruno A; Deming, Yuetiva; Cruchaga, Carlos

2016-01-01

Analyses of genome-wide association studies (GWAS) for complex disorders usually identify common variants with a relatively small effect size that only explain a small proportion of phenotypic heritability. Several studies have suggested that a significant fraction of heritability may be explained by low-frequency (minor allele frequency (MAF) of 1-5 %) and rare-variants that are not contained in the commercial GWAS genotyping arrays (Schork et al., Curr Opin Genet Dev 19:212, 2009). Rare variants can also have relatively large effects on risk for developing human diseases or disease phenotype (Cruchaga et al., PLoS One 7:e31039, 2012). However, it is necessary to perform next-generation sequencing (NGS) studies in a large population (>4,000 samples) to detect a significant rare-variant association. Several NGS methods, such as custom capture sequencing and amplicon-based sequencing, are designed to screen a small proportion of the genome, but most of these methods are limited in the number of samples that can be multiplexed (i.e. most sequencing kits only provide 96 distinct index). Additionally, the sequencing library preparation for 4,000 samples remains expensive and thus conducting NGS studies with the aforementioned methods are not feasible for most research laboratories.The need for low-cost large scale rare-variant detection makes pooled-DNA sequencing an ideally efficient and cost-effective technique to identify rare variants in target regions by sequencing hundreds to thousands of samples. Our recent work has demonstrated that pooled-DNA sequencing can accurately detect rare variants in targeted regions in multiple DNA samples with high sensitivity and specificity (Jin et al., Alzheimers Res Ther 4:34, 2012). In these studies we used a well-established pooled-DNA sequencing approach and a computational package, SPLINTER (short indel prediction by large deviation inference and nonlinear true frequency estimation by recursion) (Vallania et al., Genome Res 20:1711, 2010), for accurate identification of rare variants in large DNA pools. Given an average sequencing coverage of 30× per haploid genome, SPLINTER can detect rare variants and short indels up to 4 base pairs (bp) with high sensitivity and specificity (up to 1 haploid allele in a pool as large as 500 individuals). Step-by-step instructions on how to conduct pooled-DNA sequencing experiments and data analyses are described in this chapter.

Micropreparative capillary gel electrophoresis of DNA: rapid expressed sequence tag library construction.

PubMed

Shi, Liang; Khandurina, Julia; Ronai, Zsolt; Li, Bi-Yu; Kwan, Wai King; Wang, Xun; Guttman, András

2003-01-01

A capillary gel electrophoresis based automated DNA fraction collection technique was developed to support a novel DNA fragment-pooling strategy for expressed sequence tag (EST) library construction. The cDNA population is first cleaved by BsaJ I and EcoR I restriction enzymes, and then subpooled by selective ligation with specific adapters followed by polymerase chain reaction (PCR) amplification and labeling. Combination of this cDNA fingerprinting method with high-resolution capillary gel electrophoresis separation and precise fractionation of individual cDNA transcript representatives avoids redundant fragment selection and concomitant repetitive sequencing of abundant transcripts. Using a computer-controlled capillary electrophoresis device the transcript representatives were separated by their size and fractions were automatically collected in every 30 s into 96-well plates. The high resolving power of the sieving matrix ensured sequencing grade separation of the DNA fragments (i.e., single-base resolution) and successful fraction collection. Performance and precision of the fraction collection procedure was validated by PCR amplification of the collected DNA fragments followed by capillary electrophoresis analysis for size and purity verification. The collected and PCR-amplified transcript representatives, ranging up to several hundred base pairs, were then sequenced to create an EST library.

DNA tetrominoes: the construction of DNA nanostructures using self-organised heterogeneous deoxyribonucleic acids shapes.

PubMed

Ong, Hui San; Rahim, Mohd Syafiq; Firdaus-Raih, Mohd; Ramlan, Effirul Ikhwan

2015-01-01

The unique programmability of nucleic acids offers alternative in constructing excitable and functional nanostructures. This work introduces an autonomous protocol to construct DNA Tetris shapes (L-Shape, B-Shape, T-Shape and I-Shape) using modular DNA blocks. The protocol exploits the rich number of sequence combinations available from the nucleic acid alphabets, thus allowing for diversity to be applied in designing various DNA nanostructures. Instead of a deterministic set of sequences corresponding to a particular design, the protocol promotes a large pool of DNA shapes that can assemble to conform to any desired structures. By utilising evolutionary programming in the design stage, DNA blocks are subjected to processes such as sequence insertion, deletion and base shifting in order to enrich the diversity of the resulting shapes based on a set of cascading filters. The optimisation algorithm allows mutation to be exerted indefinitely on the candidate sequences until these sequences complied with all the four fitness criteria. Generated candidates from the protocol are in agreement with the filter cascades and thermodynamic simulation. Further validation using gel electrophoresis indicated the formation of the designed shapes. Thus, supporting the plausibility of constructing DNA nanostructures in a more hierarchical, modular, and interchangeable manner.

Novel Parvovirus and Related Variant in Human Plasma

PubMed Central

Fryer, Jacqueline F.; Kapoor, Amit; Minor, Philip D.; Delwart, Eric

2006-01-01

We report a novel parvovirus (PARV4) and related variants in pooled human plasma used in the manufacture of plasma-derived medical products. Viral DNA was detected by using highly selective polymerase chain reaction assays; 5% of pools tested positive, and amounts of DNA ranged from <500 copies/mL to >106 copies/mL plasma. PMID:16494735

A Well-Resolved Phylogeny of the Trees of Puerto Rico Based on DNA Barcode Sequence Data

PubMed Central

Muscarella, Robert; Uriarte, María; Erickson, David L.; Swenson, Nathan G.; Zimmerman, Jess K.; Kress, W. John

2014-01-01

Background The use of phylogenetic information in community ecology and conservation has grown in recent years. Two key issues for community phylogenetics studies, however, are (i) low terminal phylogenetic resolution and (ii) arbitrarily defined species pools. Methodology/principal findings We used three DNA barcodes (plastid DNA regions rbcL, matK, and trnH-psbA) to infer a phylogeny for 527 native and naturalized trees of Puerto Rico, representing the vast majority of the entire tree flora of the island (89%). We used a maximum likelihood (ML) approach with and without a constraint tree that enforced monophyly of recognized plant orders. Based on 50% consensus trees, the ML analyses improved phylogenetic resolution relative to a comparable phylogeny generated with Phylomatic (proportion of internal nodes resolved: constrained ML = 74%, unconstrained ML = 68%, Phylomatic = 52%). We quantified the phylogenetic composition of 15 protected forests in Puerto Rico using the constrained ML and Phylomatic phylogenies. We found some evidence that tree communities in areas of high water stress were relatively phylogenetically clustered. Reducing the scale at which the species pool was defined (from island to soil types) changed some of our results depending on which phylogeny (ML vs. Phylomatic) was used. Overall, the increased terminal resolution provided by the ML phylogeny revealed additional patterns that were not observed with a less-resolved phylogeny. Conclusions/significance With the DNA barcode phylogeny presented here (based on an island-wide species pool), we show that a more fully resolved phylogeny increases power to detect nonrandom patterns of community composition in several Puerto Rican tree communities. Especially if combined with additional information on species functional traits and geographic distributions, this phylogeny will (i) facilitate stronger inferences about the role of historical processes in governing the assembly and composition of Puerto Rican forests, (ii) provide insight into Caribbean biogeography, and (iii) aid in incorporating evolutionary history into conservation planning. PMID:25386879

A well-resolved phylogeny of the trees of Puerto Rico based on DNA barcode sequence data.

PubMed

Muscarella, Robert; Uriarte, María; Erickson, David L; Swenson, Nathan G; Zimmerman, Jess K; Kress, W John

2014-01-01

The use of phylogenetic information in community ecology and conservation has grown in recent years. Two key issues for community phylogenetics studies, however, are (i) low terminal phylogenetic resolution and (ii) arbitrarily defined species pools. We used three DNA barcodes (plastid DNA regions rbcL, matK, and trnH-psbA) to infer a phylogeny for 527 native and naturalized trees of Puerto Rico, representing the vast majority of the entire tree flora of the island (89%). We used a maximum likelihood (ML) approach with and without a constraint tree that enforced monophyly of recognized plant orders. Based on 50% consensus trees, the ML analyses improved phylogenetic resolution relative to a comparable phylogeny generated with Phylomatic (proportion of internal nodes resolved: constrained ML = 74%, unconstrained ML = 68%, Phylomatic = 52%). We quantified the phylogenetic composition of 15 protected forests in Puerto Rico using the constrained ML and Phylomatic phylogenies. We found some evidence that tree communities in areas of high water stress were relatively phylogenetically clustered. Reducing the scale at which the species pool was defined (from island to soil types) changed some of our results depending on which phylogeny (ML vs. Phylomatic) was used. Overall, the increased terminal resolution provided by the ML phylogeny revealed additional patterns that were not observed with a less-resolved phylogeny. With the DNA barcode phylogeny presented here (based on an island-wide species pool), we show that a more fully resolved phylogeny increases power to detect nonrandom patterns of community composition in several Puerto Rican tree communities. Especially if combined with additional information on species functional traits and geographic distributions, this phylogeny will (i) facilitate stronger inferences about the role of historical processes in governing the assembly and composition of Puerto Rican forests, (ii) provide insight into Caribbean biogeography, and (iii) aid in incorporating evolutionary history into conservation planning.

High-resolution mapping and sequence analysis of 597 cDNA clones transcribed from the 1 Mb region in human chromosome 4q16.3 containing Huntington disease gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hadano, S.; Ishida, Y.; Tomiyasu, H.

1994-09-01

To complete a transcription map of the 1 Mb region in human chromosome 4p16.3 containing the Huntington disease (HD) gene, the isolation of cDNA clones are being performed throughout. Our method relies on a direct screening of the cDNA libraries probed with single copy microclones from 3 YAC clones spanning 1 Mbp of the HD gene region. AC-DNAs were isolated by a preparative pulsed-field gel electrophoresis, amplified by both a single unique primer (SUP)-PCR and a linker ligation PCR, and 6 microclone-DNA libraries were generated. Then, 8,640 microclones from these libraries were independently amplified by PCR, and arrayed onto themore » membranes. 800-900 microclones that were not cross-hybridized with total human and yeast genomic DNA, TAC vector DNA, and ribosomal cDNA on a dot hybridization (putatively carrying single copy sequences) were pooled to make 9 probe pools. A total of {approximately}1.8x10{sup 7} plaques from the human brain cDNA libraries was screened with 9 pool-probes, and then 672 positive cDNA clones were obtained. So far, 597 cDNA clones were defined and arrayed onto a map of the 1 Mbp of the HD gene region by hybridization with HD region-specific cosmid contigs and YAC clones. Further characterization including a DNA sequencing and Northern blot analysis is currently underway.« less

A new surveillance and response tool: risk map of infected Oncomelania hupensis detected by Loop-mediated isothermal amplification (LAMP) from pooled samples.

PubMed

Tong, Qun-Bo; Chen, Rui; Zhang, Yi; Yang, Guo-Jing; Kumagai, Takashi; Furushima-Shimogawara, Rieko; Lou, Di; Yang, Kun; Wen, Li-Yong; Lu, Shao-Hong; Ohta, Nobuo; Zhou, Xiao-Nong

2015-01-01

Although schistosomiasis remains a serious health problem worldwide, significant achievements in schistosomiasis control has been made in the People's Republic of China. The disease has been eliminated in five out of 12 endemic provinces, and the prevalence in remaining endemic areas is very low and is heading toward elimination. A rapid and sensitive method for monitoring the distribution of infected Oncomelania hupensis is urgently required. We applied a loop-mediated isothermal amplification (LAMP) assay targeting 28S rDNA for the rapid and effective detection of Schistosoma japonicum DNA in infected and prepatent infected O. hupensis snails. The detection limit of the LAMP method was 100 fg of S. japonicum genomic DNA. To promote the application of the approach in the field, the LAMP assay was used to detect infection in pooled samples of field-collected snails. In the pooled sample detection, snails were collected from 28 endemic areas, and 50 snails from each area were pooled based on the maximum pool size estimation, crushed together and DNA was extracted from each pooled sample as template for the LAMP assay. Based on the formula for detection from pooled samples, the proportion of positive pooled samples and the positive proportion of O. hupensis detected by LAMP of Xima village reached 66.67% and 1.33%, while those of Heini, Hongjia, Yangjiang and Huangshan villages were 33.33% and 0.67%, and those of Tuanzhou and Suliao villages were 16.67% and 0.33%, respectively. The remaining 21 monitoring field sites gave negative results. A risk map for the transmission of schistosomiasis was constructed using ArcMap, based on the positive proportion of O. hupensis infected with S. japonicum, as detected by the LAMP assay, which will form a guide for surveillance and response strategies in high risk areas. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2014-01-01

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

PCNA appears in two populations of slow and fast diffusion with a constant ratio throughout S-phase in replicating mammalian cells.

PubMed

Zessin, Patrick J M; Sporbert, Anje; Heilemann, Mike

2016-01-13

DNA replication is a fundamental cellular process that precedes cell division. Proliferating cell nuclear antigen (PCNA) is a central scaffold protein that orchestrates DNA replication by recruiting many factors essential for the replication machinery. We studied the mobility of PCNA in live mammalian cells using single-particle tracking in combination with photoactivated-localization microscopy (sptPALM) and found two populations. The first population which is only present in cells with active DNA replication, showed slow diffusion and was found to be located in replication foci. The second population showed fast diffusion, and represents the nucleoplasmic pool of unbound PCNA not involved in DNA replication. The ratio of these two populations remained constant throughout different stages of S-phase. A fraction of molecules in both populations showed spatially constrained mobility. We determined an exploration radius of ~100 nm for 13% of the slow-diffusing PCNA molecules, and of ~600 nm for 46% of the fast-diffusing PCNA molecules.

Biompha-LAMP: A New Rapid Loop-Mediated Isothermal Amplification Assay for Detecting Schistosoma mansoni in Biomphalaria glabrata Snail Host.

PubMed

Gandasegui, Javier; Fernández-Soto, Pedro; Hernández-Goenaga, Juan; López-Abán, Julio; Vicente, Belén; Muro, Antonio

2016-12-01

Schistosomiasis remains one of the most common endemic parasitic diseases affecting over 230 million people worlwide. Schistosoma mansoni is the main species causing intestinal and hepatic schistosomiasis and the fresh water pulmonate snails of the genus Biomphalaria are best known for their role as intermediate hosts of the parasite. The development of new molecular monitoring assays for large-scale screening of snails from transmission sites to detect the presence of schistosomes is an important point to consider for snail control interventions related to schistosomiasis elimination. Our work was focussed on developing and evaluating a new LAMP assay combined with a simple DNA extraction method to detect S. mansoni in experimentally infected snails as a diagnostic tool for field conditions. A LAMP assay using a set of six primers targeting a sequence of S. mansoni ribosomal intergenic spacer 28S-18S rRNA was designed. The detection limit of the LAMP assay was 0.1 fg of S. mansoni DNA at 63°C for 50 minutes. LAMP was evaluated by examining S. mansoni DNA in B. glabrata snails experimentally exposed to miracidia at different times post-exposure: early prepatent period (before cercarial shedding), light infections (snails exposed to a low number of miracidia) and detection of infected snails in pooled samples (within a group of uninfected snails). DNA for LAMP assays was obtained by using a commercial DNA extraction kit or a simple heat NaOH extraction method. We detected S. mansoni DNA in all groups of snails by using no complicated requirement procedure for DNA obtaining. Our LAMP assay, named Biompha-LAMP, is specific, sensitive, rapid and potentially adaptable as a cost-effective method for screening of intermediate hosts infected with S. mansoni in both individual snails and pooled samples. The assay could be suitable for large-scale field surveys for schistosomes control campaigns in endemic areas.

Biompha-LAMP: A New Rapid Loop-Mediated Isothermal Amplification Assay for Detecting Schistosoma mansoni in Biomphalaria glabrata Snail Host

PubMed Central

Hernández-Goenaga, Juan; López-Abán, Julio; Vicente, Belén; Muro, Antonio

2016-01-01

Background Schistosomiasis remains one of the most common endemic parasitic diseases affecting over 230 million people worlwide. Schistosoma mansoni is the main species causing intestinal and hepatic schistosomiasis and the fresh water pulmonate snails of the genus Biomphalaria are best known for their role as intermediate hosts of the parasite. The development of new molecular monitoring assays for large-scale screening of snails from transmission sites to detect the presence of schistosomes is an important point to consider for snail control interventions related to schistosomiasis elimination. Our work was focussed on developing and evaluating a new LAMP assay combined with a simple DNA extraction method to detect S. mansoni in experimentally infected snails as a diagnostic tool for field conditions. Methodology/Principal findings A LAMP assay using a set of six primers targeting a sequence of S. mansoni ribosomal intergenic spacer 28S-18S rRNA was designed. The detection limit of the LAMP assay was 0.1 fg of S. mansoni DNA at 63°C for 50 minutes. LAMP was evaluated by examining S. mansoni DNA in B. glabrata snails experimentally exposed to miracidia at different times post-exposure: early prepatent period (before cercarial shedding), light infections (snails exposed to a low number of miracidia) and detection of infected snails in pooled samples (within a group of uninfected snails). DNA for LAMP assays was obtained by using a commercial DNA extraction kit or a simple heat NaOH extraction method. We detected S. mansoni DNA in all groups of snails by using no complicated requirement procedure for DNA obtaining. Conclusions/Significance Our LAMP assay, named Biompha-LAMP, is specific, sensitive, rapid and potentially adaptable as a cost-effective method for screening of intermediate hosts infected with S. mansoni in both individual snails and pooled samples. The assay could be suitable for large-scale field surveys for schistosomes control campaigns in endemic areas. PMID:27941967

Evaluation of Group Genetic Ancestry of Populations from Philadelphia and Dakar in the Context of Sex-Biased Admixture in the Americas

PubMed Central

Stefflova, Klara; Dulik, Matthew C.; Pai, Athma A.; Walker, Amy H.; Zeigler-Johnson, Charnita M.; Gueye, Serigne M.; Schurr, Theodore G.; Rebbeck, Timothy R.

2009-01-01

Background Population history can be reflected in group genetic ancestry, where genomic variation captured by the mitochondrial DNA (mtDNA) and non-recombining portion of the Y chromosome (NRY) can separate female- and male-specific admixture processes. Genetic ancestry may influence genetic association studies due to differences in individual admixture within recently admixed populations like African Americans. Principal Findings We evaluated the genetic ancestry of Senegalese as well as European Americans and African Americans from Philadelphia. Senegalese mtDNA consisted of ∼12% U haplotypes (U6 and U5b1b haplotypes, common in North Africa) while the NRY haplotypes belonged solely to haplogroup E. In Philadelphia, we observed varying degrees of admixture. While African Americans have 9–10% mtDNAs and ∼31% NRYs of European origin, these results are not mirrored in the mtDNA/NRY pools of European Americans: they have less than 7% mtDNAs and less than 2% NRYs from non-European sources. Additionally, there is <2% Native American contribution to Philadelphian African American ancestry and the admixture from combined mtDNA/NRY estimates is consistent with the admixture derived from autosomal genetic data. To further dissect these estimates, we have analyzed our samples in the context of different demographic groups in the Americas. Conclusions We found that sex-biased admixture in African-derived populations is present throughout the Americas, with continual influence of European males, while Native American females contribute mainly to populations of the Caribbean and South America. The high non-European female contribution to the pool of European-derived populations is consistently characteristic of Iberian colonization. These data suggest that genomic data correlate well with historical records of colonization in the Americas. PMID:19946364

Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

DOE PAGES

Freudenthal, Bret D.; Beard, William A.; Perera, Lalith; ...

2014-11-17

Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2’-deoxyguanosine found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate E. coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerase discriminates between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities,more » nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine (Cy) and 8-oxodGTP(syn) utilizes its Hoogsteen edge to base pair with adenine (Ad). Here in this paper we utilized time-lapse crystallography to follow 8-oxo-dGTP insertion opposite Ad or Cy with human DNA pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxodGTP utilizes charge modulation during insertion that can lead to a blocked DNA repair intermediate.« less

Investigation on the diagnostic sensitivity of molecular tools used for detection of koi herpesvirus.

PubMed

Bergmann, Sven M; Riechardt, Meike; Fichtner, Dieter; Lee, Peiyu; Kempter, Jolanta

2010-02-01

Previous and new PCRs for KHV detection were compared by estimation of their sensitivity in recognizing KHV DNA in plasmids, cell culture extracted KHV DNA and total DNA obtained from field tissue samples. A modified real-time PCR (Gilad et al., 2004), combined with an internal control system (IC2, Hoffmann et al., 2006) in a duplex assay, was used as a "gold standard". The lowest reliably determined virus concentration between, 5 and 10 KHV DNA genomic equivalents, was found by real-time PCR (Gilad et al., 2004), nested PCR (Bergmann et al., 2006) and one-tube semi-nested PCR. All other published and unpublished PCRs, as well as the commercial Loopamp, recognized KHV DNA at higher concentrations only. Additionally, KHV variants, newly adapted to European conditions, which could not be detected by PCR according to Bercovier et al. (2005) were found in two field samples from carp and koi from different regions of Germany. A negative influence of sample pooling was shown with field samples tested by real-time PCR. 2009 Elsevier B.V. All rights reserved.

The effect of common imaging and hot water maceration on DNA recovery from skeletal remains.

PubMed

Frank, Emilie M; Mundorff, Amy Z; Davoren, Jon M

2015-12-01

Identifying human remains often begins with cleaning and imaging the material. Hot water maceration is used to remove adherent soft tissue from bone and radiographs are taken to better visualize osseous details. Heat and radiation are known to have harmful effects on DNA, but their ability to degrade DNA when used for cleaning and imaging has not been well studied. To better understand their individual and combined effects on the recoverability of DNA from bone, skeletal samples were subjected to (1) hot water maceration (62 °C for 45 min); (2) CT scanning (0.6mm slices, 120 kV, 10.4s); (3) X-ray (50 kVp, 150 mA, 0.03 s, 40 in); and (4) all 3 treatments combined. Forty-eight DNA samples were extracted, quantified and amplified with the AmpFLSTR(®) Identifiler(®) system. Nearly all of the processed samples had reduced RFU values relative to the unprocessed samples, indicating some amount of genetic loss. This loss did not always translate into loss of profile completeness, since only a few samples had a reduction in the number of loci detected after processing. DNA yields were not significantly reduced by any one of the processing methods, however the results indicate that the damaging effects are additive. It is possible that processing may reduce a bone's DNA reservoir and as more procedures are preformed, the pool of available genetic information might be diminished. Many intrinsic and extrinsic factors can affect the recoverability of DNA from bone. Collecting a DNA sample prior to processing avoids the negative effects from hot water maceration and radiological imaging. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Multiplexed resequencing analysis to identify rare variants in pooled DNA with barcode indexing using next-generation sequencer.

PubMed

Mitsui, Jun; Fukuda, Yoko; Azuma, Kyo; Tozaki, Hirokazu; Ishiura, Hiroyuki; Takahashi, Yuji; Goto, Jun; Tsuji, Shoji

2010-07-01

We have recently found that multiple rare variants of the glucocerebrosidase gene (GBA) confer a robust risk for Parkinson disease, supporting the 'common disease-multiple rare variants' hypothesis. To develop an efficient method of identifying rare variants in a large number of samples, we applied multiplexed resequencing using a next-generation sequencer to identification of rare variants of GBA. Sixteen sets of pooled DNAs from six pooled DNA samples were prepared. Each set of pooled DNAs was subjected to polymerase chain reaction to amplify the target gene (GBA) covering 6.5 kb, pooled into one tube with barcode indexing, and then subjected to extensive sequence analysis using the SOLiD System. Individual samples were also subjected to direct nucleotide sequence analysis. With the optimization of data processing, we were able to extract all the variants from 96 samples with acceptable rates of false-positive single-nucleotide variants.

Population-based rare variant detection via pooled exome or custom hybridization capture with or without individual indexing.

PubMed

Ramos, Enrique; Levinson, Benjamin T; Chasnoff, Sara; Hughes, Andrew; Young, Andrew L; Thornton, Katherine; Li, Allie; Vallania, Francesco L M; Province, Michael; Druley, Todd E

2012-12-06

Rare genetic variation in the human population is a major source of pathophysiological variability and has been implicated in a host of complex phenotypes and diseases. Finding disease-related genes harboring disparate functional rare variants requires sequencing of many individuals across many genomic regions and comparing against unaffected cohorts. However, despite persistent declines in sequencing costs, population-based rare variant detection across large genomic target regions remains cost prohibitive for most investigators. In addition, DNA samples are often precious and hybridization methods typically require large amounts of input DNA. Pooled sample DNA sequencing is a cost and time-efficient strategy for surveying populations of individuals for rare variants. We set out to 1) create a scalable, multiplexing method for custom capture with or without individual DNA indexing that was amenable to low amounts of input DNA and 2) expand the functionality of the SPLINTER algorithm for calling substitutions, insertions and deletions across either candidate genes or the entire exome by integrating the variant calling algorithm with the dynamic programming aligner, Novoalign. We report methodology for pooled hybridization capture with pre-enrichment, indexed multiplexing of up to 48 individuals or non-indexed pooled sequencing of up to 92 individuals with as little as 70 ng of DNA per person. Modified solid phase reversible immobilization bead purification strategies enable no sample transfers from sonication in 96-well plates through adapter ligation, resulting in 50% less library preparation reagent consumption. Custom Y-shaped adapters containing novel 7 base pair index sequences with a Hamming distance of ≥2 were directly ligated onto fragmented source DNA eliminating the need for PCR to incorporate indexes, and was followed by a custom blocking strategy using a single oligonucleotide regardless of index sequence. These results were obtained aligning raw reads against the entire genome using Novoalign followed by variant calling of non-indexed pools using SPLINTER or SAMtools for indexed samples. With these pipelines, we find sensitivity and specificity of 99.4% and 99.7% for pooled exome sequencing. Sensitivity, and to a lesser degree specificity, proved to be a function of coverage. For rare variants (≤2% minor allele frequency), we achieved sensitivity and specificity of ≥94.9% and ≥99.99% for custom capture of 2.5 Mb in multiplexed libraries of 22-48 individuals with only ≥5-fold coverage/chromosome, but these parameters improved to ≥98.7 and 100% with 20-fold coverage/chromosome. This highly scalable methodology enables accurate rare variant detection, with or without individual DNA sample indexing, while reducing the amount of required source DNA and total costs through less hybridization reagent consumption, multi-sample sonication in a standard PCR plate, multiplexed pre-enrichment pooling with a single hybridization and lesser sequencing coverage required to obtain high sensitivity.

A pooling-based approach to mapping genetic variants associated with DNA methylation

PubMed Central

Kaplow, Irene M.; MacIsaac, Julia L.; Mah, Sarah M.; McEwen, Lisa M.; Kobor, Michael S.; Fraser, Hunter B.

2015-01-01

DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover <2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a truly genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified more than 2000 genetic variants associated with DNA methylation. We found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data. PMID:25910490

A pooling-based approach to mapping genetic variants associated with DNA methylation

DOE PAGES

Kaplow, Irene M.; MacIsaac, Julia L.; Mah, Sarah M.; ...

2015-04-24

DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover <2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a trulymore » genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified more than 2000 genetic variants associated with DNA methylation. Here we found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data.« less

A DNA fingerprinting procedure for ultra high-throughput genetic analysis of insects.

PubMed

Schlipalius, D I; Waldron, J; Carroll, B J; Collins, P J; Ebert, P R

2001-12-01

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular information is often non-existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability in three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced approximately 50 scoreable polymorphic DNA markers, between individuals of three independent field isolates of R. dominica. Multivariate cluster analysis using forty-nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 +/- 6.0% between clades and of 21.0 +/- 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from individual DNA samples that had been combined to create the bulked samples.

Detecting SNPs and estimating allele frequencies in clonal bacterial populations by sequencing pooled DNA.

PubMed

Holt, Kathryn E; Teo, Yik Y; Li, Heng; Nair, Satheesh; Dougan, Gordon; Wain, John; Parkhill, Julian

2009-08-15

Here, we present a method for estimating the frequencies of SNP alleles present within pooled samples of DNA using high-throughput short-read sequencing. The method was tested on real data from six strains of the highly monomorphic pathogen Salmonella Paratyphi A, sequenced individually and in a pool. A variety of read mapping and quality-weighting procedures were tested to determine the optimal parameters, which afforded > or =80% sensitivity of SNP detection and strong correlation with true SNP frequency at poolwide read depth of 40x, declining only slightly at read depths 20-40x. The method was implemented in Perl and relies on the opensource software Maq for read mapping and SNP calling. The Perl script is freely available from ftp://ftp.sanger.ac.uk/pub/pathogens/pools/.

Development, standardization and validation of molecular techniques for malaria vector species identification, trophic preferences, and detection of Plasmodium falciparum.

PubMed

Rath, Animesha; Prusty, Manas R; Barik, Sushanta K; Das, Mumani; Tripathy, Hare K; Mahapatra, Namita; Hazra, Rupenangshu K

2017-01-01

Knowledge on prevalence of malaria vector species of a certain area provides important information for implementation of appropriate control strategies. The present study describes a rapid method for screening of major Anopheline vector species and at the same time detection of Plasmodium falciparum sporozoite infection and blood meal preferences/trophic preferences. The study was carried from February 2012 to March 2013 in three seasons, i.e. rainy, winter and summer in Jhumpura PHC of Keonjhar district, Odisha, India. Processing of mosquitoes was carried out in two different methods, viz. mosquito pool (P1) and mosquito DNA pool (P2). Pool size for both the methods was standardized for DNA isolation and multiplex PCR assay. This PCR based assay was employed to screen the major vector com- position in three different seasons of four different ecotypes of Keonjhar district. Pearson's correlation coefficient was determined for a comparative analysis of the morphological identification with the pool prevalence assay for each ecotype. A pool size of 10 was standardized for DNA isolation as well as PCR. PCR assay revealed that the average pool prevalence for all ecotypes was highest for An. annularis in winter and summer whereas for An. culicifacies it was rainy season. Foothill and plain ecotypes contributed to highest and lowest vectorial abundance respectively. The results of the prevalence of vector species in pool from PCR based assay were found to be highly correlated with that of the results of morphological identification. Screening by pool based PCR assay is relatively rapid as compared to conventional identification and can be employed as an important tool in malaria control programmes.

Whole mitochondrial genome sequencing of domestic horses reveals incorporation of extensive wild horse diversity during domestication

PubMed Central

2011-01-01

Background DNA target enrichment by micro-array capture combined with high throughput sequencing technologies provides the possibility to obtain large amounts of sequence data (e.g. whole mitochondrial DNA genomes) from multiple individuals at relatively low costs. Previously, whole mitochondrial genome data for domestic horses (Equus caballus) were limited to only a few specimens and only short parts of the mtDNA genome (especially the hypervariable region) were investigated for larger sample sets. Results In this study we investigated whole mitochondrial genomes of 59 domestic horses from 44 breeds and a single Przewalski horse (Equus przewalski) using a recently described multiplex micro-array capture approach. We found 473 variable positions within the domestic horses, 292 of which are parsimony-informative, providing a well resolved phylogenetic tree. Our divergence time estimate suggests that the mitochondrial genomes of modern horse breeds shared a common ancestor around 93,000 years ago and no later than 38,000 years ago. A Bayesian skyline plot (BSP) reveals a significant population expansion beginning 6,000-8,000 years ago with an ongoing exponential growth until the present, similar to other domestic animal species. Our data further suggest that a large sample of wild horse diversity was incorporated into the domestic population; specifically, at least 46 of the mtDNA lineages observed in domestic horses (73%) already existed before the beginning of domestication about 5,000 years ago. Conclusions Our study provides a window into the maternal origins of extant domestic horses and confirms that modern domestic breeds present a wide sample of the mtDNA diversity found in ancestral, now extinct, wild horse populations. The data obtained allow us to detect a population expansion event coinciding with the beginning of domestication and to estimate both the minimum number of female horses incorporated into the domestic gene pool and the time depth of the domestic horse mtDNA gene pool. PMID:22082251

Mimivirus Circulation among Wild and Domestic Mammals, Amazon Region, Brazil

PubMed Central

Dornas, Fábio P.; Rodrigues, Felipe P.; Boratto, Paulo V.M.; Silva, Lorena C.F.; Ferreira, Paulo C.P.; Bonjardim, Cláudio A.; Trindade, Giliane S.; Kroon, Erna G.; La Scola, Bernard

2014-01-01

To investigate circulation of mimiviruses in the Amazon Region of Brazil, we surveyed 513 serum samples from domestic and wild mammals. Neutralizing antibodies were detected in 15 sample pools, and mimivirus DNA was detected in 9 pools of serum from capuchin monkeys and in 16 pools of serum from cattle. PMID:24564967

Microbiological investigations on the water of a thermal bath at Budapest.

PubMed

Szuróczki, Sára; Kéki, Zsuzsa; Káli, Szandra; Lippai, Anett; Márialigeti, Károly; Tóth, Erika

2016-06-01

Thermal baths are unique aquatic environments combining a wide variety of natural and anthropogenic ecological factors, which also appear in their microbiological state. There is limited information on the microbiology of thermal baths in their complexity, tracking community shifts from the thermal wells to the pools. In the present study, the natural microbial community of well and pool waters in Gellért bath was studied in detail by cultivation-based techniques. To isolate bacteria, 10% R2A and minimal synthetic media (with "bath water") with agar-agar and gellan gum were used after prolonged incubation time; moreover, polyurethane blocks covered with media were also applied. Strains were identified by sequencing their 16S rRNA gene after grouping them by amplified rDNA restriction analysis. From each sample, the dominance of Alphaproteobacteria was characteristic though their diversity differed among samples. Members of Actinobacteria, Firmicutes, Beta- and Gamma-proteobacteria, Deinococcus-Thermus, and Bacteroidetes were also identified. Representatives of Deinococcus-Thermus phylum appeared only in the pool water. The largest groups in the pool water belonged to the Tistrella and Chelatococcus genera. The most dominant member in the well water was a new taxon, its similarity to Hartmannibacter diazotrophicus as closest relative was 93.93%.

Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

NASA Astrophysics Data System (ADS)

Freudenthal, Bret D.; Beard, William A.; Perera, Lalith; Shock, David D.; Kim, Taejin; Schlick, Tamar; Wilson, Samuel H.

2015-01-01

Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

Metastatic site location influences the diagnostic accuracy of ctDNA EGFR- mutation testing in NSCLC patients: a pooled analysis.

PubMed

Passiglia, Francesco; Rizzo, Sergio; Rolfo, Christian; Galvano, Antonio; Bronte, Enrico; Incorvaia, Lorena; Listi, Angela; Barraco, Nadia; Castiglia, Marta; Calo, Valentina; Bazan, Viviana; Russo, Antonio

2018-03-08

Recent studies evaluated the diagnostic accuracy of circulating tumor DNA (ctDNA) in the detection of epidermal growth factor receptor (EGFR) mutations from plasma of NSCLC patients, overall showing a high concordance as compared to standard tissue genotyping. However it is less clear if the location of metastatic site may influence the ability to identify EGFR mutations in plasma. This pooled analysis aims to evaluate the association between the metastatic site location and the sensitivity of ctDNA analysis in detecting EGFR mutations in NSCLC patients. Data from all published studies, evaluating the sensitivity of plasma-based EGFR-mutation testing, stratified by metastatic site location (extrathoracic (M1b) vs intrathoracic (M1a)) were collected by searching in PubMed, Cochrane Library, American Society of Clinical Oncology, and World Conference of Lung Cancer, meeting proceedings. Pooled Odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated for the ctDNA analysis sensitivity, according to metastatic site location. A total of ten studies, with 1425 patients, were eligible. Pooled analysis showed that the sensitivity of ctDNA-based EGFR-mutation testing is significantly higher in patients with M1b vs M1a disease (OR: 5.09; 95% CIs: 2.93 - 8.84). A significant association was observed for both EGFR-activating (OR: 4.30, 95% CI: 2.35-7.88) and resistant T790M mutations (OR: 11.89, 95% CI: 1.45-97.22), regardless of the use of digital-PCR (OR: 5.85, 95% CI: 3.56-9.60) or non-digital PCR technologies (OR: 2.96, 95% CI: 2.24-3.91). These data suggest that the location of metastatic sites significantly influences the diagnostic accuracy of ctDNA analysis in detecting EGFR mutations in NSCLC patients. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Bartonella spp. DNA Associated with Biting Flies from California

PubMed Central

Chung, Crystal Y.; Kasten, Rickie W.; Paff, Sandra M.; Van Horn, Brian A.; Vayssier-Taussat, Muriel; Boulouis, Henri-Jean

2004-01-01

Bartonella DNA was investigated in 104 horn flies (Haematobia spp.), 60 stable flies (Stomoxys spp.), 11 deer flies (Chrysops spp.), and 11 horse flies (Tabanus spp.) collected on cattle in California. Partial sequencing indicated B. bovis DNA in the horn fly pool and B. henselae type M DNA in one stable fly. PMID:15324557

MTH1 deficiency selectively increases non-cytotoxic oxidative DNA damage in lung cancer cells: more bad news than good?

PubMed

Abbas, Hussein H K; Alhamoudi, Kheloud M H; Evans, Mark D; Jones, George D D; Foster, Steven S

2018-04-16

Targeted therapies are based on exploiting cancer-cell-specific genetic features or phenotypic traits to selectively kill cancer cells while leaving normal cells unaffected. Oxidative stress is a cancer hallmark phenotype. Given that free nucleotide pools are particularly vulnerable to oxidation, the nucleotide pool sanitising enzyme, MTH1, is potentially conditionally essential in cancer cells. However, findings from previous MTH1 studies have been contradictory, meaning the relevance of MTH1 in cancer is still to be determined. Here we ascertained the role of MTH1 specifically in lung cancer cell maintenance, and the potential of MTH1 inhibition as a targeted therapy strategy to improve lung cancer treatments. Using siRNA-mediated knockdown or small-molecule inhibition, we tested the genotoxic and cytotoxic effects of MTH1 deficiency on H23 (p53-mutated), H522 (p53-mutated) and A549 (wildtype p53) non-small cell lung cancer cell lines relative to normal MRC-5 lung fibroblasts. We also assessed if MTH1 inhibition augments current therapies. MTH1 knockdown increased levels of oxidatively damaged DNA and DNA damage signaling alterations in all lung cancer cell lines but not normal fibroblasts, despite no detectable differences in reactive oxygen species levels between any cell lines. Furthermore, MTH1 knockdown reduced H23 cell proliferation. However, unexpectedly, it did not induce apoptosis in any cell line or enhance the effects of gemcitabine, cisplatin or radiation in combination treatments. Contrastingly, TH287 and TH588 MTH1 inhibitors induced apoptosis in H23 and H522 cells, but only increased oxidative DNA damage levels in H23, indicating that they kill cells independently of DNA oxidation and seemingly via MTH1-distinct mechanisms. MTH1 has a NSCLC-specific p53-independent role for suppressing DNA oxidation and genomic instability, though surprisingly the basis of this may not be reactive-oxygen-species-associated oxidative stress. Despite this, overall our cell viability data indicates that targeting MTH1 will likely not be an across-the-board effective NSCLC therapeutic strategy; rather it induces non-cytotoxic DNA damage that could promote cancer heterogeneity and evolution.

DNA Precursor Metabolism and Mitochondrial Genome Stability

DTIC Science & Technology

2003-04-01

mitochondrial DNA replication , to learn how the pool sizes are regulated, and to understand how perturbations of normal dNTP metabolism within the...mitochondria raises the possibility, however unlikely, that it is serving a function in addition to its role in DNA replication . The literature on non-DNA...is below since many authors do not follow the 200 word limit 14. SUBJECT TERMS Mitochondria, Genome stability, DNA precursors, Mitochondrial DNA

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

PubMed

Deuerling, Elke; Patzelt, Holger; Vorderwülbecke, Sonja; Rauch, Thomas; Kramer, Günter; Schaffitzel, Elke; Mogk, Axel; Schulze-Specking, Agnes; Langen, Hanno; Bukau, Bernd

2003-03-01

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

Occult HBV infection in HIV-infected adults and evaluation of pooled NAT for HBV.

PubMed

Dinesha, T R; Boobalan, J; Sivamalar, S; Subashini, D; Solomon, S S; Murugavel, K G; Balakrishnan, P; Smith, D M; Saravanan, S

2018-06-01

The study aimed to determine the prevalence of occult hepatitis B virus infection among HIV-infected persons and to evaluate the use of a pooling strategy to detect occult HBV infection in the setting of HIV infection. Five hundred and two HIV-positive individuals were tested for HBV, occult HBV and hepatitis C and D with serologic and nucleic acid testing (NAT). We also evaluated a pooled NAT strategy for screening occult HBV infection among the HIV-positive individuals. The prevalence of HBV infection among HIV-positive individuals was 32 (6.4%), and occult HBV prevalence was 10%. The pooling HBV NAT had a sensitivity of 66.7% and specificity of 100%, compared to HBV DNA NAT of individual samples. In conclusion, this study found a high prevalence of occult HBV infection among our HIV-infected population. We also demonstrated that pooled HBV NAT is highly specific, moderately sensitive and cost-effective. As conventional HBV viral load assays are expensive in resource-limited settings such as India, pooled HBV DNA NAT might be a good way for detecting occult HBV infection and will reduce HBV-associated complications. © 2018 John Wiley & Sons Ltd.

Photo-triggered recognition between host and guest compounds in a giant vesicle encapsulating photo-pierceable vesicles.

PubMed

Suzuki, Kentaro; Machida, Kotaro; Yamaguchi, Kazuo; Sugawara, Tadashi

2018-01-01

Here, we used centrifugal precipitation to construct a giant vesicle (GV) encapsulating smaller giant vesicles (GV-in-GV) which comprises a photo-resistant outer GV and a photo-pierceable inner GV; the outer GV contained a fluorescent probe (SYBR Green I) in its inner water pool, and the inner GV contained double-stranded DNA (dsDNA) in its inner water pool. The phospholipid membrane of the inner GV was made photo-pierceable by inclusion of ca. 15mol% of a caged phospholipid in its membrane. Immediately after exposure of the GV-in-GVs to UV irradiation, strong fluorescence was detected in the inner water pool of the outer GV, indicating that dsDNA had been released from the inner GV and had complexed with the fluorescent probe. These dynamics can be recognized as a macroscopic representation of the molecular level function of a caged compound. Copyright © 2017 Elsevier B.V. All rights reserved.

DNA pooling: a comprehensive, multi-stage association analysis of ACSL6 and SIRT5 polymorphisms in schizophrenia.

PubMed

Chowdari, K V; Northup, A; Pless, L; Wood, J; Joo, Y H; Mirnics, K; Lewis, D A; Levitt, P R; Bacanu, S-A; Nimgaonkar, V L

2007-04-01

Many candidate gene association studies have evaluated incomplete, unrepresentative sets of single nucleotide polymorphisms (SNPs), producing non-significant results that are difficult to interpret. Using a rapid, efficient strategy designed to investigate all common SNPs, we tested associations between schizophrenia and two positional candidate genes: ACSL6 (Acyl-Coenzyme A synthetase long-chain family member 6) and SIRT5 (silent mating type information regulation 2 homologue 5). We initially evaluated the utility of DNA sequencing traces to estimate SNP allele frequencies in pooled DNA samples. The mean variances for the DNA sequencing estimates were acceptable and were comparable to other published methods (mean variance: 0.0008, range 0-0.0119). Using pooled DNA samples from cases with schizophrenia/schizoaffective disorder (Diagnostic and Statistical Manual of Mental Disorders edition IV criteria) and controls (n=200, each group), we next sequenced all exons, introns and flanking upstream/downstream sequences for ACSL6 and SIRT5. Among 69 identified SNPs, case-control allele frequency comparisons revealed nine suggestive associations (P<0.2). Each of these SNPs was next genotyped in the individual samples composing the pools. A suggestive association with rs 11743803 at ACSL6 remained (allele-wise P=0.02), with diminished evidence in an extended sample (448 cases, 554 controls, P=0.062). In conclusion, we propose a multi-stage method for comprehensive, rapid, efficient and economical genetic association analysis that enables simultaneous SNP detection and allele frequency estimation in large samples. This strategy may be particularly useful for research groups lacking access to high throughput genotyping facilities. Our analyses did not yield convincing evidence for associations of schizophrenia with ACSL6 or SIRT5.

Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments

PubMed Central

Sun, Kun; Jiang, Peiyong; Chan, K. C. Allen; Wong, John; Cheng, Yvonne K. Y.; Liang, Raymond H. S.; Chan, Wai-kong; Ma, Edmond S. K.; Chan, Stephen L.; Cheng, Suk Hang; Chan, Rebecca W. Y.; Tong, Yu K.; Ng, Simon S. M.; Wong, Raymond S. M.; Hui, David S. C.; Leung, Tse Ngong; Leung, Tak Y.; Lai, Paul B. S.; Chiu, Rossa W. K.; Lo, Yuk Ming Dennis

2015-01-01

Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma. PMID:26392541

Assessment of the role of DNA repair in damaged forensic samples.

PubMed

Ambers, Angie; Turnbough, Meredith; Benjamin, Robert; King, Jonathan; Budowle, Bruce

2014-11-01

Previous studies on DNA damage and repair have involved in vitro laboratory procedures that induce a single type of lesion in naked templates. Although repair of singular, sequestered types of DNA damage has shown some success, forensic and ancient specimens likely contain a number of different types of lesions. This study sought to (1) develop protocols to damage DNA in its native state, (2) generate a pool of candidate samples for repair that more likely emulate authentic forensic samples, and (3) assess the ability of the PreCR(TM) Repair Mix to repair the resultant lesions. Complexed, native DNA is more difficult to damage than naked DNA. Modified procedures included the use of higher concentrations and longer exposure times. Three types of samples, those that demonstrated damage based on short tandem repeat (STR) profile signals, were selected for repair experiments: environmentally damaged bloodstains, bleach-damaged whole blood, and human skeletal remains. Results showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR(TM) assay. The data suggest that the use of PreCR in casework should be considered with caution due to the assay's varied results.

Serological and molecular investigation of Ehrlichia spp. and Anaplasma spp. in ticks and blood of dogs, in the Thrace Region of Turkey.

PubMed

Çetinkaya, Handan; Matur, Erdal; Akyazi, İbrahim; Ekiz, Elif Ergul; Aydin, Levent; Toparlak, Mufit

2016-07-01

In recent years, tick-borne diseases like ehrlichiosis and anaplasmosis became widespread worldwide threatening the health of both human and companion animals. Therefore, the aim of this study was to determine the presence of Anaplasma spp., and Ehrlichia spp. in dogs and ticks in the Thrace Region of Turkey. A total of 400 blood samples and 912 ticks were collected from dogs living in shelters that are located in four cities (Istanbul, Edirne, Tekirdag and Kirklareli) of the Thrace Region. Blood and buffy coat smears were prepared for microscopic examination. Hematologic and serologic analyses were performed using cell counter and commercial Snap3Dx test kit, respectively. Eight hundred fifty of collected ticks were classified as Rhipicephalus sanguineus, 33 as Rhipicephalus turanicus and 29 as Ixodes ricinus. After DNA extraction from blood samples and pooled ticks (127 tick pools, in total), nested PCR was performed to detect the DNA of Anaplasma spp., and Ehrlichia spp. The seroprevalence of Ehrlichia canis was 27.25% (109) by Snap3Dx test and the total molecular positivity was 11.75% (47) in dog blood samples and 21.25% (27) in tick pools by nested PCR. The frequencies of the infected blood samples with E. canis, Anaplasma phagocytophilum and Anaplasma platys were detected as 6%, 4% and 6%, respectively. E. canis and A. platys were detected in R. sanguineus pools with a ratio of 15.75% and 0.7%, respectively. In addition, A. platys was also detected in R. turanicus pools (0.7%). A. phagocytophilum was found only in I. ricinus pools (3.93%). Morulae of three species were detected in buffy coat and blood smears. While anemia was observed in dogs infected with E. canis and co-infected (with one or more species), thrombocytopenia was observed only in co-infected dogs. This is the first study providing evidence for the presence of Anaplasma spp. and Ehrlichia spp. in dogs and ticks in the Thrace Region of Turkey. Based on the results of the tests used in this study, we recommend the combined use of serologic, molecular, cytologic, hematologic analyses and physical examination of tick exposure for an accurate diagnosis of ehrlichiosis and anaplasmosis. Copyright © 2016 Elsevier GmbH. All rights reserved.

Complete Mitochondrial DNA Analysis of Eastern Eurasian Haplogroups Rarely Found in Populations of Northern Asia and Eastern Europe

PubMed Central

Derenko, Miroslava; Malyarchuk, Boris; Denisova, Galina; Perkova, Maria; Rogalla, Urszula; Grzybowski, Tomasz; Khusnutdinova, Elza; Dambueva, Irina; Zakharov, Ilia

2012-01-01

With the aim of uncovering all of the most basal variation in the northern Asian mitochondrial DNA (mtDNA) haplogroups, we have analyzed mtDNA control region and coding region sequence variation in 98 Altaian Kazakhs from southern Siberia and 149 Barghuts from Inner Mongolia, China. Both populations exhibit the prevalence of eastern Eurasian lineages accounting for 91.9% in Barghuts and 60.2% in Altaian Kazakhs. The strong affinity of Altaian Kazakhs and populations of northern and central Asia has been revealed, reflecting both influences of central Asian inhabitants and essential genetic interaction with the Altai region indigenous populations. Statistical analyses data demonstrate a close positioning of all Mongolic-speaking populations (Mongolians, Buryats, Khamnigans, Kalmyks as well as Barghuts studied here) and Turkic-speaking Sojots, thus suggesting their origin from a common maternal ancestral gene pool. In order to achieve a thorough coverage of DNA lineages revealed in the northern Asian matrilineal gene pool, we have completely sequenced the mtDNA of 55 samples representing haplogroups R11b, B4, B5, F2, M9, M10, M11, M13, N9a and R9c1, which were pinpointed from a massive collection (over 5000 individuals) of northern and eastern Asian, as well as European control region mtDNA sequences. Applying the newly updated mtDNA tree to the previously reported northern Asian and eastern Asian mtDNA data sets has resolved the status of the poorly classified mtDNA types and allowed us to obtain the coalescence age estimates of the nodes of interest using different calibrated rates. Our findings confirm our previous conclusion that northern Asian maternal gene pool consists of predominantly post-LGM components of eastern Asian ancestry, though some genetic lineages may have a pre-LGM/LGM origin. PMID:22363811

Molecular genetic studies of natives on Easter Island: evidence of an early European and Amerindian contribution to the Polynesian gene pool.

PubMed

Lie, B A; Dupuy, B M; Spurkland, A; Fernández-Viña, M A; Hagelberg, E; Thorsby, E

2007-01-01

Most archaeological and linguistic evidence suggest a Polynesian origin of the population of Easter Island (Rapanui), and this view has been supported by the identification of Polynesian mitochondrial DNA (mtDNA) polymorphisms in prehistoric skeletal remains. However, some evidence of an early South American contact also exists (the sweet potato, bottle gourd etc.), but genetic studies have so far failed to show an early Amerindian contribution to the gene pool on Easter Island. To address this issue, we analyzed mtDNA and Y chromosome markers and performed high-resolution human leukocyte antigen (HLA) genotyping of DNA harvested from previously collected sera of 48 reputedly nonadmixed native Easter Islanders. All individuals carried mtDNA types and HLA alleles previously found in Polynesia, and most men carried Y chromosome markers of Polynesian origin, providing further evidence of a Polynesian origin of the population of Easter Island. A few individuals carried HLA alleles and/or Y chromosome markers of European origin. More interestingly, some individuals carried the HLA alleles A*0212 and B*3905, which are of typical Amerindian origin. The genealogy of some of the individuals carrying these non-Polynesian HLA alleles and their haplotypic backgrounds suggest an introduction into Easter Island in the early 1800s, or earlier. Thus, there may have been an early European and Amerindian contribution to the Polynesian gene pool of Easter Island.

Systematic investigation of the relationship between high myopia and polymorphisms of the MMP2, TIMP2, and TIMP3 genes by a DNA pooling approach.

PubMed

Leung, Kim Hung; Yiu, Wai Chi; Yap, Maurice K H; Ng, Po Wah; Fung, Wai Yan; Sham, Pak Chung; Yip, Shea Ping

2011-06-01

This study examined the relationship between high myopia and three myopia candidate genes--matrix metalloproteinase 2 (MMP2) and tissue inhibitor of metalloproteinase-2 and -3 (TIMP2 and TIMP3)--involved in scleral remodeling. Recruited for the study were unrelated adult Han Chinese who were high myopes (spherical equivalent, ≤ -6.0 D in both eyes; cases) and emmetropes (within ±1.0 D in both eyes; controls). Sample set 1 had 300 cases and 300 controls, and sample set 2 had 356 cases and 354 controls. Forty-nine tag single-nucleotide polymorphisms (SNPs) were selected from these candidate genes. The first stage was an initial screen of six case pools and six control pools constructed from sample set 1, each pool consisting of 50 distinct subjects of the same affection status. In the second stage, positive SNPs from the first stage were confirmed by genotyping individual samples forming the DNA pools. In the third stage, positive SNPs from stage 2 were replicated, with sample set 2 genotyped individually. Of the 49 SNPs screened by DNA pooling, three passed the lenient threshold of P < 0.10 (nested ANOVA) and were followed up by individual genotyping. Of the three SNPs genotyped, two TIMP3 SNPs were found to be significantly associated with high myopia by single-marker or haplotype analysis. However, the initial positive results could not be replicated by sample set 2. MMP2, TIPM2, and TIMP3 genes were not associated with high myopia in this Chinese sample and hence are unlikely to play a major role in the genetic susceptibility to high myopia.

Using Pool-seq to Search for Genomic Regions Affected by Hybrid Inviability in the copepod T. californicus.

PubMed

Lima, Thiago G; Willett, Christopher S

2018-05-11

The formation of reproductive barriers between allopatric populations involves the accumulation of incompatibilities that lead to intrinsic postzygotic isolation. The evolution of these incompatibilities is usually explained by the Dobzhansky-Muller model, where epistatic interactions that arise within the diverging populations, lead to deleterious interactions when they come together in a hybrid genome. These incompatibilities can lead to hybrid inviability, killing individuals with certain genotypic combinations, and causing the population's allele frequency to deviate from Mendelian expectations. Traditionally, hybrid inviability loci have been detected by genotyping individuals at different loci across the genome. However, this method becomes time consuming and expensive as the number of markers or individuals increases. Here, we test if a Pool-seq method can be used to scan the genome of F2 hybrids to detect genomic regions that are affected by hybrid inviability. We survey the genome of hybrids between 2 populations of the copepod Tigriopus californicus, and show that this method has enough power to detect even small changes in allele frequency caused by hybrid inviability. We show that allele frequency estimates in Pool-seq can be affected by the sampling of alleles from the pool of DNA during the library preparation and sequencing steps and that special considerations must be taken when aligning hybrid reads to a reference when the populations/species are divergent.

Macromolecular Synthesis During the Germination of Saccharomyces cerevisiae Spores

PubMed Central

Rousseau, Paul; Halvorson, Harlyn O.

1973-01-01

After the dormancy of Saccharomyces cerevisiae ascospores had been broken, the synthesis of proteins was observed first, followed rapidly by synthesis of ribonucleic acid (RNA) and much later by deoxyribonucleic acid (DNA) synthesis. Phosphoglucomutase activity increased in a periodic (step) fashion, whereas the activity of five other enzymes increased linearly during germination and outgrowth. The rate of synthesis of these enzymes was highest at about the period of DNA replication. The amino acid pools of dormant spores contained high levels of proline, glutamic acid, and histidine. At 2 h after onset of germination, the pools of phenylalanine and methionine had disappeared and the other components had decreased significantly. By 3.5 h, with the exception of proline and cystine, most amino acid pool components had significantly increased. PMID:4570780

Role of Escherichia coli dnaG function in coliphage M13 DNA synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dasgupta, S.; Mitra, S.

Examination of the role of Escherichia coli dnaG function in different stages of M13 phage DNA synthesis by ultracentrifugal analysis of intracellular phage DNA in a thermosensitive dnaG mutant shows that: (a) the formation of parental double-strand replicative-form DNA (rfDNA) from the infecting virus is independent of dnaG function; (b) the synthesis of progeny rfDNA requires dnaG product; (c) after a pool of rfDNA is made up, dnaG function is not required for the progeny single-strand DNA (ssDNA) synthesis. The ssDNAs produced under nonpermissive condition are mostly circular and biologically functional.

Gene pools in wild Lima bean (Phaseolus lunatus L.) from the Americas: evidences for an Andean origin and past migrations.

PubMed

Serrano-Serrano, Martha L; Hernández-Torres, Jorge; Castillo-Villamizar, Genis; Debouck, Daniel G; Sánchez, María I Chacón

2010-01-01

The aims of this research were to assess the genetic structure of wild Phaseolus lunatus L. in the Americas and the hypothesis of a relatively recent Andean origin of the species. For this purpose, nuclear and non-coding chloroplast DNA markers were analyzed in a collection of 59 wild Lima bean accessions and six allied species. Twenty-three chloroplast and 28 nuclear DNA haplotypes were identified and shown to be geographically structured. Three highly divergent wild Lima bean gene pools, AI, MI, and MII, with mostly non-overlapping geographic ranges, are proposed. The results support an Andean origin of wild Lima beans during Pleistocene times and an early divergence of the three gene pools at an age that is posterior to completion of the Isthmus of Panama and major Andean orogeny. Gene pools would have evolved and reached their current geographic distribution mainly in isolation and therefore are of high priority for conservation and breeding programs.

A statistical method for the detection of variants from next-generation resequencing of DNA pools.

PubMed

Bansal, Vikas

2010-06-15

Next-generation sequencing technologies have enabled the sequencing of several human genomes in their entirety. However, the routine resequencing of complete genomes remains infeasible. The massive capacity of next-generation sequencers can be harnessed for sequencing specific genomic regions in hundreds to thousands of individuals. Sequencing-based association studies are currently limited by the low level of multiplexing offered by sequencing platforms. Pooled sequencing represents a cost-effective approach for studying rare variants in large populations. To utilize the power of DNA pooling, it is important to accurately identify sequence variants from pooled sequencing data. Detection of rare variants from pooled sequencing represents a different challenge than detection of variants from individual sequencing. We describe a novel statistical approach, CRISP [Comprehensive Read analysis for Identification of Single Nucleotide Polymorphisms (SNPs) from Pooled sequencing] that is able to identify both rare and common variants by using two approaches: (i) comparing the distribution of allele counts across multiple pools using contingency tables and (ii) evaluating the probability of observing multiple non-reference base calls due to sequencing errors alone. Information about the distribution of reads between the forward and reverse strands and the size of the pools is also incorporated within this framework to filter out false variants. Validation of CRISP on two separate pooled sequencing datasets generated using the Illumina Genome Analyzer demonstrates that it can detect 80-85% of SNPs identified using individual sequencing while achieving a low false discovery rate (3-5%). Comparison with previous methods for pooled SNP detection demonstrates the significantly lower false positive and false negative rates for CRISP. Implementation of this method is available at http://polymorphism.scripps.edu/~vbansal/software/CRISP/.

DNA-Catalyzed DNA Cleavage by a Radical Pathway with Well-Defined Products.

PubMed

Lee, Yujeong; Klauser, Paul C; Brandsen, Benjamin M; Zhou, Cong; Li, Xinyi; Silverman, Scott K

2017-01-11

We describe an unprecedented DNA-catalyzed DNA cleavage process in which a radical-based reaction pathway cleanly results in excision of most atoms of a specific guanosine nucleoside. Two new deoxyribozymes (DNA enzymes) were identified by in vitro selection from N 40 or N 100 random pools initially seeking amide bond hydrolysis, although they both cleave simple single-stranded DNA oligonucleotides. Each deoxyribozyme generates both superoxide (O 2 -• or HOO • ) and hydrogen peroxide (H 2 O 2 ) and leads to the same set of products (3'-phosphoglycolate, 5'-phosphate, and base propenal) as formed by the natural product bleomycin, with product assignments by mass spectrometry and colorimetric assay. We infer the same mechanistic pathway, involving formation of the C4' radical of the guanosine nucleoside that is subsequently excised. Consistent with a radical pathway, glutathione fully suppresses catalysis. Conversely, adding either superoxide or H 2 O 2 from the outset strongly enhances catalysis. The mechanism of generation and involvement of superoxide and H 2 O 2 by the deoxyribozymes is not yet defined. The deoxyribozymes do not require redox-active metal ions and function with a combination of Zn 2+ and Mg 2+ , although including Mn 2+ increases the activity, and Mn 2+ alone also supports catalysis. In contrast to all of these observations, unrelated DNA-catalyzed radical DNA cleavage reactions require redox-active metals and lead to mixtures of products. This study reports an intriguing example of a well-defined, DNA-catalyzed, radical reaction process that cleaves single-stranded DNA and requires only redox-inactive metal ions.

Detection of Leishmania amazonensis and Leishmania braziliensis in Culicoides (Diptera, Ceratopogonidae) in an endemic area of cutaneous leishmaniasis in the Brazilian Amazonia.

PubMed

Rebêlo, José Manuel Macário; Rodrigues, Bruno Leite; Bandeira, Maria da Conceição Abreu; Moraes, Jorge Luiz Pinto; Fonteles, Raquel Silva; Pereira, Silma Regina Ferreira

2016-12-01

Biting midges in the genus Culicoides act as vectors of arboviruses throughout the world and as vectors of filariasis in Latin America, the Caribbean, and parts of Africa. Although Culicoides spp. are currently not considered to be vectors of Leishmania protozoa, the high abundance of biting midges in areas with active cutaneous leishmaniasis transmission points to the possibility of Culicoides infection by these pathogens. We used PCR to test captured Culicoides species for natural infection with Leishmania spp. We tested 450 Culicoides females, divided into 30 pools of 15 individuals each, as follows: nine pools of C. foxi (135 specimens), seven pools of C. filariferus (105), seven pools of C. insignis (105), five pools of C. ignacioi (75), and two pools of C. flavivenula (30). PCR confirmed the presence of Leishmania braziliensis DNA in C. ignacioi (0.14%), C. insignis (0.14%), and C. foxi (0.11); and Le. amazonensis DNA in C. filariferus (0.14%) and C. flavivenula (0.50%). We conclude that these Culicoides species can be naturally infected, but vector competence and transmission capability must be confirmed in future studies. Our results warrant further investigation into the role of these biting midge species in the leishmaniasis epidemiological cycle. © 2016 The Society for Vector Ecology.

Mitochondrial DNA variation in the Viking age population of Norway.

PubMed

Krzewińska, Maja; Bjørnstad, Gro; Skoglund, Pontus; Olason, Pall Isolfur; Bill, Jan; Götherström, Anders; Hagelberg, Erika

2015-01-19

The medieval Norsemen or Vikings had an important biological and cultural impact on many parts of Europe through raids, colonization and trade, from about AD 793 to 1066. To help understand the genetic affinities of the ancient Norsemen, and their genetic contribution to the gene pool of other Europeans, we analysed DNA markers in Late Iron Age skeletal remains from Norway. DNA was extracted from 80 individuals, and mitochondrial DNA polymorphisms were detected by next-generation sequencing. The sequences of 45 ancient Norwegians were verified as genuine through the identification of damage patterns characteristic of ancient DNA. The ancient Norwegians were genetically similar to previously analysed ancient Icelanders, and to present-day Shetland and Orkney Islanders, Norwegians, Swedes, Scots, English, German and French. The Viking Age population had higher frequencies of K*, U*, V* and I* haplogroups than their modern counterparts, but a lower proportion of T* and H* haplogroups. Three individuals carried haplotypes that are rare in Norway today (U5b1b1, Hg A* and an uncommon variant of H*). Our combined analyses indicate that Norse women were important agents in the overseas expansion and settlement of the Vikings, and that women from the Orkneys and Western Isles contributed to the colonization of Iceland. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Mitochondrial DNA variation in the Viking age population of Norway

PubMed Central

Krzewińska, Maja; Bjørnstad, Gro; Skoglund, Pontus; Olason, Pall Isolfur; Bill, Jan; Götherström, Anders; Hagelberg, Erika

2015-01-01

The medieval Norsemen or Vikings had an important biological and cultural impact on many parts of Europe through raids, colonization and trade, from about AD 793 to 1066. To help understand the genetic affinities of the ancient Norsemen, and their genetic contribution to the gene pool of other Europeans, we analysed DNA markers in Late Iron Age skeletal remains from Norway. DNA was extracted from 80 individuals, and mitochondrial DNA polymorphisms were detected by next-generation sequencing. The sequences of 45 ancient Norwegians were verified as genuine through the identification of damage patterns characteristic of ancient DNA. The ancient Norwegians were genetically similar to previously analysed ancient Icelanders, and to present-day Shetland and Orkney Islanders, Norwegians, Swedes, Scots, English, German and French. The Viking Age population had higher frequencies of K*, U*, V* and I* haplogroups than their modern counterparts, but a lower proportion of T* and H* haplogroups. Three individuals carried haplotypes that are rare in Norway today (U5b1b1, Hg A* and an uncommon variant of H*). Our combined analyses indicate that Norse women were important agents in the overseas expansion and settlement of the Vikings, and that women from the Orkneys and Western Isles contributed to the colonization of Iceland. PMID:25487335

A Specialized Histone H1 Variant Is Required for Adaptive Responses to Complex Abiotic Stress and Related DNA Methylation in Arabidopsis1[OPEN

PubMed Central

Rutowicz, Kinga; Puzio, Marcin; Halibart-Puzio, Joanna; Lirski, Maciej; Kotliński, Maciej; Kroteń, Magdalena A.; Knizewski, Lukasz; Lange, Bartosz; Muszewska, Anna; Śniegowska-Świerk, Katarzyna; Kościelniak, Janusz; Iwanicka-Nowicka, Roksana; Buza, Krisztián; Janowiak, Franciszek; Żmuda, Katarzyna; Jõesaar, Indrek; Laskowska-Kaszub, Katarzyna; Fogtman, Anna; Kollist, Hannes; Zielenkiewicz, Piotr; Tiuryn, Jerzy; Siedlecki, Paweł; Swiezewski, Szymon; Ginalski, Krzysztof; Koblowska, Marta; Archacki, Rafał; Wilczynski, Bartek; Rapacz, Marcin; Jerzmanowski, Andrzej

2015-01-01

Linker (H1) histones play critical roles in chromatin compaction in higher eukaryotes. They are also the most variable of the histones, with numerous nonallelic variants cooccurring in the same cell. Plants contain a distinct subclass of minor H1 variants that are induced by drought and abscisic acid and have been implicated in mediating adaptive responses to stress. However, how these variants facilitate adaptation remains poorly understood. Here, we show that the single Arabidopsis (Arabidopsis thaliana) stress-inducible variant H1.3 occurs in plants in two separate and most likely autonomous pools: a constitutive guard cell-specific pool and a facultative environmentally controlled pool localized in other tissues. Physiological and transcriptomic analyses of h1.3 null mutants demonstrate that H1.3 is required for both proper stomatal functioning under normal growth conditions and adaptive developmental responses to combined light and water deficiency. Using fluorescence recovery after photobleaching analysis, we show that H1.3 has superfast chromatin dynamics, and in contrast to the main Arabidopsis H1 variants H1.1 and H1.2, it has no stable bound fraction. The results of global occupancy studies demonstrate that, while H1.3 has the same overall binding properties as the main H1 variants, including predominant heterochromatin localization, it differs from them in its preferences for chromatin regions with epigenetic signatures of active and repressed transcription. We also show that H1.3 is required for a substantial part of DNA methylation associated with environmental stress, suggesting that the likely mechanism underlying H1.3 function may be the facilitation of chromatin accessibility by direct competition with the main H1 variants. PMID:26351307

Unique Prokaryotic Consortia in Geochemically Distinct Sediments from Red Sea Atlantis II and Discovery Deep Brine Pools

PubMed Central

Siam, Rania; Mustafa, Ghada A.; Sharaf, Hazem; Moustafa, Ahmed; Ramadan, Adham R.; Antunes, Andre; Bajic, Vladimir B.; Stingl, Uli; Marsis, Nardine G. R.; Coolen, Marco J. L.; Sogin, Mitchell; Ferreira, Ari J. S.; Dorry, Hamza El

2012-01-01

The seafloor is a unique environment, which allows insights into how geochemical processes affect the diversity of biological life. Among its diverse ecosystems are deep-sea brine pools - water bodies characterized by a unique combination of extreme conditions. The ‘polyextremophiles’ that constitute the microbial assemblage of these deep hot brines have not been comprehensively studied. We report a comparative taxonomic analysis of the prokaryotic communities of the sediments directly below the Red Sea brine pools, namely, Atlantis II, Discovery, Chain Deep, and an adjacent brine-influenced site. Analyses of sediment samples and high-throughput pyrosequencing of PCR-amplified environmental 16S ribosomal RNA genes (16S rDNA) revealed that one sulfur (S)-rich Atlantis II and one nitrogen (N)-rich Discovery Deep section contained distinct microbial populations that differed from those found in the other sediment samples examined. Proteobacteria, Actinobacteria, Cyanobacteria, Deferribacteres, and Euryarchaeota were the most abundant bacterial and archaeal phyla in both the S- and N-rich sections. Relative abundance-based hierarchical clustering of the 16S rDNA pyrotags assigned to major taxonomic groups allowed us to categorize the archaeal and bacterial communities into three major and distinct groups; group I was unique to the S-rich Atlantis II section (ATII-1), group II was characteristic for the N-rich Discovery sample (DD-1), and group III reflected the composition of the remaining sediments. Many of the groups detected in the S-rich Atlantis II section are likely to play a dominant role in the cycling of methane and sulfur due to their phylogenetic affiliations with bacteria and archaea involved in anaerobic methane oxidation and sulfate reduction. PMID:22916172

Genotoxic Effects in Swimmers Exposed to Disinfection By-products in Indoor Swimming Pools

PubMed Central

Kogevinas, Manolis; Villanueva, Cristina M.; Font-Ribera, Laia; Liviac, Danae; Bustamante, Mariona; Espinoza, Felicidad; Nieuwenhuijsen, Mark J.; Espinosa, Aina; Fernandez, Pilar; DeMarini, David M.; Grimalt, Joan O.; Grummt, Tamara; Marcos, Ricard

2010-01-01

Background Exposure to disinfection by-products (DBPs) in drinking water has been associated with cancer risk. A recent study (Villanueva et al. 2007; Am J Epidemiol 165:148–156) found an increased bladder cancer risk among subjects attending swimming pools relative to those not attending. Objectives We evaluated adults who swam in chlorinated pools to determine whether exposure to DBPs in pool water is associated with biomarkers of genotoxicity. Methods We collected blood, urine, and exhaled air samples from 49 nonsmoking adult volunteers before and after they swam for 40 min in an indoor chlorinated pool. We estimated associations between the concentrations of four trihalomethanes (THMs) in exhaled breath and changes in micronuclei (MN) and DNA damage (comet assay) in peripheral blood lymphocytes before and 1 hr after swimming; urine mutagenicity (Ames assay) before and 2 hr after swimming; and MN in exfoliated urothelial cells before and 2 weeks after swimming. We also estimated associations and interactions with polymorphisms in genes related to DNA repair or to DBP metabolism. Results After swimming, the total concentration of the four THMs in exhaled breath was seven times higher than before swimming. The change in the frequency of micronucleated lymphocytes after swimming increased in association with higher exhaled concentrations of the brominated THMs (p = 0.03 for bromodichloromethane, p = 0.05 for chlorodibromomethane, p = 0.01 for bromoform) but not chloroform. Swimming was not associated with DNA damage detectable by the comet assay. Urine mutagenicity increased significantly after swimming, in association with the higher concentration of exhaled bromoform (p = 0.004). We found no significant associations with changes in micronucleated urothelial cells. Conclusions Our findings support potential genotoxic effects of exposure to DBPs from swimming pools. The positive health effects gained by swimming could be increased by reducing the potential health risks of pool water. PMID:20833606

Synthesis of Mitochondrial DNA Precursors during Myogenesis, an Analysis in Purified C2C12 Myotubes*

PubMed Central

Frangini, Miriam; Franzolin, Elisa; Chemello, Francesco; Laveder, Paolo; Romualdi, Chiara; Bianchi, Vera; Rampazzo, Chiara

2013-01-01

During myogenesis, myoblasts fuse into multinucleated myotubes that acquire the contractile fibrils and accessory structures typical of striated skeletal muscle fibers. To support the high energy requirements of muscle contraction, myogenesis entails an increase in mitochondrial (mt) mass with stimulation of mtDNA synthesis and consumption of DNA precursors (dNTPs). Myotubes are quiescent cells and as such down-regulate dNTP production despite a high demand for dNTPs. Although myogenesis has been studied extensively, changes in dNTP metabolism have not been examined specifically. In differentiating cultures of C2C12 myoblasts and purified myotubes, we analyzed expression and activities of enzymes of dNTP biosynthesis, dNTP pools, and the expansion of mtDNA. Myotubes exibited pronounced post-mitotic modifications of dNTP synthesis with a particularly marked down-regulation of de novo thymidylate synthesis. Expression profiling revealed the same pattern of enzyme down-regulation in adult murine muscles. The mtDNA increased steadily after myoblast fusion, turning over rapidly, as revealed after treatment with ethidium bromide. We individually down-regulated p53R2 ribonucleotide reductase, thymidine kinase 2, and deoxyguanosine kinase by siRNA transfection to examine how a further reduction of these synthetic enzymes impacted myotube development. Silencing of p53R2 had little effect, but silencing of either mt kinase caused 50% mtDNA depletion and an unexpected decrease of all four dNTP pools independently of the kinase specificity. We suggest that during development of myotubes the shortage of even a single dNTP may affect all four pools through dysregulation of ribonucleotide reduction and/or dissipation of the non-limiting dNTPs during unproductive elongation of new DNA chains. PMID:23297407

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

PubMed Central

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

2014-01-01

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

Studies on the Mechanism of Action of Hydrazine-Induced Methylation of DNA Guanne

DTIC Science & Technology

1984-10-03

potent methylating agent , diazomethane (-CH -N+-N). Several in vivo studies were carried out to determine the role of aldehydes in the alkylation of DNA...methylating agent available to interact with DNA. If such a mechanism occurs, it may explain why disulfiram appears to inhibit the alkylation of DNA...a much slower/poorer alkylating agent for DNA. Effect of the 1-Carbon Pool on DNA Methylation in Hydrazine Toxicity: In Vitro In vitro studies were

Multisubstrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities

PubMed Central

Verastegui, Y.; Cheng, J.; Engel, K.; Kolczynski, D.; Mortimer, S.; Lavigne, J.; Montalibet, J.; Romantsov, T.; Hall, M.; McConkey, B. J.; Rose, D. R.; Tomashek, J. J.; Scott, B. R.

2014-01-01

ABSTRACT Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon (12C) or stable-isotope-labeled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the 13C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. PMID:25028422

Diagnostic accuracy of droplet digital PCR for detection of EGFR T790M mutation in circulating tumor DNA

PubMed Central

Tong, Xiang; Wang, Ye; Wang, Chengdi; Jin, Jing; Tian, Panwen; Li, Weimin

2018-01-01

Objectives Although different methods have been established to detect epidermal growth factor receptor (EGFR) T790M mutation in circulating tumor DNA (ctDNA), a wide range of diagnostic accuracy values were reported in previous studies. The aim of this meta-analysis was to provide pooled diagnostic accuracy measures for droplet digital PCR (ddPCR) in the diagnosis of EGFR T790M mutation based on ctDNA. Materials and methods A systematic review and meta-analysis were carried out based on resources from Pubmed, Web of Science, Embase and Cochrane Library up to October 11, 2017. Data were extracted to assess the pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio (NLR), diagnostic OR (DOR), and areas under the summary receiver-operating characteristic curve (SROC). Results Eleven of 311 studies identified have met the including criteria. The sensitivity and specificity of ddPCR for the detection of T790M mutation in ctDNA ranged from 0.0% to 100.0% and 63.2% to 100.0%, respectively. For the pooled analysis, ddPCR had a performance of 70.1% (95% CI, 62.7%–76.7%) sensitivity, 86.9 % (95% CI, 80.6%–91.7%) specificity, 3.67 (95% CI, 2.33–5.79) PLR, 0.41 (95% CI, 0.32–0.55) NLR, and 10.83 (95% CI, 5.86–20.03) DOR, with the area under the SROC curve being 0.82. Conclusion The ddPCR harbored a good performance for detection of EGFR T790M mutation in ctDNA. PMID:29844700

Thymidine kinase 2 (H126N) knockin mice show the essential role of balanced deoxynucleotide pools for mitochondrial DNA maintenance.

PubMed

Akman, Hasan O; Dorado, Beatriz; López, Luis C; García-Cazorla, Angeles; Vilà, Maya R; Tanabe, Lauren M; Dauer, William T; Bonilla, Eduardo; Tanji, Kurenai; Hirano, Michio

2008-08-15

Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2(-/-)) mice developed rapidly progressive weakness after age 10 days and died between ages 2 and 3 weeks. Tk2(-/-) animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype.

Thymidine kinase 2 (H126N) knockin mice show the essential role of balanced deoxynucleotide pools for mitochondrial DNA maintenance

PubMed Central

Akman, Hasan O.; Dorado, Beatriz; López, Luis C.; García-Cazorla, Ángeles; Vilà, Maya R.; Tanabe, Lauren M.; Dauer, William T.; Bonilla, Eduardo; Tanji, Kurenai; Hirano, Michio

2008-01-01

Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2−/−) mice developed rapidly progressive weakness after age 10 days and died between ages 2 and 3 weeks. Tk2−/− animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype. PMID:18467430

Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

PubMed

Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

2014-01-15

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

Large-scale pattern of genetic differentiation within African rainforest trees: insights on the roles of ecological gradients and past climate changes on the evolution of Erythrophleum spp (Fabaceae).

PubMed

Duminil, Jerome; Brown, Richard P; Ewédjè, Eben-Ezer B K; Mardulyn, Patrick; Doucet, Jean-Louis; Hardy, Olivier J

2013-09-12

The evolutionary events that have shaped biodiversity patterns in the African rainforests are still poorly documented. Past forest fragmentation and ecological gradients have been advocated as important drivers of genetic differentiation but their respective roles remain unclear. Using nuclear microsatellites (nSSRs) and chloroplast non-coding sequences (pDNA), we characterised the spatial genetic structure of Erythrophleum (Fabaceae) forest trees in West and Central Africa (Guinea Region, GR). This widespread genus displays a wide ecological amplitude and taxonomists recognize two forest tree species, E. ivorense and E. suaveolens, which are difficult to distinguish in the field and often confused. Bayesian-clustering applied on nSSRs of a blind sample of 648 specimens identified three major gene pools showing no or very limited introgression. They present parapatric distributions correlated to rainfall gradients and forest types. One gene pool is restricted to coastal evergreen forests and corresponds to E. ivorense; a second one is found in gallery forests from the dry forest zone of West Africa and North-West Cameroon and corresponds to West-African E. suaveolens; the third gene pool occurs in semi-evergreen forests and corresponds to Central African E. suaveolens. These gene pools have mostly unique pDNA haplotypes but they do not form reciprocally monophyletic clades. Nevertheless, pDNA molecular dating indicates that the divergence between E. ivorense and Central African E. suaveolens predates the Pleistocene. Further Bayesian-clustering applied within each major gene pool identified diffuse genetic discontinuities (minor gene pools displaying substantial introgression) at a latitude between 0 and 2°N in Central Africa for both species, and at a longitude between 5° and 8°E for E. ivorense. Moreover, we detected evidence of past population declines which are consistent with historical habitat fragmentation induced by Pleistocene climate changes. Overall, deep genetic differentiation (major gene pools) follows ecological gradients that may be at the origin of speciation, while diffuse differentiation (minor gene pools) are tentatively interpreted as the signature of past forest fragmentation induced by past climate changes.

Large-scale pattern of genetic differentiation within African rainforest trees: insights on the roles of ecological gradients and past climate changes on the evolution of Erythrophleum spp (Fabaceae)

PubMed Central

2013-01-01

Background The evolutionary events that have shaped biodiversity patterns in the African rainforests are still poorly documented. Past forest fragmentation and ecological gradients have been advocated as important drivers of genetic differentiation but their respective roles remain unclear. Using nuclear microsatellites (nSSRs) and chloroplast non-coding sequences (pDNA), we characterised the spatial genetic structure of Erythrophleum (Fabaceae) forest trees in West and Central Africa (Guinea Region, GR). This widespread genus displays a wide ecological amplitude and taxonomists recognize two forest tree species, E. ivorense and E. suaveolens, which are difficult to distinguish in the field and often confused. Results Bayesian-clustering applied on nSSRs of a blind sample of 648 specimens identified three major gene pools showing no or very limited introgression. They present parapatric distributions correlated to rainfall gradients and forest types. One gene pool is restricted to coastal evergreen forests and corresponds to E. ivorense; a second one is found in gallery forests from the dry forest zone of West Africa and North-West Cameroon and corresponds to West-African E. suaveolens; the third gene pool occurs in semi-evergreen forests and corresponds to Central African E. suaveolens. These gene pools have mostly unique pDNA haplotypes but they do not form reciprocally monophyletic clades. Nevertheless, pDNA molecular dating indicates that the divergence between E. ivorense and Central African E. suaveolens predates the Pleistocene. Further Bayesian-clustering applied within each major gene pool identified diffuse genetic discontinuities (minor gene pools displaying substantial introgression) at a latitude between 0 and 2°N in Central Africa for both species, and at a longitude between 5° and 8°E for E. ivorense. Moreover, we detected evidence of past population declines which are consistent with historical habitat fragmentation induced by Pleistocene climate changes. Conclusions Overall, deep genetic differentiation (major gene pools) follows ecological gradients that may be at the origin of speciation, while diffuse differentiation (minor gene pools) are tentatively interpreted as the signature of past forest fragmentation induced by past climate changes. PMID:24028582

Genome-wide Association Study for Ovarian Cancer Susceptibility using Pooled DNA

PubMed Central

Lu, Yi; Chen, Xiaoqing; Beesley, Jonathan; Johnatty, Sharon E.; deFazio, Anna; Lambrechts, Sandrina; Lambrechts, Diether; Despierre, Evelyn; Vergotes, Ignace; Chang-Claude, Jenny; Hein, Rebecca; Nickels, Stefan; Wang-Gohrke, Shan; Dörk, Thilo; Dürst, Matthias; Antonenkova, Natalia; Bogdanova, Natalia; Goodman, Marc T.; Lurie, Galina; Wilkens, Lynne R.; Carney, Michael E.; Butzow, Ralf; Nevanlinna, Heli; Heikkinen, Tuomas; Leminen, Arto; Kiemeney, Lambertus A.; Massuger, Leon F.A.G.; van Altena, Anne M.; Aben, Katja K.; Kjaer, Susanne Krüger; Høgdall, Estrid; Jensen, Allan; Brooks-Wilson, Angela; Le, Nhu; Cook, Linda; Earp, Madalene; Kelemen, Linda; Easton, Douglas; Pharoah, Paul; Song, Honglin; Tyrer, Jonathan; Ramus, Susan; Menon, Usha; Gentry-Maharaj, Alexandra; Gayther, Simon A.; Bandera, Elisa V.; Olson, Sara H.; Orlow, Irene; Rodriguez-Rodriguez, Lorna

2013-01-01

Recent genome-wide association studies (GWAS) have identified four low-penetrance ovarian cancer susceptibility loci. We hypothesized that further moderate or low penetrance variants exist among the subset of SNPs not well tagged by the genotyping arrays used in the previous studies which would account for some of the remaining risk. We therefore conducted a time- and cost-effective stage 1 GWAS on 342 invasive serous cases and 643 controls genotyped on pooled DNA using the high density Illumina 1M-Duo array. We followed up 20 of the most significantly associated SNPs, which are not well tagged by the lower density arrays used by the published GWAS, and genotyping them on individual DNA. Most of the top 20 SNPs were clearly validated by individually genotyping the samples used in the pools. However, none of the 20 SNPs replicated when tested for association in a much larger stage 2 set of 4,651 cases and 6,966 controls from the Ovarian Cancer Association Consortium. Given that most of the top 20 SNPs from pooling were validated in the same samples by individual genotyping, the lack of replication is likely to be due to the relatively small sample size in our stage 1 GWAS rather than due to problems with the pooling approach. We conclude that there are unlikely to be any moderate or large effects on ovarian cancer risk untagged by the less dense arrays. However our study lacked power to make clear statements on the existence of hitherto untagged small effect variants. PMID:22794196

Diagnosis of duck plague in waterfowl by polymerase chain reaction

USGS Publications Warehouse

Hansen, W.R.; Nashold, S.W.; Docherty, D.E.; Brown, S.E.; Knudson, D.L.

2000-01-01

A recently developed polymerase chain reaction (PCR) assay was used for diagnosis of duck plague in waterfowl tissues from past and current cases of waterfowl mortality and to identify duck plague virus in combined cloacal/oral-pharyngeal swab samples from healthy mallards (Anas platyrhynchos) after a disease outbreak. The PCR was able to detect viral DNA from all the individual or pooled tissues assayed from 10 waterfowl, including liver and spleen samples from three Muscovy ducks (Cairina moschata domesticus) that did not yield virus isolates. The strong staining intensity of the PCR products from the waterfowl tissues indicated that large amounts of virus were present, even when virus was not isolated. Duck plague DNA was also detected in a cloacal swab sample from a wood duck (Aix sponsa) carcass submitted for diagnosis. The PCR assay identified duck plague DNA in 13 swab samples that produced virus isolates from carrier mallards sampled in 1981 after a duck plague die-off. The duck plague PCR clearly demonstrated the ability to quickly diagnose duck plague in suspect mortality cases and to detect virus shed by carrier waterfowl.

High-sensitivity stable-isotope probing by a quantitative terminal restriction fragment length polymorphism protocol.

PubMed

Andeer, Peter; Strand, Stuart E; Stahl, David A

2012-01-01

Stable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)-TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli, Rhodococcus, Variovorax, and Microbacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.

Chromosomal damage and EROD induction in tree swallows (Tachycineta bicolor) along the Upper Mississippi River, Minnesota, USA

USGS Publications Warehouse

Emilie Bigorgne,; Custer, Thomas W.; Dummer, Paul; Erickson, Richard A.; Karouna-Renier, Natalie K.; Schultz, Sandra; Custer, Christine M.; Thogmartin, Wayne E.; Cole W. Matson,

2015-01-01

The health of tree swallows, Tachycineta bicolor, on the Upper Mississippi River (UMR) was assessed in 2010 and 2011 using biomarkers at six sites downriver of Minneapolis/St. Paul, MN metropolitan area, a tributary into the UMR, and a nearby lake. Chromosomal damage was evaluated in nestling blood by measuring the coefficient of variation of DNA content (DNA CV) using flow cytometry. Cytochrome P450 1A activity in nestling liver was measured using the ethoxyresorufin-O-dealkylase (EROD) assay, and oxidative stress was estimated in nestling livers via determination of thiobarbituric acid reacting substances (TBARS), reduced glutathione (GSH), oxidized glutathione (GSSG), the ratio GSSG/GSH, total sulfhydryl, and protein bound sulfhydryl (PBSH). A multilevel regression model (DNA CV) and simple regressions (EROD and oxidative stress) were used to evaluate biomarker responses for each location. Chromosomal damage was significantly elevated at two sites on the UMR (Pigs Eye and Pool 2) relative to the Green Mountain Lake reference site, while the induction of EROD activity was only observed at Pigs Eye. No measures of oxidative stress differed among sites. Multivariate analysis confirmed an increased DNA CV at Pigs Eye and Pool 2, and elevated EROD activity at Pigs Eye. These results suggest that the health of tree swallows has been altered at the DNA level at Pigs Eye and Pool 2 sites, and at the physiological level at Pigs Eye site only.

Induction of Strain-Transcending Immunity against Plasmodium chabaudi adami Malaria with a Multiepitope DNA Vaccine

PubMed Central

Scorza, T.; Grubb, K.; Smooker, P.; Rainczuk, A.; Proll, D.; Spithill, T. W.

2005-01-01

A major goal of current malaria vaccine programs is to develop multivalent vaccines that will protect humans against the many heterologous malaria strains that circulate in endemic areas. We describe a multiepitope DNA vaccine, derived from a genomic Plasmodium chabaudi adami DS DNA expression library of 30,000 plasmids, which induces strain-transcending immunity in mice against challenge with P. c. adami DK. Segregation of this library and DNA sequence analysis identified vaccine subpools encoding open reading frames (ORFs)/peptides of >9 amino acids [aa] (the V9+ pool, 303 plasmids) and >50 aa (V50+ pool, 56 plasmids), respectively. The V9+ and V50+ plasmid vaccine subpools significantly cross-protected mice against heterologous P. c. adami DK challenge, and protection correlated with the induction of both specific gamma interferon production by splenic cells and opsonizing antibodies. Bioinformatic analysis showed that 22 of the V50+ ORFs were polypeptides conserved among three or more Plasmodium spp., 13 of which are predicted hypothetical proteins. Twenty-nine of these ORFs are orthologues of predicted Plasmodium falciparum sequences known to be expressed in the blood stage, suggesting that this vaccine pool encodes multiple blood-stage antigens. The results have implications for malaria vaccine design by providing proof-of-principle that significant strain-transcending immunity can be induced using multiepitope blood-stage DNA vaccines and suggest that both cellular responses and opsonizing antibodies are necessary for optimal protection against P. c. adami. PMID:15845504

Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae).

PubMed

Zhao, Ya-E; Xu, Ji-Ru; Hu, Li; Wu, Li-Ping; Wang, Zheng-Hang

2012-05-01

The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ≥ 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs. Copyright © 2012 Elsevier Inc. All rights reserved.

Genome-Wide Requirements for Resistance to Functionally Distinct DNA-Damaging Agents

PubMed Central

Proctor, Michael; Flaherty, Patrick; Jordan, Michael I; Arkin, Adam P; Davis, Ronald W; Nislow, Corey; Giaever, Guri

2005-01-01

The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of ~4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups. PMID:16121259

Discovering Protein-Coding Genes from the Environment: Time for the Eukaryotes?

PubMed

Marmeisse, Roland; Kellner, Harald; Fraissinet-Tachet, Laurence; Luis, Patricia

2017-09-01

Eukaryotic microorganisms from diverse environments encompass a large number of taxa, many of them still unknown to science. One strategy to mine these organisms for genes of biotechnological relevance is to use a pool of eukaryotic mRNA directly extracted from environmental samples. Recent reports demonstrate that the resulting metatranscriptomic cDNA libraries can be screened by expression in yeast for a wide range of genes and functions from many of the different eukaryotic taxa. In combination with novel emerging high-throughput technologies, we anticipate that this approach should contribute to exploring the functional diversity of the eukaryotic microbiota. Copyright © 2017 Elsevier Ltd. All rights reserved.

Performance of prenatal screening using maternal serum and ultrasound markers for Down syndrome in Chinese women: a systematic review and meta-analysis.

PubMed

Tu, S; Rosenthal, M; Wang, D; Huang, J; Chen, Y

2016-09-01

Controversies about the performance of conventional prenatal screening using maternal serum and ultrasound markers (PSMSUM) in detecting Down syndrome (DS) have been raised as a result of a recently available noninvasive prenatal test based on cell-free fetal DNA sequencing. To evaluate the screening performance of PSMSUM in detecting DS in Chinese women. An exhaustive literature search of MEDLINE, Embase, the Cochrane Library, ISI Web of Science and China BioMedical Disc. Primary studies, published from January 2004 to November 2014, which examined the screening accuracy of PSMSUM in pregnant Chinese women, compared with a reference standard, either chromosomal verification or inspection of the newborn. Data were extracted as screening positive/negative results for Down and non-Down syndrome pregnancies, allowing estimation of sensitivities and specificities. Risks of bias within and across studies were assessed. Screening accuracy measures were pooled using a bivariate random effects regression model. Seventy-eight studies, involving six categories of PSMSUM, were included. Second-trimester double serum [pooled sensitivity (SEN) = 0.80, pooled specificity (SPE) = 0.95] and triple-serum (pooled SEN = 0.79, pooled SPE = 0.96) screening were the predominant PSMSUM methods. The screening performances of these methods achieved the national standard but varied enormously across studies. First-trimester combined screening (pooled SEN = 0.92, pooled SPE = 0.93) and second-trimester quadruple serum screening (median SEN = 0.86, median SPE = 0.96) performed better, but were rarely used. Second-trimester maternal serum screening has the potential to achieve satisfactory screening performance in middle- and low-income countries. The reported enormous range in screening performance of second-trimester PSMSUM calls for urgent implementation of methods for performance optimization. Meta-analysis results show good accuracy of maternal serum and ultrasound screening for trisomy 21 in Chinese women. © 2016 Royal College of Obstetricians and Gynaecologists.

Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

PubMed

Makarova, Alena V; Grabow, Corinn; Gening, Leonid V; Tarantul, Vyacheslav Z; Tahirov, Tahir H; Bessho, Tadayoshi; Pavlov, Youri I

2011-01-31

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+) ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA"). We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

[Genetic ecological monitoring in human populations: heterozygosity, mtDNA haplotype variation, and genetic load].

PubMed

Balanovskiĭ, O P; Koshel', S M; Zaporozhchenko, V V; Pshenichnov, A S; Frolova, S A; Kuznetsova, M A; Baranova, E E; Teuchezh, I E; Kuznetsova, A A; Romashkina, M V; Utevskaia, O M; Churnosov, M I; Villems, R; Balanovskaia, E V

2011-11-01

Yu. P. Altukhov suggested that heterozygosity is an indicator of the state of the gene pool. The idea and a linked concept of genetic ecological monitoring were applied to a new dataset on mtDNA variation in East European ethnic groups. Haplotype diversity (an analog of the average heterozygosity) was shown to gradually decrease northwards. Since a similar trend is known for population density, interlinked changes were assumed for a set of parameters, which were ordered to form a causative chain: latitude increases, land productivity decreases, population density decreases, effective population size decreases, isolation of subpopulations increases, genetic drift increases, and mtDNA haplotype diversity decreases. An increase in genetic drift increases the random inbreeding rate and, consequently, the genetic load. This was confirmed by a significant correlation observed between the incidence of autosomal recessive hereditary diseases and mtDNA haplotype diversity. Based on the findings, mtDNA was assumed to provide an informative genetic system for genetic ecological monitoring; e.g., analyzing the ecology-driven changes in the gene pool.

Coupling Spore Traps and Quantitative PCR Assays for Detection of the Downy Mildew Pathogens of Spinach (Peronospora effusa) and Beet (P. schachtii)

PubMed Central

Klosterman, Steven J.; Anchieta, Amy; McRoberts, Neil; Koike, Steven T.; Subbarao, Krishna V.; Voglmayr, Hermann; Choi, Young-Joon; Thines, Marco; Martin, Frank N.

2016-01-01

Downy mildew of spinach (Spinacia oleracea), caused by Peronospora effusa, is a production constraint on production worldwide, including in California, where the majority of U.S. spinach is grown. The aim of this study was to develop a real-time quantitative polymerase chain reaction (qPCR) assay for detection of airborne inoculum of P. effusa in California. Among oomycete ribosomal DNA (rDNA) sequences examined for assay development, the highest nucleotide sequence identity was observed between rDNA sequences of P. effusa and P. schachtii, the cause of downy mildew on sugar beet and Swiss chard in the leaf beet group (Beta vulgaris subsp. vulgaris). Single-nucleotide polymorphisms were detected between P. effusa and P. schachtii in the 18S rDNA regions for design of P. effusa- and P. schachtii-specific TaqMan probes and reverse primers. An allele-specific probe and primer amplification method was applied to determine the frequency of both P. effusa and P. schachtii rDNA target sequences in pooled DNA samples, enabling quantification of rDNA of P. effusa from impaction spore trap samples collected from spinach production fields. The rDNA copy numbers of P. effusa were, on average, ≈3,300-fold higher from trap samples collected near an infected field compared with those levels recorded at a site without a nearby spinach field. In combination with disease-conducive weather forecasting, application of the assays may be helpful to time fungicide applications for disease management. PMID:24964150

Deoxyribonucleoside kinases in mitochondrial DNA depletion.

PubMed

Saada-Reisch, Ann

2004-10-01

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are a heterogeneous group of mitochondrial disorders, manifested by a decreased mtDNA copy number and respiratory chain dysfunction. Primary MDS are inherited autosomally and may affect a single organ or multiple tissues. Mutated mitochondrial deoxyribonucleoside kinases; deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2), were associated with the hepatocerebral and myopathic forms of MDS respectively. dGK and TK2 are key enzymes in the mitochondrial nucleotide salvage pathway, providing the mitochondria with deoxyribonucleotides (dNP) essential for mtDNA synthesis. Although the mitochondrial dNP pool is physically separated from the cytosolic one, dNP's may still be imported through specific transport. Non-replicating tissues, where cytosolic dNP supply is down regulated, are thus particularly vulnerable to dGK and TK2 deficiency. The overlapping substrate specificity of deoxycytidine kinase (dCK) may explain the relative sparing of muscle in dGK deficiency, while low basal TK2 activity render this tissue susceptible to TK2 deficiency. The precise pathophysiological mechanisms of mtDNA depletion due to dGK and TK2 deficiencies remain to be determined, though recent findings confirm that it is attributed to imbalanced dNTP pools.

Origins of domestic dog in southern East Asia is supported by analysis of Y-chromosome DNA.

PubMed

Ding, Z-L; Oskarsson, M; Ardalan, A; Angleby, H; Dahlgren, L-G; Tepeli, C; Kirkness, E; Savolainen, P; Zhang, Y-P

2012-05-01

Global mitochondrial DNA (mtDNA) data indicates that the dog originates from domestication of wolf in Asia South of Yangtze River (ASY), with minor genetic contributions from dog-wolf hybridisation elsewhere. Archaeological data and autosomal single nucleotide polymorphism data have instead suggested that dogs originate from Europe and/or South West Asia but, because these datasets lack data from ASY, evidence pointing to ASY may have been overlooked. Analyses of additional markers for global datasets, including ASY, are therefore necessary to test if mtDNA phylogeography reflects the actual dog history and not merely stochastic events or selection. Here, we analyse 14,437 bp of Y-chromosome DNA sequence in 151 dogs sampled worldwide. We found 28 haplotypes distributed in five haplogroups. Two haplogroups were universally shared and included three haplotypes carried by 46% of all dogs, but two other haplogroups were primarily restricted to East Asia. Highest genetic diversity and virtually complete phylogenetic coverage was found within ASY. The 151 dogs were estimated to originate from 13-24 wolf founders, but there was no indication of post-domestication dog-wolf hybridisations. Thus, Y-chromosome and mtDNA data give strikingly similar pictures of dog phylogeography, most importantly that roughly 50% of the gene pools are shared universally but only ASY has nearly the full range of genetic diversity, such that the gene pools in all other regions may derive from ASY. This corroborates that ASY was the principal, and possibly sole region of wolf domestication, that a large number of wolves were domesticated, and that subsequent dog-wolf hybridisation contributed modestly to the dog gene pool.

Origins of domestic dog in Southern East Asia is supported by analysis of Y-chromosome DNA

PubMed Central

Ding, Z-L; Oskarsson, M; Ardalan, A; Angleby, H; Dahlgren, L-G; Tepeli, C; Kirkness, E; Savolainen, P; Zhang, Y-P

2012-01-01

Global mitochondrial DNA (mtDNA) data indicates that the dog originates from domestication of wolf in Asia South of Yangtze River (ASY), with minor genetic contributions from dog–wolf hybridisation elsewhere. Archaeological data and autosomal single nucleotide polymorphism data have instead suggested that dogs originate from Europe and/or South West Asia but, because these datasets lack data from ASY, evidence pointing to ASY may have been overlooked. Analyses of additional markers for global datasets, including ASY, are therefore necessary to test if mtDNA phylogeography reflects the actual dog history and not merely stochastic events or selection. Here, we analyse 14 437 bp of Y-chromosome DNA sequence in 151 dogs sampled worldwide. We found 28 haplotypes distributed in five haplogroups. Two haplogroups were universally shared and included three haplotypes carried by 46% of all dogs, but two other haplogroups were primarily restricted to East Asia. Highest genetic diversity and virtually complete phylogenetic coverage was found within ASY. The 151 dogs were estimated to originate from 13–24 wolf founders, but there was no indication of post-domestication dog–wolf hybridisations. Thus, Y-chromosome and mtDNA data give strikingly similar pictures of dog phylogeography, most importantly that roughly 50% of the gene pools are shared universally but only ASY has nearly the full range of genetic diversity, such that the gene pools in all other regions may derive from ASY. This corroborates that ASY was the principal, and possibly sole region of wolf domestication, that a large number of wolves were domesticated, and that subsequent dog–wolf hybridisation contributed modestly to the dog gene pool. PMID:22108628

Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA.

PubMed

Weiberg, Arne; Pöhler, Dirk; Morgenstern, Burkhard; Karlovsky, Petr

2008-10-13

cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool. The method is based on selective amplification of cDNA fragments generated by restriction endonucleases, electrophoretic separation of the products and comparison of the band patterns between treated samples and controls. Unequal distribution of restriction sites used to generate cDNA fragments negatively affects the performance of cDNA-AFLP. Some transcripts are represented by more than one fragment while other escape detection, causing redundancy and reducing the coverage of the analysis, respectively. With the goal of improving the coverage of cDNA-AFLP without increasing its redundancy, we designed a modified cDNA-AFLP protocol. Immobilized cDNA is sequentially digested with several restriction endonucleases and the released DNA fragments are collected in mutually exclusive pools. To investigate the performance of the protocol, software tool MECS (Multiple Enzyme cDNA-AFLP Simulation) was written in Perl. cDNA-AFLP protocols described in the literature and the new sequential digestion protocol were simulated on sets of cDNA sequences from mouse, human and Arabidopsis thaliana. The redundancy and coverage, the total number of PCR reactions, and the average fragment length were calculated for each protocol and cDNA set. Simulation revealed that sequential digestion of immobilized cDNA followed by the partitioning of released fragments into mutually exclusive pools outperformed other cDNA-AFLP protocols in terms of coverage, redundancy, fragment length, and the total number of PCRs. Primers generating 30 to 70 amplicons per PCR provided the highest fraction of electrophoretically distinguishable fragments suitable for normalization. For A. thaliana, human and mice transcriptome, the use of two marking enzymes and three sequentially applied releasing enzymes for each of the marking enzymes is recommended.

DNA: Polymer and molecular code

NASA Astrophysics Data System (ADS)

Shivashankar, G. V.

1999-10-01

The thesis work focusses upon two aspects of DNA, the polymer and the molecular code. Our approach was to bring single molecule micromanipulation methods to the study of DNA. It included a home built optical microscope combined with an atomic force microscope and an optical tweezer. This combined approach led to a novel method to graft a single DNA molecule onto a force cantilever using the optical tweezer and local heating. With this method, a force versus extension assay of double stranded DNA was realized. The resolution was about 10 picoN. To improve on this force measurement resolution, a simple light backscattering technique was developed and used to probe the DNA polymer flexibility and its fluctuations. It combined the optical tweezer to trap a DNA tethered bead and the laser backscattering to detect the beads Brownian fluctuations. With this technique the resolution was about 0.1 picoN with a millisecond access time, and the whole entropic part of the DNA force-extension was measured. With this experimental strategy, we measured the polymerization of the protein RecA on an isolated double stranded DNA. We observed the progressive decoration of RecA on the l DNA molecule, which results in the extension of l , due to unwinding of the double helix. The dynamics of polymerization, the resulting change in the DNA entropic elasticity and the role of ATP hydrolysis were the main parts of the study. A simple model for RecA assembly on DNA was proposed. This work presents a first step in the study of genetic recombination. Recently we have started a study of equilibrium binding which utilizes fluorescence polarization methods to probe the polymerization of RecA on single stranded DNA. In addition to the study of material properties of DNA and DNA-RecA, we have developed experiments for which the code of the DNA is central. We studied one aspect of DNA as a molecular code, using different techniques. In particular the programmatic use of template specificity makes gene expression a prime example of a biological code. We developed a novel method of making DNA micro- arrays, the so-called DNA chip. Using the optical tweezer concept, we were able to pattern biomolecules on a solid substrate, developing a new type of sub-micron laser lithography. A laser beam is focused onto a thin gold film on a glass substrate. Laser ablation of gold results in local aggregation of nanometer scale beads conjugated with small DNA oligonucleotides, with sub-micron resolution. This leads to specific detection of cDNA and RNA molecules. We built a simple micro-array fabrication and detection in the laboratory, based on this method, to probe addressable pools (genes, proteins or antibodies). We have lately used molecular beacons (single stranded DNA with a stem-loop structure containing a fluorophore and quencher), for the direct detection of unlabelled mRNA. As a first step towards a study of the dynamics of the biological code, we have begun to examine the patterns of gene expression during virus (T7 phage) infection of E-coli bacteria.

Optimized DNA extraction methods for encysted embryos of the endangered fairy shrimp, Branchinecta sandiegonensis

USGS Publications Warehouse

Steele, A.N.; Simovich, M.A.; Pepino, D.; Schroeder, K.M.; Vandergast, A.G.; Bohonak, A.J.

2009-01-01

The San Diego fairy shrimp Branchinecta sandiegonensis is a federally endangered species endemic to vernal pools in southern California, USA. Filling events in these habitats are highly variable, with some pools failing to hold water long enough for reproduction over many successive years. Studies of this species are thus hindered by the relatively rare appearance of aquatically active life history phases. Because diapausing cysts are abundant and present at all times, they provide an underutilized opportunity for both species identification and genetic studies. However, methods for extracting DNA from cysts are technically challenging because of their structure and size. Here we present a protocol for extracting DNA from B. sandiegonensis cysts in sufficient quantities for "DNA Barcoding", microsatellite analysis and other genotyping and sequencing applications. The technique will aid in population genetic studies and species identification (since taxonomic keys only distinguish among adults), and will be applicable to other crustaceans with similar diapausing cysts. ?? Springer Science+Business Media B.V. 2008.

A novel pooled-sample multiplex luminex assay for high-throughput measurement of relative telomere length.

PubMed

Jasmine, Farzana; Shinkle, Justin; Sabarinathan, Mekala; Ahsan, Habibul; Pierce, Brandon L; Kibriya, Muhammad G

2018-03-12

Relative telomere length (RTL) is a potential biomarker of aging and risk for chronic disease. Previously, we developed a probe-based RTL assay on Luminex platform, where probes for Telomere (T) and reference gene (R) for a given DNA sample were tested in a single well. Here, we describe a method of pooling multiple samples in one well to increase the throughput and cost-effectiveness. We used four different microbeads for the same T-probe and four different microbeads for the same R-probe. Each pair of probe sets were hybridized to DNA in separate plates and then pooled in a single plate for all the subsequent steps. We used DNA samples from 60 independent individuals and repeated in multiple batches to test the precision. The precision was good to excellent with Intraclass correlation coefficient (ICC) of 0.908 (95% CI 0.856-0.942). More than 67% of the variation in the RTL could be explained by sample-to-sample variation; less than 0.1% variation was due to batch-to-batch variation and 0.3% variation was explained by bead-to-bead variation. We increased the throughput of RTL Luminex assay from 60 to 240 samples per run. The new assay was validated against the original Luminex assay without pooling (r = 0.79, P = 1.44 × 10 -15 ). In an independent set of samples (n = 550), the new assay showed a negative correlation of RTL with age (r = -0.41), a result providing external validation for the method. We describe a novel high throughput pooled-sample multiplex Luminex assay for RTL with good to excellent precision suitable for large-scale studies. © 2018 Wiley Periodicals, Inc.

Ancient DNA evidence supports the contribution of Di-Qiang people to the han Chinese gene pool.

PubMed

Zhao, Yong-Bin; Li, Hong-Jie; Li, Sheng-Nan; Yu, Chang-Chun; Gao, Shi-Zhu; Xu, Zhi; Jin, Li; Zhu, Hong; Zhou, Hui

2011-02-01

Han Chinese is the largest ethnic group in the world. During its development, it gradually integrated with many neighboring populations. To uncover the origin of the Han Chinese, ancient DNA analysis was performed on the remains of 46 humans (1700 to 1900 years ago) excavated from the Taojiazhai site in Qinghai province, northwest of China, where the Di-Qiang populations had previously lived. In this study, eight mtDNA haplogroups (A, B, D, F, M*, M10, N9a, and Z) and one Y-chromosome haplogroup (O3) were identified. All analyses show that the Taojiazhai population presents close genetic affinity to Tibeto-Burman populations (descendants of Di-Qiang populations) and Han Chinese, suggesting that the Di-Qiang populations may have contributed to the Han Chinese genetic pool. 2010 Wiley-Liss, Inc.

Co-circulation of West Nile virus and distinct insect-specific flaviviruses in Turkey.

PubMed

Ergünay, Koray; Litzba, Nadine; Brinkmann, Annika; Günay, Filiz; Sarıkaya, Yasemen; Kar, Sırrı; Örsten, Serra; Öter, Kerem; Domingo, Cristina; Erisoz Kasap, Özge; Özkul, Aykut; Mitchell, Luke; Nitsche, Andreas; Alten, Bülent; Linton, Yvonne-Marie

2017-03-20

Active vector surveillance provides an efficient tool for monitoring the presence or spread of emerging or re-emerging vector-borne viruses. This study was undertaken to investigate the circulation of flaviviruses. Mosquitoes were collected from 58 locations in 10 provinces across the Aegean, Thrace and Mediterranean Anatolian regions of Turkey in 2014 and 2015. Following morphological identification, mosquitoes were pooled and screened by nested and real-time PCR assays. Detected viruses were further characterised by sequencing. Positive pools were inoculated onto cell lines for virus isolation. Next generation sequencing was employed for genomic characterisation of the isolates. A total of 12,711 mosquito specimens representing 15 species were screened in 594 pools. Eleven pools (2%) were reactive in the virus screening assays. Sequencing revealed West Nile virus (WNV) in one Culex pipiens (s.l.) pool from Thrace. WNV sequence corresponded to lineage one clade 1a but clustered distinctly from the Turkish prototype isolate. In 10 pools, insect-specific flaviviruses were characterised as Culex theileri flavivirus in 5 pools of Culex theileri and one pool of Cx. pipiens (s.l.), Ochlerotatus caspius flavivirus in two pools of Aedes (Ochlerotatus) caspius, Flavivirus AV-2011 in one pool of Culiseta annulata, and an undetermined flavivirus in one pool of Uranotaenia unguiculata from the Aegean and Thrace regions. DNA forms or integration of the detected insect-specific flaviviruses were not observed. A virus strain, tentatively named as "Ochlerotatus caspius flavivirus Turkey", was isolated from an Ae. caspius pool in C6/36 cells. The viral genome comprised 10,370 nucleotides with a putative polyprotein of 3,385 amino acids that follows the canonical flavivirus polyprotein organisation. Sequence comparisons and phylogenetic analyses revealed the close relationship of this strain with Ochlerotatus caspius flavivirus from Portugal and Hanko virus from Finland. Several conserved structural and amino acid motifs were identified. We identified WNV and several distinct insect-specific flaviviruses during an extensive biosurveillance study of mosquitoes in various regions of Turkey in 2014 and 2015. Ongoing circulation of WNV is revealed, with an unprecedented genetic diversity. A probable replicating form of an insect flavivirus identified only in DNA form was detected.

From the Cover: Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins

NASA Astrophysics Data System (ADS)

Ito, Takashi; Tashiro, Kosuke; Muta, Shigeru; Ozawa, Ritsuko; Chiba, Tomoko; Nishizawa, Mayumi; Yamamoto, Kiyoshi; Kuhara, Satoru; Sakaki, Yoshiyuki

2000-02-01

Protein-protein interactions play pivotal roles in various aspects of the structural and functional organization of the cell, and their complete description is indispensable to thorough understanding of the cell. As an approach toward this goal, here we report a comprehensive system to examine two-hybrid interactions in all of the possible combinations between proteins of Saccharomyces cerevisiae. We cloned all of the yeast ORFs individually as a DNA-binding domain fusion ("bait") in a MATa strain and as an activation domain fusion ("prey") in a MATα strain, and subsequently divided them into pools, each containing 96 clones. These bait and prey clone pools were systematically mated with each other, and the transformants were subjected to strict selection for the activation of three reporter genes followed by sequence tagging. Our initial examination of ≈4 × 106 different combinations, constituting ≈10% of the total to be tested, has revealed 183 independent two-hybrid interactions, more than half of which are entirely novel. Notably, the obtained binary data allow us to extract more complex interaction networks, including the one that may explain a currently unsolved mechanism for the connection between distinct steps of vesicular transport. The approach described here thus will provide many leads for integration of various cellular functions and serve as a major driving force in the completion of the protein-protein interaction map.

Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML.

PubMed

Liyanage, Sanduni U; Hurren, Rose; Voisin, Veronique; Bridon, Gaëlle; Wang, Xiaoming; Xu, ChangJiang; MacLean, Neil; Siriwardena, Thirushi P; Gronda, Marcela; Yehudai, Dana; Sriskanthadevan, Shrivani; Avizonis, Daina; Shamas-Din, Aisha; Minden, Mark D; Bader, Gary D; Laposa, Rebecca; Schimmer, Aaron D

2017-05-11

Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2'3'-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML. © 2017 by The American Society of Hematology.

Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML

PubMed Central

Liyanage, Sanduni U.; Hurren, Rose; Voisin, Veronique; Bridon, Gaëlle; Wang, Xiaoming; Xu, ChangJiang; MacLean, Neil; Siriwardena, Thirushi P.; Gronda, Marcela; Yehudai, Dana; Sriskanthadevan, Shrivani; Avizonis, Daina; Shamas-Din, Aisha; Minden, Mark D.; Bader, Gary D.; Laposa, Rebecca

2017-01-01

Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2′3′-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2′3′-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML. PMID:28283480

MPV17 Loss Causes Deoxynucleotide Insufficiency and Slow DNA Replication in Mitochondria

PubMed Central

Dalla Rosa, Ilaria; Cámara, Yolanda; Durigon, Romina; Moss, Chloe F.; Vidoni, Sara; Akman, Gokhan; Hunt, Lilian; Johnson, Mark A.; Grocott, Sarah; Wang, Liya; Thorburn, David R.; Hirano, Michio; Poulton, Joanna; Taylor, Robert W.; Elgar, Greg; Martí, Ramon; Voshol, Peter; Holt, Ian J.; Spinazzola, Antonella

2016-01-01

MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients’ quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway. PMID:26760297

Swimming Pools.

ERIC Educational Resources Information Center

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

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

Effects of Sulfur Mustard on Intracellular Calcium and Synthesis of Basement Membrane Zone Proteins in Human Skin

DTIC Science & Technology

1993-08-04

development of blisters. They postulated that DNA single strand breaks (SSB) due to spontaneous or enzymatically induced depurination of alkylated DNA ... bases activate the chromosomal enzyme poly(ADP-ribose)transferase (PADPRT). The latter would deplete cellular NAD÷ pools. In literature, this

DNA methylation changes measured in pre-diagnostic peripheral blood samples are associated with smoking and lung cancer risk.

PubMed

Baglietto, Laura; Ponzi, Erica; Haycock, Philip; Hodge, Allison; Bianca Assumma, Manuela; Jung, Chol-Hee; Chung, Jessica; Fasanelli, Francesca; Guida, Florence; Campanella, Gianluca; Chadeau-Hyam, Marc; Grankvist, Kjell; Johansson, Mikael; Ala, Ugo; Provero, Paolo; Wong, Ee Ming; Joo, Jihoon; English, Dallas R; Kazmi, Nabila; Lund, Eiliv; Faltus, Christian; Kaaks, Rudolf; Risch, Angela; Barrdahl, Myrto; Sandanger, Torkjel M; Southey, Melissa C; Giles, Graham G; Johansson, Mattias; Vineis, Paolo; Polidoro, Silvia; Relton, Caroline L; Severi, Gianluca

2017-01-01

DNA methylation changes are associated with cigarette smoking. We used the Illumina Infinium HumanMethylation450 array to determine whether methylation in DNA from pre-diagnostic, peripheral blood samples is associated with lung cancer risk. We used a case-control study nested within the EPIC-Italy cohort and a study within the MCCS cohort as discovery sets (a total of 552 case-control pairs). We validated the top signals in 429 case-control pairs from another 3 studies. We identified six CpGs for which hypomethylation was associated with lung cancer risk: cg05575921 in the AHRR gene (p-value pooled  = 4 × 10 -17 ), cg03636183 in the F2RL3 gene (p-value pooled  = 2 × 10 - 13 ), cg21566642 and cg05951221 in 2q37.1 (p-value pooled  = 7 × 10 -16 and 1 × 10 -11 respectively), cg06126421 in 6p21.33 (p-value pooled  = 2 × 10 -15 ) and cg23387569 in 12q14.1 (p-value pooled  = 5 × 10 -7 ). For cg05951221 and cg23387569 the strength of association was virtually identical in never and current smokers. For all these CpGs except for cg23387569, the methylation levels were different across smoking categories in controls (p-values heterogeneity  ≤ 1.8 x10 - 7 ), were lowest for current smokers and increased with time since quitting for former smokers. We observed a gain in discrimination between cases and controls measured by the area under the ROC curve of at least 8% (p-values ≥ 0.003) in former smokers by adding methylation at the 6 CpGs into risk prediction models including smoking status and number of pack-years. Our findings provide convincing evidence that smoking and possibly other factors lead to DNA methylation changes measurable in peripheral blood that may improve prediction of lung cancer risk. © 2016 UICC.

In vivo insertion pool sequencing identifies virulence factors in a complex fungal–host interaction

PubMed Central

Uhse, Simon; Pflug, Florian G.; Stirnberg, Alexandra; Ehrlinger, Klaus; von Haeseler, Arndt

2018-01-01

Large-scale insertional mutagenesis screens can be powerful genome-wide tools if they are streamlined with efficient downstream analysis, which is a serious bottleneck in complex biological systems. A major impediment to the success of next-generation sequencing (NGS)-based screens for virulence factors is that the genetic material of pathogens is often underrepresented within the eukaryotic host, making detection extremely challenging. We therefore established insertion Pool-Sequencing (iPool-Seq) on maize infected with the biotrophic fungus U. maydis. iPool-Seq features tagmentation, unique molecular barcodes, and affinity purification of pathogen insertion mutant DNA from in vivo-infected tissues. In a proof of concept using iPool-Seq, we identified 28 virulence factors, including 23 that were previously uncharacterized, from an initial pool of 195 candidate effector mutants. Because of its sensitivity and quantitative nature, iPool-Seq can be applied to any insertional mutagenesis library and is especially suitable for genetically complex setups like pooled infections of eukaryotic hosts. PMID:29684023

Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase.

PubMed

Liew, Li Phing; Lim, Zun Yi; Cohen, Matan; Kong, Ziqing; Marjavaara, Lisette; Chabes, Andrei; Bell, Stephen D

2016-11-01

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Properties of targeted preamplification in DNA and cDNA quantification.

PubMed

Andersson, Daniel; Akrap, Nina; Svec, David; Godfrey, Tony E; Kubista, Mikael; Landberg, Göran; Ståhlberg, Anders

2015-01-01

Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR). To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR. The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification. On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.

Accessing the Soil Metagenome for Studies of Microbial Diversity▿ †

PubMed Central

Delmont, Tom O.; Robe, Patrick; Cecillon, Sébastien; Clark, Ian M.; Constancias, Florentin; Simonet, Pascal; Hirsch, Penny R.; Vogel, Timothy M.

2011-01-01

Soil microbial communities contain the highest level of prokaryotic diversity of any environment, and metagenomic approaches involving the extraction of DNA from soil can improve our access to these communities. Most analyses of soil biodiversity and function assume that the DNA extracted represents the microbial community in the soil, but subsequent interpretations are limited by the DNA recovered from the soil. Unfortunately, extraction methods do not provide a uniform and unbiased subsample of metagenomic DNA, and as a consequence, accurate species distributions cannot be determined. Moreover, any bias will propagate errors in estimations of overall microbial diversity and may exclude some microbial classes from study and exploitation. To improve metagenomic approaches, investigate DNA extraction biases, and provide tools for assessing the relative abundances of different groups, we explored the biodiversity of the accessible community DNA by fractioning the metagenomic DNA as a function of (i) vertical soil sampling, (ii) density gradients (cell separation), (iii) cell lysis stringency, and (iv) DNA fragment size distribution. Each fraction had a unique genetic diversity, with different predominant and rare species (based on ribosomal intergenic spacer analysis [RISA] fingerprinting and phylochips). All fractions contributed to the number of bacterial groups uncovered in the metagenome, thus increasing the DNA pool for further applications. Indeed, we were able to access a more genetically diverse proportion of the metagenome (a gain of more than 80% compared to the best single extraction method), limit the predominance of a few genomes, and increase the species richness per sequencing effort. This work stresses the difference between extracted DNA pools and the currently inaccessible complete soil metagenome. PMID:21183646

Pivotal Role of Inosine Triphosphate Pyrophosphatase in Maintaining Genome Stability and the Prevention of Apoptosis in Human Cells

PubMed Central

Lopez-Bertoni, Hernando; Luo, Xu; Pavlov, Youri I.

2012-01-01

Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPases are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPase, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C>A, leads to null ITPase activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPase encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPase are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevated risk for degenerative diseases and cancer. PMID:22384212

The Polynesian gene pool: an early contribution by Amerindians to Easter Island.

PubMed

Thorsby, Erik

2012-03-19

It is now generally accepted that Polynesia was first settled by peoples from southeast Asia. An alternative that eastern parts of Polynesia were first inhabited by Amerindians has found little support. There are, however, many indications of a 'prehistoric' (i.e. before Polynesia was discovered by Europeans) contact between Polynesia and the Americas, but genetic evidence of a prehistoric Amerindian contribution to the Polynesian gene pool has been lacking. We recently carried out genomic HLA (human leucocyte antigen) typing as well as typing for mitochondrial DNA (mtDNA) and Y chromosome markers of blood samples collected in 1971 and 2008 from reputedly non-admixed Easter Islanders. All individuals carried HLA alleles and mtDNA types previously found in Polynesia, and most of the males carried Y chromosome markers of Polynesian origin (a few had European Y chromosome markers), further supporting an initial Polynesian population on Easter Island. The HLA investigations revealed, however, that some individuals also carried HLA alleles which have previously almost only been found in Amerindians. We could trace the introduction of these Amerindian alleles to before the Peruvian slave trades, i.e. before the 1860s, and provide suggestive evidence that they were introduced already in prehistoric time. Our results demonstrate an early Amerindian contribution to the Polynesian gene pool on Easter Island, and illustrate the usefulness of typing for immunogenetic markers such as HLA to complement mtDNA and Y chromosome analyses in anthropological investigations.

A genome-wide association study for fat-related traits computed by image analysis in Japanese Black cattle.

PubMed

Nakajima, Ayaka; Kawaguchi, Fuki; Uemoto, Yoshinobu; Fukushima, Moriyuki; Yoshida, Emi; Iwamoto, Eiji; Akiyama, Takayuki; Kohama, Namiko; Kobayashi, Eiji; Honda, Takeshi; Oyama, Kenji; Mannen, Hideyuki; Sasazaki, Shinji

2018-05-01

The objective of this study was to identify genomic regions associated with fat-related traits using a Japanese Black cattle population in Hyogo. From 1836 animals, those with high or low values were selected on the basis of corrected phenotype and then pooled into high and low groups (n = 100 each), respectively. DNA pool-based genome-wide association study (GWAS) was performed using Illumina BovineSNP50 BeadChip v2 with three replicate assays for each pooled sample. GWAS detected that two single nucleotide polymorphisms (SNPs) on BTA7 (ARS-BFGL-NGS-35463 and Hapmap23838-BTA-163815) and one SNP on BTA12 (ARS-BFGL-NGS-2915) significantly affected fat percentage (FAR). The significance of ARS-BFGL-NGS-35463 on BTA7 was confirmed by individual genotyping in all pooled samples. Moreover, association analysis between SNP and FAR in 803 Japanese Black cattle revealed a significant effect of SNP on FAR. Thus, further investigation of these regions is required to identify FAR-associated genes and mutations, which can lead to the development of DNA markers for marker-assisted selection for the genetic improvement of beef quality. © 2018 Japanese Society of Animal Science.

Detection of Helicobacter and Campylobacter spp. from the aquatic environment of marine mammals.

PubMed

Goldman, C G; Matteo, M J; Loureiro, J D; Degrossi, J; Teves, S; Heredia, S Rodriguez; Alvarez, K; González, A Beltrán; Catalano, M; Boccio, J; Cremaschi, G; Solnick, J V; Zubillaga, M B

2009-01-13

The mechanism by which Helicobacter species are transmitted remains unclear. To examine the possible role of environmental transmission in marine mammals, we sought the presence of Helicobacter spp. and non-Helicobacter bacteria within the order Campylobacterales in water from the aquatic environment of marine mammals, and in fish otoliths regurgitated by dolphins. Water was collected from six pools, two inhabited by dolphins and four inhabited by seals. Regurgitated otoliths were collected from the bottom of dolphins' pools. Samples were evaluated by culture, PCR and DNA sequence analysis. Sequences from dolphins' water and from regurgitated otoliths clustered with 99.8-100% homology with sequences from gastric fluids, dental plaque and saliva from dolphins living in those pools, and with 99.5% homology with H. cetorum. Sequences from seals' water clustered with 99.5% homology with a sequence amplified from a Northern sea lion (AY203900). Control PCR on source water for the pools and from otoliths dissected from feeder fish were negative. The findings of Helicobacter spp. DNA in the aquatic environment suggests that contaminated water from regurgitated fish otoliths and perhaps other tissues may play a role in Helicobacter transmission among marine mammals.

Prenatal Ambient Air Pollution, Placental Mitochondrial DNA Content, and Birth Weight in the INMA (Spain) and ENVIRONAGE (Belgium) Birth Cohorts.

PubMed

Clemente, Diana B P; Casas, Maribel; Vilahur, Nadia; Begiristain, Haizea; Bustamante, Mariona; Carsin, Anne-Elie; Fernández, Mariana F; Fierens, Frans; Gyselaers, Wilfried; Iñiguez, Carmen; Janssen, Bram G; Lefebvre, Wouter; Llop, Sabrina; Olea, Nicolás; Pedersen, Marie; Pieters, Nicky; Santa Marina, Loreto; Souto, Ana; Tardón, Adonina; Vanpoucke, Charlotte; Vrijheid, Martine; Sunyer, Jordi; Nawrot, Tim S

2016-05-01

Mitochondria are sensitive to environmental toxicants due to their lack of repair capacity. Changes in mitochondrial DNA (mtDNA) content may represent a biologically relevant intermediate outcome in mechanisms linking air pollution and fetal growth restriction. We investigated whether placental mtDNA content is a possible mediator of the association between prenatal nitrogen dioxide (NO2) exposure and birth weight. We used data from two independent European cohorts: INMA (n = 376; Spain) and ENVIRONAGE (n = 550; Belgium). Relative placental mtDNA content was determined as the ratio of two mitochondrial genes (MT-ND1 and MTF3212/R3319) to two control genes (RPLP0 and ACTB). Effect estimates for individual cohorts and the pooled data set were calculated using multiple linear regression and mixed models. We also performed a mediation analysis. Pooled estimates indicated that a 10-μg/m3 increment in average NO2 exposure during pregnancy was associated with a 4.9% decrease in placental mtDNA content (95% CI: -9.3, -0.3%) and a 48-g decrease (95% CI: -87, -9 g) in birth weight. However, the association with birth weight was significant for INMA (-66 g; 95% CI: -111, -23 g) but not for ENVIRONAGE (-20 g; 95% CI: -101, 62 g). Placental mtDNA content was associated with significantly higher mean birth weight (pooled analysis, interquartile range increase: 140 g; 95% CI: 43, 237 g). Mediation analysis estimates, which were derived for the INMA cohort only, suggested that 10% (95% CI: 6.6, 13.0 g) of the association between prenatal NO2 and birth weight was mediated by changes in placental mtDNA content. Our results suggest that mtDNA content can be one of the potential mediators of the association between prenatal air pollution exposure and birth weight. Clemente DB, Casas M, Vilahur N, Begiristain H, Bustamante M, Carsin AE, Fernández MF, Fierens F, Gyselaers W, Iñiguez C, Janssen BG, Lefebvre W, Llop S, Olea N, Pedersen M, Pieters N, Santa Marina L, Souto A, Tardón A, Vanpoucke C, Vrijheid M, Sunyer J, Nawrot TS. 2016. Prenatal ambient air pollution, placental mitochondrial DNA content, and birth weight in the INMA (Spain) and ENVIRONAGE (Belgium) birth cohorts. Environ Health Perspect 124:659-665; http://dx.doi.org/10.1289/ehp.1408981.

Adaptation of Drosophila to a novel laboratory environment reveals temporally heterogeneous trajectories of selected alleles.

PubMed

Orozco-terWengel, Pablo; Kapun, Martin; Nolte, Viola; Kofler, Robert; Flatt, Thomas; Schlötterer, Christian

2012-10-01

The genomic basis of adaptation to novel environments is a fundamental problem in evolutionary biology that has gained additional importance in the light of the recent global change discussion. Here, we combined laboratory natural selection (experimental evolution) in Drosophila melanogaster with genome-wide next generation sequencing of DNA pools (Pool-Seq) to identify alleles that are favourable in a novel laboratory environment and traced their trajectories during the adaptive process. Already after 15 generations, we identified a pronounced genomic response to selection, with almost 5000 single nucleotide polymorphisms (SNP; genome-wide false discovery rates < 0.005%) deviating from neutral expectation. Importantly, the evolutionary trajectories of the selected alleles were heterogeneous, with the alleles falling into two distinct classes: (i) alleles that continuously rise in frequency; and (ii) alleles that at first increase rapidly but whose frequencies then reach a plateau. Our data thus suggest that the genomic response to selection can involve a large number of selected SNPs that show unexpectedly complex evolutionary trajectories, possibly due to nonadditive effects. © 2012 Blackwell Publishing Ltd.

Belimumab in the treatment of systemic lupus erythematosus: high disease activity predictors of response.

PubMed

van Vollenhoven, Ronald F; Petri, Michelle A; Cervera, Ricard; Roth, David A; Ji, Beulah N; Kleoudis, Christi S; Zhong, Z John; Freimuth, William

2012-08-01

To identify factors that predict response to belimumab treatment in the phase 3 BLISS trials of autoantibody-positive systemic lupus erythematosus (SLE) and further analyse clinical efficacy in various patient subsets. The BLISS trials compared belimumab 1 and 10 mg/kg versus placebo, all plus standard SLE therapy, over 52 or 76 weeks. Pooled subgroup analyses of week 52 SLE responder index rates (the primary endpoint in both trials) were performed based on demographic characteristics and baseline disease activity indicators. Pooled multivariate analysis was performed to determine predictors of response and treatment effect. Pooled univariate and multivariate analyses (N=1684) identified baseline factors associated with an increased benefit of belimumab versus placebo. These factors included the Safety Of Estrogens In Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI) ≥10, low complement, anti-dsDNA positivity and corticosteroid use. Efficacy outcomes were assessed in the low complement/anti-dsDNA-positive and SELENA-SLEDAI ≥10 subgroups. Week 52 SLE Responder Index rates in the low complement/anti-dsDNA-positive subgroup were 31.7%, 41.5% (p=0.002) and 51.5% (p<0.001) with placebo and belimumab 1 mg/kg and 10 mg/kg, respectively; corresponding rates in the SELENA-SLEDAI ≥10 subgroup were 44.3%, 58.0% (p<0.001) and 63.2% (p<0.001). Further analysis of secondary endpoints in the low complement/anti-dsDNA-positive subgroup showed that compared with placebo, belimumab produced greater benefits regarding severe flares, corticosteroid use and health-related quality of life. These findings suggest that belimumab has greater therapeutic benefit than standard therapy alone in patients with higher disease activity, anti-dsDNA positivity, low complement or corticosteroid treatment at baseline. CLINICALTRIALS.GOV: identifiers NCT00424476 and NCT00410384.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaplow, Irene M.; MacIsaac, Julia L.; Mah, Sarah M.

DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover <2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a trulymore » genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified more than 2000 genetic variants associated with DNA methylation. Here we found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data.« less

Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

PubMed Central

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

2013-01-01

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

Combinatorial pooling enables selective sequencing of the barley gene space.

PubMed

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

2013-04-01

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

Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells

PubMed Central

Yoneshima, Yasuto; Abolhassani, Nona; Iyama, Teruaki; Sakumi, Kunihiko; Shiomi, Naoko; Mori, Masahiko; Shiomi, Tadahiro; Noda, Tetsuo; Tsuchimoto, Daisuke; Nakabeppu, Yusaku

2016-01-01

Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity. PMID:27618981

The impact of natural transformation on adaptation in spatially structured bacterial populations.

PubMed

Moradigaravand, Danesh; Engelstädter, Jan

2014-06-20

Recent studies have demonstrated that natural transformation and the formation of highly structured populations in bacteria are interconnected. In spite of growing evidence about this connection, little is known about the dynamics of natural transformation in spatially structured bacterial populations. In this work, we model the interdependency between the dynamics of the bacterial gene pool and those of environmental DNA in space to dissect the effect of transformation on adaptation. Our model reveals that even with only a single locus under consideration, transformation with a free DNA fragment pool results in complex adaptation dynamics that do not emerge in previous models focusing only on the gene shuffling effect of transformation at multiple loci. We demonstrate how spatial restriction on population growth and DNA diffusion in the environment affect the impact of transformation on adaptation. We found that in structured bacterial populations intermediate DNA diffusion rates predominantly cause transformation to impede adaptation by spreading deleterious alleles in the population. Overall, our model highlights distinctive evolutionary consequences of bacterial transformation in spatially restricted compared to planktonic bacterial populations.

Enabling multiplexed testing of pooled donor cells through whole-genome sequencing.

PubMed

Chan, Yingleong; Chan, Ying Kai; Goodman, Daniel B; Guo, Xiaoge; Chavez, Alejandro; Lim, Elaine T; Church, George M

2018-04-19

We describe a method that enables the multiplex screening of a pool of many different donor cell lines. Our method accurately predicts each donor proportion from the pool without requiring the use of unique DNA barcodes as markers of donor identity. Instead, we take advantage of common single nucleotide polymorphisms, whole-genome sequencing, and an algorithm to calculate the proportions from the sequencing data. By testing using simulated and real data, we showed that our method robustly predicts the individual proportions from a mixed-pool of numerous donors, thus enabling the multiplexed testing of diverse donor cells en masse.More information is available at https://pgpresearch.med.harvard.edu/poolseq/.

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

PubMed Central

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

2012-01-01

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

Precise Estimation of Allele Frequencies of Single-Nucleotide Polymorphisms by a Quantitative SSCP Analysis of Pooled DNA

PubMed Central

Sasaki, Tomonari; Tahira, Tomoko; Suzuki, Akari; Higasa, Koichiro; Kukita, Yoji; Baba, Shingo; Hayashi, Kenshi

2001-01-01

We show that single-nucleotide polymorphisms (SNPs) of moderate to high heterozygosity (minor allele frequencies >10%) can be efficiently detected, and their allele frequencies accurately estimated, by pooling the DNA samples and applying a capillary-based SSCP analysis. In this method, alleles are separated into peaks, and their frequencies can be reliably and accurately quantified from their peak heights (SD <1.8%). We found that as many as 40% of publicly available SNPs that were analyzed by this method have widely differing allele frequency distributions among groups of different ethnicity (parents of Centre d'Etude Polymorphisme Humaine families vs. Japanese individuals). These results demonstrate the effectiveness of the present pooling method in the reevaluation of candidate SNPs that have been collected by examination of limited numbers of individuals. The method should also serve as a robust quantitative technique for studies in which a precise estimate of SNP allele frequencies is essential—for example, in linkage disequilibrium analysis. PMID:11083945

Getting it Right: How DNA Polymerases Select the Right Nucleotide.

PubMed

Ludmann, Samra; Marx, Andreas

2016-01-01

All living organisms are defined by their genetic code encrypted in their DNA. DNA polymerases are the enzymes that are responsible for all DNA syntheses occurring in nature. For DNA replication, repair and recombination these enzymes have to read the parental DNA and recognize the complementary nucleotide out of a pool of four structurally similar deoxynucleotide triphosphates (dNTPs) for a given template. The selection of the nucleotide is in accordance with the Watson-Crick rule. In this process the accuracy of DNA synthesis is crucial for the maintenance of the genome stability. However, to spur evolution a certain degree of freedom must be allowed. This brief review highlights the mechanistic basis for selecting the right nucleotide by DNA polymerases.

Singular and combined effects of blowdown, salvage logging, and wildfire on forest floor and soil mercury pools.

PubMed

Mitchell, Carl P J; Kolka, Randall K; Fraver, Shawn

2012-08-07

A number of factors influence the amount of mercury (Hg) in forest floors and soils, including deposition, volatile emission, leaching, and disturbances such as fire. Currently the impact on soil Hg pools from other widespread forest disturbances such as blowdown and management practices like salvage logging are unknown. Moreover, ecological and biogeochemical responses to disturbances are generally investigated within a single-disturbance context, with little currently known about the impact of multiple disturbances occurring in rapid succession. In this study we capitalize on a combination of blowdown, salvage logging and fire events in the sub-boreal region of northern Minnesota to assess both the singular and combined effects of these disturbances on forest floor and soil total Hg concentrations and pools. Although none of the disturbance combinations affected Hg in mineral soil, we did observe significant effects on both Hg concentrations and pools in the forest floor. Blowdown increased the mean Hg pool in the forest floor by 0.76 mg Hg m(-2) (223%). Salvage logging following blowdown created conditions leading to a significantly more severe forest floor burn during wildfire, which significantly enhanced Hg emission. This sequence of combined events resulted in a mean loss of approximately 0.42 mg Hg m(-2) (68% of pool) from the forest floor, after conservatively accounting for potential losses via enhanced soil leaching and volatile emissions between the disturbance and sampling dates. Fire alone or blowdown followed by fire did not significantly affect the total Hg concentrations or pools in the forest floor. Overall, unexpected consequences for soil Hg accumulation and by extension, atmospheric Hg emission and risk to aquatic biota, may result when combined impacts are considered in addition to singular forest floor and soil disturbances.

Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis.

PubMed

Lardizabal, K D; Metz, J G; Sakamoto, T; Hutton, W C; Pollard, M R; Lassner, M W

2000-03-01

Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.

Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

PubMed Central

Makarova, Alena V.; Grabow, Corinn; Gening, Leonid V.; Tarantul, Vyacheslav Z.; Tahirov, Tahir H.; Bessho, Tadayoshi; Pavlov, Youri I.

2011-01-01

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts. PMID:21304950

The mitochondrial DNA makeup of Romanians: A forensic mtDNA control region database and phylogenetic characterization.

PubMed

Turchi, Chiara; Stanciu, Florin; Paselli, Giorgia; Buscemi, Loredana; Parson, Walther; Tagliabracci, Adriano

2016-09-01

To evaluate the pattern of Romanian population from a mitochondrial perspective and to establish an appropriate mtDNA forensic database, we generated a high-quality mtDNA control region dataset from 407 Romanian subjects belonging to four major historical regions: Moldavia, Transylvania, Wallachia and Dobruja. The entire control region (CR) was analyzed by Sanger-type sequencing assays and the resulting 306 different haplotypes were classified into haplogroups according to the most updated mtDNA phylogeny. The Romanian gene pool is mainly composed of West Eurasian lineages H (31.7%), U (12.8%), J (10.8%), R (10.1%), T (9.1%), N (8.1%), HV (5.4%),K (3.7%), HV0 (4.2%), with exceptions of East Asian haplogroup M (3.4%) and African haplogroup L (0.7%). The pattern of mtDNA variation observed in this study indicates that the mitochondrial DNA pool is geographically homogeneous across Romania and that the haplogroup composition reveals signals of admixture of populations of different origin. The PCA scatterplot supported this scenario, with Romania located in southeastern Europe area, close to Bulgaria and Hungary, and as a borderland with respect to east Mediterranean and other eastern European countries. High haplotype diversity (0.993) and nucleotide diversity indices (0.00838±0.00426), together with low random match probability (0.0087) suggest the usefulness of this control region dataset as a forensic database in routine forensic mtDNA analysis and in the investigation of maternal genetic lineages in the Romanian population. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells.

PubMed

Kang, Shin-Young; Kim, Yeon-Gu; Kang, Seunghee; Lee, Hong Weon; Lee, Eun Gyo

2016-05-01

Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Uniparental genetic heritage of belarusians: encounter of rare middle eastern matrilineages with a central European mitochondrial DNA pool.

PubMed

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

2013-01-01

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

Suppression of the E. coli SOS response by dNTP pool changes.

PubMed

Maslowska, Katarzyna H; Makiela-Dzbenska, Karolina; Fijalkowska, Iwona J; Schaaper, Roel M

2015-04-30

The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30(+)-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction. Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Modification of CMV DNA detection from dried blood spots for diagnosing congenital CMV infection.

PubMed

Binda, Sandro; Caroppo, Simona; Didò, Patrizia; Primache, Valeria; Veronesi, Licia; Calvario, Agata; Piana, Andrea; Barbi, Maria

2004-07-01

Detection of viral DNA in dried blood spots using the Guthrie card (DBS test) is a reliable and practical method of diagnosing congenital cytomegalovirus (CMV) infection. The test lends itself to epidemiological studies to establish the prevalence of the infection, but also to neonatal screening for secondary prevention of sequelae. These applications would be facilitated if it were possible to use smaller samples and do the test on pools of individual cases. To ascertain whether doing the test on smaller, pooled samples still accurately identifies neonates with congenital CMV infection. We tested DBS from: (A) 39 laboratory reference cases; (B) 156 neonates suspected of having congenital CMV infection; (C) 119 children examined for the retrospective diagnosis of congenital CMV; (D) mock specimens prepared with known amounts of viral DNA. The test using only one third of the usual amount of dried blood was 100% sensitive and specific compared to the standard DBS test (A) and to viral isolation (A and B). Pools of three single cases gave the same results as viral isolation (B) and the small-sample test (B and C). All the versions of the test gave a detection limit of 400 copies/ml. The modified procedure can accurately diagnose congenital CMV infection. It achieves savings in both the patient material and the costs of testing.

Prenatal Ambient Air Pollution, Placental Mitochondrial DNA Content, and Birth Weight in the INMA (Spain) and ENVIRONAGE (Belgium) Birth Cohorts

PubMed Central

Clemente, Diana B.P.; Casas, Maribel; Vilahur, Nadia; Begiristain, Haizea; Bustamante, Mariona; Carsin, Anne-Elie; Fernández, Mariana F.; Fierens, Frans; Gyselaers, Wilfried; Iñiguez, Carmen; Janssen, Bram G.; Lefebvre, Wouter; Llop, Sabrina; Olea, Nicolás; Pedersen, Marie; Pieters, Nicky; Santa Marina, Loreto; Souto, Ana; Tardón, Adonina; Vanpoucke, Charlotte; Vrijheid, Martine; Sunyer, Jordi; Nawrot, Tim S.

2015-01-01

Background: Mitochondria are sensitive to environmental toxicants due to their lack of repair capacity. Changes in mitochondrial DNA (mtDNA) content may represent a biologically relevant intermediate outcome in mechanisms linking air pollution and fetal growth restriction. Objective: We investigated whether placental mtDNA content is a possible mediator of the association between prenatal nitrogen dioxide (NO2) exposure and birth weight. Methods: We used data from two independent European cohorts: INMA (n = 376; Spain) and ENVIRONAGE (n = 550; Belgium). Relative placental mtDNA content was determined as the ratio of two mitochondrial genes (MT-ND1 and MTF3212/R3319) to two control genes (RPLP0 and ACTB). Effect estimates for individual cohorts and the pooled data set were calculated using multiple linear regression and mixed models. We also performed a mediation analysis. Results: Pooled estimates indicated that a 10-μg/m3 increment in average NO2 exposure during pregnancy was associated with a 4.9% decrease in placental mtDNA content (95% CI: –9.3, –0.3%) and a 48-g decrease (95% CI: –87, –9 g) in birth weight. However, the association with birth weight was significant for INMA (–66 g; 95% CI: –111, –23 g) but not for ENVIRONAGE (–20 g; 95% CI: –101, 62 g). Placental mtDNA content was associated with significantly higher mean birth weight (pooled analysis, interquartile range increase: 140 g; 95% CI: 43, 237 g). Mediation analysis estimates, which were derived for the INMA cohort only, suggested that 10% (95% CI: 6.6, 13.0 g) of the association between prenatal NO2 and birth weight was mediated by changes in placental mtDNA content. Conclusion: Our results suggest that mtDNA content can be one of the potential mediators of the association between prenatal air pollution exposure and birth weight. Citation: Clemente DB, Casas M, Vilahur N, Begiristain H, Bustamante M, Carsin AE, Fernández MF, Fierens F, Gyselaers W, Iñiguez C, Janssen BG, Lefebvre W, Llop S, Olea N, Pedersen M, Pieters N, Santa Marina L, Souto A, Tardón A, Vanpoucke C, Vrijheid M, Sunyer J, Nawrot TS. 2016. Prenatal ambient air pollution, placental mitochondrial DNA content, and birth weight in the INMA (Spain) and ENVIRONAGE (Belgium) birth cohorts. Environ Health Perspect 124:659–665; http://dx.doi.org/10.1289/ehp.1408981 PMID:26317635

Comprehensive analysis of correlation coefficients estimated from pooling heterogeneous microarray data

PubMed Central

2013-01-01

Background The synthesis of information across microarray studies has been performed by combining statistical results of individual studies (as in a mosaic), or by combining data from multiple studies into a large pool to be analyzed as a single data set (as in a melting pot of data). Specific issues relating to data heterogeneity across microarray studies, such as differences within and between labs or differences among experimental conditions, could lead to equivocal results in a melting pot approach. Results We applied statistical theory to determine the specific effect of different means and heteroskedasticity across 19 groups of microarray data on the sign and magnitude of gene-to-gene Pearson correlation coefficients obtained from the pool of 19 groups. We quantified the biases of the pooled coefficients and compared them to the biases of correlations estimated by an effect-size model. Mean differences across the 19 groups were the main factor determining the magnitude and sign of the pooled coefficients, which showed largest values of bias as they approached ±1. Only heteroskedasticity across the pool of 19 groups resulted in less efficient estimations of correlations than did a classical meta-analysis approach of combining correlation coefficients. These results were corroborated by simulation studies involving either mean differences or heteroskedasticity across a pool of N > 2 groups. Conclusions The combination of statistical results is best suited for synthesizing the correlation between expression profiles of a gene pair across several microarray studies. PMID:23822712

Multiplexed direct genomic selection (MDiGS): a pooled BAC capture approach for highly accurate CNV and SNP/INDEL detection.

PubMed

Alvarado, David M; Yang, Ping; Druley, Todd E; Lovett, Michael; Gurnett, Christina A

2014-06-01

Despite declining sequencing costs, few methods are available for cost-effective single-nucleotide polymorphism (SNP), insertion/deletion (INDEL) and copy number variation (CNV) discovery in a single assay. Commercially available methods require a high investment to a specific region and are only cost-effective for large samples. Here, we introduce a novel, flexible approach for multiplexed targeted sequencing and CNV analysis of large genomic regions called multiplexed direct genomic selection (MDiGS). MDiGS combines biotinylated bacterial artificial chromosome (BAC) capture and multiplexed pooled capture for SNP/INDEL and CNV detection of 96 multiplexed samples on a single MiSeq run. MDiGS is advantageous over other methods for CNV detection because pooled sample capture and hybridization to large contiguous BAC baits reduces sample and probe hybridization variability inherent in other methods. We performed MDiGS capture for three chromosomal regions consisting of ∼ 550 kb of coding and non-coding sequence with DNA from 253 patients with congenital lower limb disorders. PITX1 nonsense and HOXC11 S191F missense mutations were identified that segregate in clubfoot families. Using a novel pooled-capture reference strategy, we identified recurrent chromosome chr17q23.1q23.2 duplications and small HOXC 5' cluster deletions (51 kb and 12 kb). Given the current interest in coding and non-coding variants in human disease, MDiGS fulfills a niche for comprehensive and low-cost evaluation of CNVs, coding, and non-coding variants across candidate regions of interest. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

DNA capture elements for rapid detection and identification of biological agents

NASA Astrophysics Data System (ADS)

Kiel, Johnathan L.; Parker, Jill E.; Holwitt, Eric A.; Vivekananda, Jeeva

2004-08-01

DNA capture elements (DCEs; aptamers) are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. Reporter molecules were attached to the finished sequences. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. These DCEs have demonstrated specificity and sensitivity equal to or better than antibody.

Design of DNA pooling to allow incorporation of covariates in rare variants analysis.

PubMed

Guan, Weihua; Li, Chun

2014-01-01

Rapid advances in next-generation sequencing technologies facilitate genetic association studies of an increasingly wide array of rare variants. To capture the rare or less common variants, a large number of individuals will be needed. However, the cost of a large scale study using whole genome or exome sequencing is still high. DNA pooling can serve as a cost-effective approach, but with a potential limitation that the identity of individual genomes would be lost and therefore individual characteristics and environmental factors could not be adjusted in association analysis, which may result in power loss and a biased estimate of genetic effect. For case-control studies, we propose a design strategy for pool creation and an analysis strategy that allows covariate adjustment, using multiple imputation technique. Simulations show that our approach can obtain reasonable estimate for genotypic effect with only slight loss of power compared to the much more expensive approach of sequencing individual genomes. Our design and analysis strategies enable more powerful and cost-effective sequencing studies of complex diseases, while allowing incorporation of covariate adjustment.

Whole Genome Association Study in a Homogenous Population in Shandong Peninsula of China Reveals JARID2 as a Susceptibility Gene for Schizophrenia

PubMed Central

Liu, Yang; Chen, Gang; Norton, Nadine; Liu, Wenmin; Zhu, Haining; Zhou, Peng; Luan, Meng; Yang, Shulin; Chen, Xing; Carroll, Liam; Williams, Nigel M.; O'Donovan, Michael C.; Kirov, George; Owen, Michael J.

2009-01-01

DNA pooling can provide an economic and efficient way to detect susceptibility loci to complex diseases. We carried out a genome screen with 400 microsatellite markers spaced at approximately 10 cm in two DNA pools consisting of 119 schizophrenia (SZ) patients and 119 controls recruited from a homogenous population in the Chang Le area of the Shandong peninsula of China. Association of D6S289, a dinucleotide repeat polymorphism in the JARID2 gene with SZ, was found and confirmed by individual genotyping (X2 = 17.89; P = .047). In order to refine the signal, we genotyped 14 single nucleotide polymorphisms (SNPs) covering JARID2 and the neighboring gene, DNTBP1, in an extended sample of 309 cases and 309 controls from Shandong peninsula (including the samples from the pools). However, rs2235258 and rs9654600 in JARID2 showed association in allelic, genotypic and haplotypic tests with SZ patients from Chang Le area. This was not replicates in the extended sample, we conclude that JARID2 could be a susceptibility gene for SZ. PMID:19884986

Intra- and Interlaboratory Variabilities of Results Obtained with the Quantiplex Human Immunodeficiency Virus Type 1 RNA bDNA Assay, Version 3.0

PubMed Central

Kellogg, James A.; Atria, Peter V.; Sanders, Jeffrey C.; Eyster, M. Elaine

2001-01-01

Normal assay variation associated with bDNA tests for human immunodeficiency virus type 1 (HIV-1) RNA performed at two laboratories with different levels of test experience was investigated. Two 5-ml aliquots of blood in EDTA tubes were collected from each patient for whom the HIV-1 bDNA test was ordered. Blood was stored for no more than 4 h at room temperature prior to plasma separation. Plasma was stored at −70°C until transported to the Central Pennsylvania Alliance Laboratory (CPAL; York, Pa.) and to the Hershey Medical Center (Hershey, Pa.) on dry ice. Samples were stored at ≤−70°C at both laboratories prior to testing. Pools of negative (donor), low-HIV-1-RNA-positive, and high-HIV-1-RNA-positive plasma samples were also repeatedly tested at CPAL to determine both intra- and interrun variation. From 11 August 1999 until 14 September 2000, 448 patient specimens were analyzed in parallel at CPAL and Hershey. From 206 samples with results of ≥1,000 copies/ml at CPAL, 148 (72%) of the results varied by ≤0.20 log10 when tested at Hershey and none varied by >0.50 log10. However, of 242 specimens with results of <1,000 copies/ml at CPAL, 11 (5%) of the results varied by >0.50 log10 when tested at Hershey. Of 38 aliquots of HIV-1 RNA pool negative samples included in 13 CPAL bDNA runs, 37 (97%) gave results of <50 copies/ml and 1 (3%) gave a result of 114 copies/ml. Low-positive HIV-1 RNA pool intrarun variation ranged from 0.06 to 0.26 log10 while the maximum interrun variation was 0.52 log10. High-positive HIV-1 RNA pool intrarun variation ranged from 0.04 to 0.32 log10, while the maximum interrun variation was 0.55 log10. In our patient population, a change in bDNA HIV-1 RNA results of ≤0.50 log10 over time most likely represents normal laboratory test variation. However, a change of >0.50 log10, especially if the results are >1,000 copies/ml, is likely to be significant. PMID:11329458

Intra- and interlaboratory variabilities of results obtained with the Quantiplex human immunodeficiency virus type 1 RNA bDNA assay, version 3.0.

PubMed

Kellogg, J A; Atria, P V; Sanders, J C; Eyster, M E

2001-05-01

Normal assay variation associated with bDNA tests for human immunodeficiency virus type 1 (HIV-1) RNA performed at two laboratories with different levels of test experience was investigated. Two 5-ml aliquots of blood in EDTA tubes were collected from each patient for whom the HIV-1 bDNA test was ordered. Blood was stored for no more than 4 h at room temperature prior to plasma separation. Plasma was stored at -70 degrees C until transported to the Central Pennsylvania Alliance Laboratory (CPAL; York, Pa.) and to the Hershey Medical Center (Hershey, Pa.) on dry ice. Samples were stored at < or =-70 degrees C at both laboratories prior to testing. Pools of negative (donor), low-HIV-1-RNA-positive, and high-HIV-1-RNA-positive plasma samples were also repeatedly tested at CPAL to determine both intra- and interrun variation. From 11 August 1999 until 14 September 2000, 448 patient specimens were analyzed in parallel at CPAL and Hershey. From 206 samples with results of > or =1,000 copies/ml at CPAL, 148 (72%) of the results varied by < or =0.20 log(10) when tested at Hershey and none varied by >0.50 log(10). However, of 242 specimens with results of <1,000 copies/ml at CPAL, 11 (5%) of the results varied by >0.50 log(10) when tested at Hershey. Of 38 aliquots of HIV-1 RNA pool negative samples included in 13 CPAL bDNA runs, 37 (97%) gave results of <50 copies/ml and 1 (3%) gave a result of 114 copies/ml. Low-positive HIV-1 RNA pool intrarun variation ranged from 0.06 to 0.26 log(10) while the maximum interrun variation was 0.52 log(10). High-positive HIV-1 RNA pool intrarun variation ranged from 0.04 to 0.32 log(10), while the maximum interrun variation was 0.55 log(10). In our patient population, a change in bDNA HIV-1 RNA results of < or =0.50 log(10) over time most likely represents normal laboratory test variation. However, a change of >0.50 log(10), especially if the results are >1,000 copies/ml, is likely to be significant.

West Nile virus, Anopheles flavivirus, a novel flavivirus as well as Merida-like rhabdovirus Turkey in field-collected mosquitoes from Thrace and Anatolia.

PubMed

Öncü, Ceren; Brinkmann, Annika; Günay, Filiz; Kar, Sırrı; Öter, Kerem; Sarıkaya, Yasemen; Nitsche, Andreas; Linton, Yvonne-Marie; Alten, Bülent; Ergünay, Koray

2018-01-01

Mosquitoes are involved in the transmission and maintenance of several viral diseases with significant health impact. Biosurveillance efforts have also revealed insect-specific viruses, observed to cocirculate with pathogenic strains. This report describes the findings of flavivirus and rhabdovirus screening, performed in eastern Thrace and Aegean region of Anatolia during 2016, including and expanding on locations with previously-documented virus activity. A mosquito cohort of 1545 individuals comprising 14 species were collected and screened in 108 pools via generic and specific amplification and direct metagenomics by next generation sequencing. Seven mosquito pools (6.4%) were positive in the flavivirus screening. West Nile virus lineage 1 clade 1a sequences were characterized in a pool Culex pipiens sensu lato specimens, providing the initial virus detection in Aegean region following 2010 outbreak. In an Anopheles maculipennis sensu lato pool, sequences closely-related to Anopheles flaviviruses were obtained, with similarities to several African and Australian strains of this new insect-specific flavivirus clade. In pools comprising Uranotaenia unguiculata (n=3), Cx. pipiens s.l. (n=1) and Aedes caspius (n=1) mosquitoes, sequences of a novel flavivirus, distantly-related to Flavivirus AV2011, identified previously in Spain and Turkey, were characterized. Moreover, DNA forms of the novel flavivirus were detected in two Ur. unguiculata pools. These sequences were highly-similar to the sequences amplified from viral RNA, with undisrupted reading frames, suggest the occurrence of viral DNA forms in natural conditions within mosquito hosts. Rhabdovirus screening revealed sequences of a recently-described novel virus, named the Merida-like virus Turkey (MERDLVT) in 5 Cx. pipiens s.l. pools (4.6%). Partial L and N gene sequences of MERDLVT were well-conserved among strains, with evidence for geographical clustering in phylogenetic analyses. Metagenomics provided the near-full genomic sequence in a specimen, revealing an identical genome organization and limited divergence from the prototype MERDLVT isolate. Copyright © 2017 Elsevier B.V. All rights reserved.

Human parvovirus PARV4 in plasma pools of Chinese origin.

PubMed

Ma, Y-Y; Guo, Y; Zhao, X; Wang, Z; Lv, M-M; Yan, Q-P; Zhang, J-G

2012-10-01

Human parvovirus 4 (PARV4) is present in blood and blood products. As the presence and levels of PARV4 in Chinese source plasma pools have never been determined, we implemented real-time quantitative PCR to investigate the presence of PARV4 in source plasma pools in China. Results showed that 26·15% (51/195) of lots tested positive for PARV4. The amounts of DNA ranged from 2·83 × 10(3) copies/ml to 2·35×10(7) copies/ml plasma. The high level of PARV4 in plasma pools may pose a potential risk to recipients. Further studies on the pathogenesis of PARV4 are urgently required. © 2012 The Author(s). Vox Sanguinis © 2012 International Society of Blood Transfusion.

Associations between polymorphisms in the NICD domain of bovine NOTCH1 gene and growth traits in Chinese Qinchuan cattle.

PubMed

Liu, Mei; Zhang, Chenge; Lai, Xinsheng; Xue, Jing; Lan, Xianyong; Lei, Chuzhao; Jia, Yutang; Chen, Hong

2017-05-01

NOTCH1 is one of the four mammalian Notch receptors, which is involved in the Notch signaling pathway. Specifically, NOTCH1 promotes the proliferation of myogenic precursor cells, and the NICD domain of NOTCH1 can impair regeneration of skeletal muscles. However, similar research on the bovine NOTCH1 gene is lacking. In this study, we detected the polymorphisms of the bovine NOTCH1 gene in a total of 448 individuals from Chinese Qinchuan cattle with DNA pooling, forced PCR-RFLP, and DNA sequencing methods. Five novel SNPs were identified within the NICD domain, and eight haplotypes comprising combinations of these five SNPs were studied as well. The association analysis of SNPs' effects with growth traits revealed that g.A48250G was significantly associated with body height, body weight, and height at hip cross, and that g.A49239C only showed significant associations with body height. This suggests that the NOTCH1 gene is a strong candidate gene that could be utilized as a promising marker in beef cattle breeding programs.

Defects in maintenance of mitochondrial DNA are associated with intramitochondrial nucleotide imbalances.

PubMed

Ashley, Neil; Adams, Susan; Slama, Abdelhamid; Zeviani, Massimo; Suomalainen, Anu; Andreu, Antonio L; Naviaux, Robert K; Poulton, Joanna

2007-06-15

Defects in mtDNA maintenance range from fatal multisystem childhood diseases, such as Alpers syndrome, to milder diseases in adults, including mtDNA depletion syndromes (MDS) and familial progressive external ophthalmoplegia (AdPEO). Most are associated with defects in genes involved in mitochondrial deoxynucleotide metabolism or utilization, such as mutations in thymidine kinase 2 (TK2) as well as the mtDNA replicative helicase, Twinkle and gamma polymerase (POLG). We have developed an in vitro system to measure incorporation of radiolabelled dNTPs into mitochondria of saponin permeabilized cells. We used this to compare the rates of mtDNA synthesis in cells from 12 patients with diseases of mtDNA maintenance. We observed reduced incorporation of exogenous alpha (32)P-dTTP in fibroblasts from a patient with Alpers syndrome associated with the A467T substitution in POLG, a patient with dGK mutations, and a patient with mtDNA depletion of unknown origin compared to controls. However, incorporation of alpha (32)P-dTTP relative to either cell doubling time or alpha (32)P-dCTP incorporation was increased in patients with thymidine kinase deficiency or PEO as the result of TWINKLE mutations compared with controls. The specific activity of newly synthesized mtDNA depends on the size of the endogenous pool diluting the exogenous labelled nucleotide. Our result is consistent with a deficiency in the intramitochondrial pool of dTTP relative to dCTP in cells from patients with TK2 deficiency and TWINKLE mutations. Such DNA precursor asymmetry could cause pausing of the replication complex and hence exacerbate the propensity for age-related mtDNA mutations. Because deviations from the normal concentrations of dNTPs are known to be mutagenic, we suggest that intramitochondrial nucleotide imbalance could underlie the multiple mtDNA mutations observed in these patients.

Comparative evaluation of hepatitis C virus RNA quantitation by branched DNA, NASBA, and monitor assays.

PubMed

Lunel, F; Cresta, P; Vitour, D; Payan, C; Dumont, B; Frangeul, L; Reboul, D; Brault, C; Piette, J C; Huraux, J M

1999-02-01

Several studies have shown a relationship between pretreatment hepatitis C virus (HCV) viral load and the response to interferon (IFN) therapy, creating a need for quantitative HCV-RNA assays. Here, we compared three commercial methods: nucleic acid sequence-based amplification NASBA (Organon), branched DNA 2.0 (bDNA) (Chiron), and Monitor (Roche), with reverse-transcription polymerase chain reaction (RT-PCR) as the reference. We assessed sensitivity and reproducibility on a well-characterized panel of sera (EUROHEP), a Chimp Rodney plasma pool, and samples from IFN-treated and -untreated patients with chronic hepatitis C caused by different HCV genotypes. The reproducibility of the NASBA and bDNA methods was slightly better than that of Monitor, especially for genotypes 2 and 4. NASBA had the highest sensitivity (99% vs. 94% and 88% with Monitor and bDNA, respectively), especially for the follow-up of patients on IFN. NASBA gave the highest HCV-RNA concentrations, which were approximately 10-fold more than with the bDNA assay and 100-fold more than with the Monitor kit. The linearity, tested on the chimp Rodney plasma pool, was better with bDNA for high viral load than with NASBA and Monitor, although for low concentration of HCV RNA, bDNA was negative. Pretreatment viral load was lower in patients who had a sustained virological response to IFN, although the bDNA method was not sensitive enough to quantify all pretreatment samples. This study indicates that gene amplification methods (NASBA or Monitor) have better sensitivity than bDNA assays for quantification of HCV RNA in patients with chronic HCV infection, although the bDNA and NASBA methods are more likely to quantify all genotypes. Prospective studies are needed to demonstrate the usefulness of quantitative assays for the follow-up of patients with chronic hepatitis C.

Phylogeographic Analysis of Mitochondrial DNA in Northern Asian Populations

PubMed Central

Derenko, Miroslava ; Malyarchuk, Boris ; Grzybowski, Tomasz ; Denisova, Galina ; Dambueva, Irina ; Perkova, Maria ; Dorzhu, Choduraa ; Luzina, Faina ; Lee, Hong Kyu ; Vanecek, Tomas ; Villems, Richard ; Zakharov, Ilia 

2007-01-01

To elucidate the human colonization process of northern Asia and human dispersals to the Americas, a diverse subset of 71 mitochondrial DNA (mtDNA) lineages was chosen for complete genome sequencing from the collection of 1,432 control-region sequences sampled from 18 autochthonous populations of northern, central, eastern, and southwestern Asia. On the basis of complete mtDNA sequencing, we have revised the classification of haplogroups A, D2, G1, M7, and I; identified six new subhaplogroups (I4, N1e, G1c, M7d, M7e, and J1b2a); and fully characterized haplogroups N1a and G1b, which were previously described only by the first hypervariable segment (HVS1) sequencing and coding-region restriction-fragment–length polymorphism analysis. Our findings indicate that the southern Siberian mtDNA pool harbors several lineages associated with the Late Upper Paleolithic and/or early Neolithic dispersals from both eastern Asia and southwestern Asia/southern Caucasus. Moreover, the phylogeography of the D2 lineages suggests that southern Siberia is likely to be a geographical source for the last postglacial maximum spread of this subhaplogroup to northern Siberia and that the expansion of the D2b branch occurred in Beringia ∼7,000 years ago. In general, a detailed analysis of mtDNA gene pools of northern Asians provides the additional evidence to rule out the existence of a northern Asian route for the initial human colonization of Asia. PMID:17924343

Phylogeographic analysis of mitochondrial DNA in northern Asian populations.

PubMed

Derenko, Miroslava; Malyarchuk, Boris; Grzybowski, Tomasz; Denisova, Galina; Dambueva, Irina; Perkova, Maria; Dorzhu, Choduraa; Luzina, Faina; Lee, Hong Kyu; Vanecek, Tomas; Villems, Richard; Zakharov, Ilia

2007-11-01

To elucidate the human colonization process of northern Asia and human dispersals to the Americas, a diverse subset of 71 mitochondrial DNA (mtDNA) lineages was chosen for complete genome sequencing from the collection of 1,432 control-region sequences sampled from 18 autochthonous populations of northern, central, eastern, and southwestern Asia. On the basis of complete mtDNA sequencing, we have revised the classification of haplogroups A, D2, G1, M7, and I; identified six new subhaplogroups (I4, N1e, G1c, M7d, M7e, and J1b2a); and fully characterized haplogroups N1a and G1b, which were previously described only by the first hypervariable segment (HVS1) sequencing and coding-region restriction-fragment-length polymorphism analysis. Our findings indicate that the southern Siberian mtDNA pool harbors several lineages associated with the Late Upper Paleolithic and/or early Neolithic dispersals from both eastern Asia and southwestern Asia/southern Caucasus. Moreover, the phylogeography of the D2 lineages suggests that southern Siberia is likely to be a geographical source for the last postglacial maximum spread of this subhaplogroup to northern Siberia and that the expansion of the D2b branch occurred in Beringia ~7,000 years ago. In general, a detailed analysis of mtDNA gene pools of northern Asians provides the additional evidence to rule out the existence of a northern Asian route for the initial human colonization of Asia.

Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villarroya, Joan, E-mail: joanvillarroya@gmail.com; Institut de Recerca l'Hospital de la Santa Creu i Sant Pau, Barcelona; Lara, Mari-Carmen

Highlights: {yields} We impaired TK2 expression in Ost TK1{sup -} cells via siRNA-mediated interference (TK2{sup -}). {yields} TK2 impairment caused severe mitochondrial DNA (mtDNA) depletion in quiescent cells. {yields} Despite mtDNA depletion, TK2{sup -} cells show high cytochrome oxidase activity. {yields} Depletion of mtDNA occurs without imbalance in the mitochondrial dNTP pool. {yields} Nuclear-encoded ENT1, DNA-pol {gamma}, TFAM and TP gene expression is lowered in TK2{sup -} cells. -- Abstract: The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed themore » first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1{sup -} cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase {gamma}, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity despite profound depletion in mtDNA levels.« less

Distribution and identification of sand flies naturally infected with Leishmania from the Southeastern Peruvian Amazon.

PubMed

Zorrilla, Victor; De Los Santos, Maxy B; Espada, Liz; Santos, Rocío Del Pilar; Fernandez, Roberto; Urquia, Albino; Stoops, Craig A; Ballard, Sarah-Blythe; Lescano, Andres G; Vásquez, Gissella M; Valdivia, Hugo O

2017-11-01

Cutaneous leishmaniasis (CL) is an important health problem in the New World affecting civilian and military populations that are frequently exposed in endemic settings. The Peruvian region of Madre de Dios located near the border with Brazil is one of the most endemic CL regions in South America with more than 4,451 reported cases between 2010 and 2015 according to the Peruvian epidemiology directorate. However, little is known regarding the diversity and distribution of sand fly vectors in this region. In this study, we aimed to characterize the sand fly fauna in this endemic setting and identify sand fly species naturally infected with Leishmania possibly involved in pathogen transmission. Sand fly collections were carried out during 2014 and 2015 in the communities of Flor de Acre, Villa Primavera, Mavila and Arca Pacahuara using CDC light traps and Shannon traps. Collected specimens were identified and non-blood-fed females were selected for Leishmania infection screening using kinetoplastid DNA-PCR (kDNA-PCR) and nested Real time PCR for species identification. A total of 10,897 phlebotomines belonging to the genus Lutzomyia (58 species) and Brumptomyia (2 species) were collected. Our study confirmed the widespread distribution and abundance of Lutzomyia (Trichophoromyia) spp. (24%), Lu. whitmani (19.4%) and Lu. yucumensis (15.8%) in the region. Analysis of Shannon diversity index indicates variability in sand fly composition across sites with Villa Primavera presenting the highest sand fly diversity and abundance. Leishmania screening by kDNA-PCR resulted in 45 positive pools collected from Flor de Acre (34 pools), Mavila (10 pools) and Arca Pacahuara (1 pool) and included 14 species: Lu. yucumensis, Lu. aragoi, Lu. sallesi, Lu. sherlocki, Lu. shawi, Lu. walkeri, Lu nevesi, Lu. migonei, Lu. davisi, Lu. carrerai, Lu. hirsuta, Lu. (Trichophoromyia) spp., Lu. llanosmartinsi and Lu. whitmani. Lutzomyia sherlocki, Lu. walkeri and Lu. llanosmartinsi had the highest infection rates (8%, 7% and 6%, respectively). We identified Leishmania guyanensis in two Lu. whitmani pools, and L. braziliensis in two Lu. llanosmartinsi pools and one Lu. davisi pool. Based on our collections there is high sand fly diversity in Madre de Dios, with differences in sand fly abundance and species composition across sites. We identified 14 sand fly species naturally infected with Leishmania spp., having detected natural infection with L. (V.) guyanensis and L. (V.) braziliensis in three sand fly species. These results suggest the presence of several potential vectors that vary in their spatial and geographical distribution, which could explain the high prevalence of CL cases in this region.

Multifunctional Nanomaterials Utilizing Hybridization Chain Reaction for Molecular Diagnostics and Bioanalytical Applications

NASA Astrophysics Data System (ADS)

Rana, Md. Muhit

DNA nanotechnology has shown great promise in molecular diagnostic, bioanalytical and biomedical applications. The great challenge of detecting target analytes, biomarkers and small molecules, in molecular diagnostics is low yield sensitivity. To address this challenge, different nanomaterials have been used for a long time and to date there is no such cost-effective bioanalytical technique which can detect these target biomarkers (DNA, RNA, circulating DNA/miRNA) or environmental heavy metal ions (Hg2+ and Ag+) in a cost-effective and efficient manner. Herein, we initially discuss two possible bioanalytical detection methods- a) colorimetric and b) fluorometric assays which are very popular nowadays due to their distinctive spectroscopic properties. Finally, we report the promising colorimetric assay using a novel DNA based amplification strategy know as hybridization chain reaction (HCR) for potential application in the visual detection of low copies of biomarkers (miRNAs as little as 20 femtomole in an RNA pool and cell extracts in seven different combinations and Ebola virus DNA as low as 400 attomoles in liquid biopsy mimics in sixteen different combinations), environmental and biological heavy metal ions (mercury and silver concentrations as low as 10 pM in water, soil and urine samples) and also successfully applied to a molecular logic gate operation to distinguish OR and AND logic gates. No results showed any false-positive or false-negative information. On the other hand, we also discuss the future possibilities of HCR amplification technology, which is very promising for fluorometric bioanalysis. The HCR based nanoprobe technology has numerous remarkable advantages over other methods. It is re-programmable, simple, inexpensive, easy to assemble and operate and can be performed with visual and spectroscopic read-outs upon recognition of the target analytes. This rapid, specific and sensitive approach for biomarkers and heavy metal ion detection generates an on-site signal while eliminating the use of sophisticated high-maintenance instrumentation. We demonstrate that this state-of-the-art technology and methodology can potentially serve as an alternative approach to detect novel disease biomarkers, small molecules and inorganic compounds. This approach can be combined with the current existing methods for real-time point-of-care molecular diagnostics and is significant for preclinical or clinical studies.

The lod score method.

PubMed

Rice, J P; Saccone, N L; Corbett, J

2001-01-01

The lod score method originated in a seminal article by Newton Morton in 1955. The method is broadly concerned with issues of power and the posterior probability of linkage, ensuring that a reported linkage has a high probability of being a true linkage. In addition, the method is sequential, so that pedigrees or lod curves may be combined from published reports to pool data for analysis. This approach has been remarkably successful for 50 years in identifying disease genes for Mendelian disorders. After discussing these issues, we consider the situation for complex disorders, where the maximum lod score (MLS) statistic shares some of the advantages of the traditional lod score approach but is limited by unknown power and the lack of sharing of the primary data needed to optimally combine analytic results. We may still learn from the lod score method as we explore new methods in molecular biology and genetic analysis to utilize the complete human DNA sequence and the cataloging of all human genes.

[Screening specific recognition motif of RNA-binding proteins by SELEX in combination with next-generation sequencing technique].

PubMed

Zhang, Lu; Xu, Jinhao; Ma, Jinbiao

2016-07-25

RNA-binding protein exerts important biological function by specifically recognizing RNA motif. SELEX (Systematic evolution of ligands by exponential enrichment), an in vitro selection method, can obtain consensus motif with high-affinity and specificity for many target molecules from DNA or RNA libraries. Here, we combined SELEX with next-generation sequencing to study the protein-RNA interaction in vitro. A pool of RNAs with 20 bp random sequences were transcribed by T7 promoter, and target protein was inserted into plasmid containing SBP-tag, which can be captured by streptavidin beads. Through only one cycle, the specific RNA motif can be obtained, which dramatically improved the selection efficiency. Using this method, we found that human hnRNP A1 RRMs domain (UP1 domain) bound RNA motifs containing AGG and AG sequences. The EMSA experiment indicated that hnRNP A1 RRMs could bind the obtained RNA motif. Taken together, this method provides a rapid and effective method to study the RNA binding specificity of proteins.

Mitochondrial DNA variants of Podolian cattle breeds testify for a dual maternal origin.

PubMed

Di Lorenzo, Piera; Lancioni, Hovirag; Ceccobelli, Simone; Colli, Licia; Cardinali, Irene; Karsli, Taki; Capodiferro, Marco Rosario; Sahin, Emine; Ferretti, Luca; Ajmone Marsan, Paolo; Sarti, Francesca Maria; Lasagna, Emiliano; Panella, Francesco; Achilli, Alessandro

2018-01-01

Over the past 15 years, 300 out of 6000 breeds of all farm animal species identified by the Food and Agriculture Organization of the United Nations (FAO) have gone extinct. Among cattle, many Podolian breeds are seriously endangered in various European areas. Podolian cattle include a group of very ancient European breeds, phenotypically close to the aurochs ancestors (Bos primigenius). The aim of the present study was to assess the genetic diversity of Podolian breeds and to reconstruct their origin. The mitochondrial DNA (mtDNA) control-regions of 18 Podolian breeds have been phylogenetically assessed. Nine non-Podolian breeds have been also included for comparison. The overall analysis clearly highlights some peculiarities in the mtDNA gene pool of some Podolian breeds. In particular, a principal component analysis point to a genetic proximity between five breeds (Chianina, Marchigiana, Maremmana, Podolica Italiana and Romagnola) reared in Central Italy and the Turkish Grey. We here propose the suggestive hypothesis of a dual ancestral contribution to the present gene pool of Podolian breeds, one deriving from Eastern European cattle; the other arising from the arrival of Middle Eastern cattle into Central Italy through a different route, perhaps by sea, ferried by Etruscan boats. The historical migration of Podolian cattle from North Eastern Europe towards Italy has not cancelled the mtDNA footprints of this previous ancient migration.

Application of subtracted gDNA microarray-assisted Bulked Segregant Analysis for rapid discovery of molecular markers associated with day-neutrality in strawberry (Fragaria x ananassa)

PubMed Central

Gor, Mian Chee; Mantri, Nitin; Pang, Edwin

2016-01-01

A Fragaria Discovery Panel (FDP; strawberry-specific SDA) containing 287 features was constructed by subtracting the pooled gDNA of nine non-angiosperm species from the pooled gDNA of five strawberry genotypes. This FDP was used for Bulk Segregant Analysis (BSA) to enable identification of molecular markers associated with day-neutrality. Analysis of hybridisation patterns of a short day (SD) DNA bulk and three day-neutral (DN) DNA bulks varying in flowering strength allowed identification of a novel feature, FaP2E11, closely linked to CYTOKININ OXIDASE 1 (CKX1) gene possibly involved in promoting flowering under non-inductive condition. The signal intensities of FaP2E11 feature obtained from the strong DN bulk (DN1) is three fold higher than the short day bulk (SD), indicating that the putative marker may linked to a CKX1 variant allele with lower enzyme activity. We propose a model for flowering regulation based on the hypothesis that flowering strength may be regulated by the copy number of FaP2E11-linked CKX1 alleles. This study demonstrates the feasibility of the SDA-based BSA approach for the identification of molecular markers associated with day-neutrality in strawberry. This innovative strategy is an efficient and cost-effective approach for molecular marker discovery. PMID:27586242

Most of the extant mtDNA boundaries in South and Southwest Asia were likely shaped during the initial settlement of Eurasia by anatomically modern humans

PubMed Central

Metspalu, Mait; Kivisild, Toomas; Metspalu, Ene; Parik, Jüri; Hudjashov, Georgi; Kaldma, Katrin; Serk, Piia; Karmin, Monika; Behar, Doron M; Gilbert, M Thomas P; Endicott, Phillip; Mastana, Sarabjit; Papiha, Surinder S; Skorecki, Karl; Torroni, Antonio; Villems, Richard

2004-01-01

Background Recent advances in the understanding of the maternal and paternal heritage of south and southwest Asian populations have highlighted their role in the colonization of Eurasia by anatomically modern humans. Further understanding requires a deeper insight into the topology of the branches of the Indian mtDNA phylogenetic tree, which should be contextualized within the phylogeography of the neighboring regional mtDNA variation. Accordingly, we have analyzed mtDNA control and coding region variation in 796 Indian (including both tribal and caste populations from different parts of India) and 436 Iranian mtDNAs. The results were integrated and analyzed together with published data from South, Southeast Asia and West Eurasia. Results Four new Indian-specific haplogroup M sub-clades were defined. These, in combination with two previously described haplogroups, encompass approximately one third of the haplogroup M mtDNAs in India. Their phylogeography and spread among different linguistic phyla and social strata was investigated in detail. Furthermore, the analysis of the Iranian mtDNA pool revealed patterns of limited reciprocal gene flow between Iran and the Indian sub-continent and allowed the identification of different assemblies of shared mtDNA sub-clades. Conclusions Since the initial peopling of South and West Asia by anatomically modern humans, when this region may well have provided the initial settlers who colonized much of the rest of Eurasia, the gene flow in and out of India of the maternally transmitted mtDNA has been surprisingly limited. Specifically, our analysis of the mtDNA haplogroups, which are shared between Indian and Iranian populations and exhibit coalescence ages corresponding to around the early Upper Paleolithic, indicates that they are present in India largely as Indian-specific sub-lineages. In contrast, other ancient Indian-specific variants of M and R are very rare outside the sub-continent. PMID:15339343

Purification of a Jojoba Embryo Wax Synthase, Cloning of its cDNA, and Production of High Levels of Wax in Seeds of Transgenic Arabidopsis

PubMed Central

Lardizabal, Kathryn D.; Metz, James G.; Sakamoto, Tetsuo; Hutton, William C.; Pollard, Michael R.; Lassner, Michael W.

2000-01-01

Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a β-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. 13C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds. PMID:10712527

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

ERIC Educational Resources Information Center

Lee, HwaYoung; Dodd, Barbara G.

2012-01-01

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

Recombinant protein production from stable mammalian cell lines and pools.

PubMed

Hacker, David L; Balasubramanian, Sowmya

2016-06-01

We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

Recent thymic emigrants and mature naive T cells exhibit differential DNA methylation at key cytokine loci.

PubMed

Berkley, Amy M; Hendricks, Deborah W; Simmons, Kalynn B; Fink, Pamela J

2013-06-15

Recent thymic emigrants (RTEs) are the youngest T cells in the lymphoid periphery and exhibit phenotypic and functional characteristics distinct from those of their more mature counterparts in the naive peripheral T cell pool. We show in this study that the Il2 and Il4 promoter regions of naive CD4(+) RTEs are characterized by site-specific hypermethylation compared with those of both mature naive (MN) T cells and the thymocyte precursors of RTEs. Thus, RTEs do not merely occupy a midpoint between the thymus and the mature T cell pool, but represent a distinct transitional T cell population. Furthermore, RTEs and MN T cells exhibit distinct CpG DNA methylation patterns both before and after activation. Compared with MN T cells, RTEs express higher levels of several enzymes that modify DNA methylation, and inhibiting methylation during culture allows RTEs to reach MN T cell levels of cytokine production. Collectively, these data suggest that the functional differences that distinguish RTEs from MN T cells are influenced by epigenetic mechanisms and provide clues to a mechanistic basis for postthymic maturation.

75 FR 65313 - Combined Notice of Filings #2

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-22

... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission Combined Notice of Filings 2 October 15...: 10/12/2010. Accession Number: 20101012-5363. Comment Date: 5 p.m. Eastern Time on Tuesday, November 2... Power Pool, Inc. Description: Southwest Power Pool, Inc. submits tariff filing per 35.13(a)(2)(iii...

76 FR 37802 - Combined Notice of Filings #2

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-28

... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission Combined Notice of Filings 2 Take notice that the Commission received the following electric rate filings: Docket Numbers: ER11-3243-001. Applicants: Southwest Power Pool, Inc. Description: Southwest Power Pool, Inc. submits tariff filing per: Amendment to Submission of Response to Reques...

The role and diagnostic value of cell-free DNA in systemic lupus erythematosus.

PubMed

Truszewska, Anna; Foroncewicz, Bartosz; Pączek, Leszek

2017-01-01

Cell-free DNA (cfDNA) represents a small fraction of total DNA pool that circulates freely in the blood both in normal and pathological conditions. Data indicate that cfDNA plays an important role in the pathogenesis of systemic lupus erythematosus (SLE) and hypomethylation may be crucial for its immunogenic properties. Although differences in quantification methodology hinder the comparison of results between the studies, it appears that levels of cfDNA are abnormally elevated in SLE patients and correlate with various antibody titers, but not with disease activity. Increased cfDNA concentration, however, may be associated with active lupus nephritis. Most of the studies confirmed apoptosis as the major cfDNA release mechanism in various conditions, but formation of neutrophil extracellular traps may significantly contribute to the cfDNA generation in SLE patients. In this review, we summarise current knowledge about the role and possible origin of cfDNA in SLE patients, and discuss why cfDNA testing for diagnostic and prognosis of SLE remains questionable.

Development of goose- and duck-specific DNA markers to determine sources of Escherichia coli in waterways.

PubMed

Hamilton, Matthew J; Yan, Tao; Sadowsky, Michael J

2006-06-01

The contamination of waterways with fecal material is a persistent threat to public health. Identification of the sources of fecal contamination is a vital component for abatement strategies and for determination of total maximum daily loads. While phenotypic and genotypic techniques have been used to determine potential sources of fecal bacteria in surface waters, most methods require construction of large known-source libraries, and they often fail to adequately differentiate among environmental isolates originating from different animal sources. In this study, we used pooled genomic tester and driver DNAs in suppression subtractive hybridizations to enrich for host source-specific DNA markers for Escherichia coli originating from locally isolated geese. Seven markers were identified. When used as probes in colony hybridization studies, the combined marker DNAs identified 76% of the goose isolates tested and cross-hybridized, on average, with 5% of the human E. coli strains and with less than 10% of the strains obtained from other animal hosts. In addition, the combined probes identified 73% of the duck isolates examined, suggesting that they may be useful for determining the contribution of waterfowl to fecal contamination. However, the hybridization probes reacted mainly with E. coli isolates obtained from geese in the upper midwestern United States, indicating that there is regional specificity of the markers identified. Coupled with high-throughput, automated macro- and microarray screening, these markers may provide a quantitative, cost-effective, and accurate library-independent method for determining the sources of genetically diverse E. coli strains for use in source-tracking studies. However, future efforts to generate DNA markers specific for E. coli must include isolates obtained from geographically diverse animal hosts.

Phylogenomics of Reichenowia parasitica, an Alphaproteobacterial Endosymbiont of the Freshwater Leech Placobdella parasitica

PubMed Central

Kvist, Sebastian; Narechania, Apurva; Oceguera-Figueroa, Alejandro; Fuks, Bella; Siddall, Mark E.

2011-01-01

Although several commensal alphaproteobacteria form close relationships with plant hosts where they aid in (e.g.,) nitrogen fixation and nodulation, only a few inhabit animal hosts. Among these, Reichenowia picta, R. ornata and R. parasitica, are currently the only known mutualistic, alphaproteobacterial endosymbionts to inhabit leeches. These bacteria are harbored in the epithelial cells of the mycetomal structures of their freshwater leech hosts, Placobdella spp., and these structures have no other obvious function than housing bacterial symbionts. However, the function of the bacterial symbionts has remained unclear. Here, we focused both on exploring the genomic makeup of R. parasitica and on performing a robust phylogenetic analysis, based on more data than previous hypotheses, to test its position among related bacteria. We sequenced a combined pool of host and symbiont DNA from 36 pairs of mycetomes and performed an in silico separation of the different DNA pools through subtractive scaffolding. The bacterial contigs were compared to 50 annotated bacterial genomes and the genome of the freshwater leech Helobdella robusta using a BLASTn protocol. Further, amino acid sequences inferred from the contigs were used as queries against the 50 bacterial genomes to establish orthology. A total of 358 orthologous genes were used for the phylogenetic analyses. In part, results suggest that R. parasitica possesses genes coding for proteins related to nitrogen fixation, iron/vitamin B translocation and plasmid survival. Our results also indicate that R. parasitica interacts with its host in part by transmembrane signaling and that several of its genes show orthology across Rhizobiaceae. The phylogenetic analyses support the nesting of R. parasitica within the Rhizobiaceae, as sister to a group containing Agrobacterium and Rhizobium species. PMID:22132238

Effect of cooling rate on sperm quality of cryopreserved Andalusian donkey spermatozoa.

PubMed

Demyda-Peyrás, S; Bottrel, M; Acha, D; Ortiz, I; Hidalgo, M; Carrasco, J J; Gómez-Arrones, V; Gósalvez, J; Dorado, J

2018-06-01

The aim of this study was to evaluate the effect of different cooling rates on post-thaw quality of cryopreserved donkey spermatozoa. Eighteen ejaculates from six adult Andalusian donkeys (three ejaculates per donkey) were collected using an artificial vagina. Pooled semen samples (two ejaculates per pool) were divided into three aliquots, and frozen in Gent freezing extender using three different cryopreservation protocols (P): P1 (conventional slow freezing, as control): semen pre-cooled in an Equitainer for 2 h and frozen in liquid nitrogen (LN 2 ) vapour; P2 (controlled pre-freeze cooling rate): semen pre-cooled at a controlled rate for 73 min and frozen in LN 2 vapour; and P3 (rapid freezing) semen frozen immediately in LN 2 vapour. After thawing at 37 °C for 30 s, semen samples were assessed for motility, morphology, acrosome and plasma membrane integrity; spermatozoa were also tested for DNA integrity. Significant (P < 0.01) differences were found between the cryopreservation protocols for all sperm parameters evaluated, except for DNA integrity. Semen samples frozen using P2 showed significantly (P < 0.01) higher values for sperm motility, morphology, sperm membrane integrity, and acrosome integrity. On the contrary, P3 reduced sperm motility (P < 0.01) and increased the percentage of spermatozoa with damaged plasma membrane (P < 0.001). In our study, we demonstrated that the sperm of Andalusian donkey is particularly sensitive to the cooling rate used before freezing. Furthermore, Andalusian donkey semen can be successfully cryopreserved using controlled cooling rates combined with freezing in LN 2 vapour. Copyright © 2018 Elsevier B.V. All rights reserved.

On the origins and admixture of Malagasy: new evidence from high-resolution analyses of paternal and maternal lineages.

PubMed

Tofanelli, Sergio; Bertoncini, Stefania; Castrì, Loredana; Luiselli, Donata; Calafell, Francesc; Donati, Giuseppe; Paoli, Giorgio

2009-09-01

The Malagasy have been shown to be a genetically admixed population combining parental lineages with African and South East Asian ancestry. In the present paper, we fit the Malagasy admixture history in a highly resolved phylogeographic framework by typing a large set of mitochondrial DNA and Y DNA markers in unrelated individuals from inland (Merina) and coastal (Antandroy, Antanosy, and Antaisaka) ethnic groups. This allowed performance of a multilevel analysis in which the diversity among main ethnic divisions, lineage ancestries, and modes of inheritance could be concurrently evaluated. Admixture was confirmed to result from the encounter of African and Southeast Asian people with minor recent male contributions from Europe. However, new scenarios are depicted about Malagasy admixture history. The distribution of ancestral components was ethnic and sex biased, with the Asian ancestry appearing more conserved in the female than in the male gene pool and in inland than in coastal groups. A statistic based on haplotype sharing (D(HS)), showing low sampling error and time linearity over the last 200 generations, was introduced here for the first time and helped to integrate our results with linguistic and archeological data. The focus about the origin of Malagasy lineages was enlarged in space and pushed back in time. Homelands could not be pinpointed but appeared to comprise two vast areas containing different populations from sub-Saharan Africa and South East Asia. The pattern of diffusion of uniparental lineages was compatible with at least two events: a primary admixture of proto-Malay people with Bantu speakers bearing a western-like pool of haplotypes, followed by a secondary flow of Southeastern Bantu speakers unpaired for gender (mainly male driven) and geography (mainly coastal).

First detections of Rickettsia helvetica and R. monacensis in ectoparasitic mites (Laelapidae and Trombiculidae) infesting rodents in south-western Slovakia.

PubMed

Miťková, Katarína; Berthová, Lenka; Kalúz, Stanislav; Kazimírová, Mária; Burdová, Lenka; Kocianová, Elena

2015-07-01

Information on circulation of rickettsiae between small mammals and their ectoparasitic mites is scarce. In this study, we investigated infestation rates of rodents with mites in some areas of SW Slovakia and the role of mites as possible vectors of pathogenic rickettsiae. A total of 615 rodents of five species were caught during 2010-2012. All individuals were examined for ectoparasites which resulted in 2821 mites belonging to three species of Laelapidae and six species of Trombiculidae. The most common Laelapidae species was Laelaps agilis (81.25 %), followed by Haemogamasus nidi, and Eulaelaps stabularis. Hirsutiella zachvatkini (16.52 %) was the dominant species of the family Trombiculidae. DNA extracted from rodent blood and ectoparasitic mites was examined for the presence of rickettsiae by PCR. By pooling mites, 345 pool samples were created, of which 112 (32.46 %) were found to be positive for the rickettsial DNA. From 487 examined rodent blood samples, rickettsial DNA was found in 46 (9.44 %). Sequencing DNA from the positive blood samples and mites revealed the identity of Rickettsia helvetica and Rickettsia monacensis. The results of the study suggest that ectoparasitic mites may be reservoirs as well as vectors of some pathogenic rickettsiae.

The Diagnostic Performance of Stool DNA Testing for Colorectal Cancer: A Systematic Review and Meta-Analysis.

PubMed

Zhai, Rong-Lin; Xu, Fei; Zhang, Pei; Zhang, Wan-Li; Wang, Hui; Wang, Ji-Liang; Cai, Kai-Lin; Long, Yue-Ping; Lu, Xiao-Ming; Tao, Kai-Xiong; Wang, Guo-Bin

2016-02-01

This meta-analysis was designed to evaluate the diagnostic performance of stool DNA testing for colorectal cancer (CRC) and compare the performance between single-gene and multiple-gene tests.MEDLINE, Cochrane, EMBASE databases were searched using keywords colorectal cancers, stool/fecal, sensitivity, specificity, DNA, and screening. Sensitivity analysis, quality assessments, and performance bias were performed for the included studies.Fifty-three studies were included in the analysis with a total sample size of 7524 patients. The studies were heterogeneous with regard to the genes being analyzed for fecal genetic biomarkers of CRC, as well as the laboratory methods being used for each assay. The sensitivity of the different assays ranged from 2% to 100% and the specificity ranged from 81% to 100%. The meta-analysis found that the pooled sensitivities for single- and multigene assays were 48.0% and 77.8%, respectively, while the pooled specificities were 97.0% and 92.7%. Receiver operator curves and diagnostic odds ratios showed no significant difference between both tests with regard to sensitivity or specificity.This meta-analysis revealed that using assays that evaluated multiple genes compared with single-gene assays did not increase the sensitivity or specificity of stool DNA testing in detecting CRC.

Antiretroviral Therapy-Induced Mitochondrial Toxicity: Potential Mechanisms Beyond Polymerase-γ Inhibition

PubMed Central

Selvaraj, Shanmugapriya; Ghebremichael, Musie; Li, Min; Foli, Yram; Langs-Barlow, Allison; Ogbuagu, Arit; Barakat, Lydia; Tubridy, Elizabeth; Edifor, Regina; Lam, Wing; Cheng, Yung-Chi; Paintsil, Elijah

2014-01-01

We hypothesized that competition between NRTI-triphosphate and endogenous deoxyribonucleoside triphosphate (dNTP) may lead to depletion of dNTP pools and mitochondrial dysfunction independent of Pol-γ inhibition. We collected peripheral blood mononuclear cells from 75 adults (25 cases: HIV-infected with mitochondrial toxicity, 25 HIV-infected positive controls, and 25 HIV-negative controls). We observed statistically significant individual and group differences in ribonucleotide (RN) and deoxyribonucleotide (dRN) pools. The median RN pool was 10062 (IQR, 7090 – 12590), 4360 (IQR, 3058 –6838), and 2968 (IQR, 2538 – 4436) pmol/106 cells for negative controls, positive controls, and cases, respectively. Cases had significantly higher absolute mtDNA copy number compared to negative controls (p<0.05). Cases had significantly higher expression of Pol-γ, nucleoside transporters, cellular kinases, and ABC compared to controls. Antiretroviral therapy perturbs ribonucleotide and deoxyribonucleotide pools. Depletion of RN and dRN pools may be associated with ART-induced mitochondrial toxicity independent of Pol-γ inhibition. PMID:24637942

Mitochondrial DNA perspective of Serbian genetic diversity.

PubMed

Davidovic, Slobodan; Malyarchuk, Boris; Aleksic, Jelena M; Derenko, Miroslava; Topalovic, Vladanka; Litvinov, Andrey; Stevanovic, Milena; Kovacevic-Grujicic, Natasa

2015-03-01

Although south-Slavic populations have been studied to date from various aspects, the population of Serbia, occupying the central part of the Balkan Peninsula, is still genetically understudied at least at the level of mitochondrial DNA (mtDNA) variation. We analyzed polymorphisms of the first and the second mtDNA hypervariable segments (HVS-I and HVS-II) and informative coding-region markers in 139 Serbians to shed more light on their mtDNA variability, and used available data on other Slavic and neighboring non-Slavic populations to assess their interrelations in a broader European context. The contemporary Serbian mtDNA profile is consistent with the general European maternal landscape having a substantial proportion of shared haplotypes with eastern, central, and southern European populations. Serbian population was characterized as an important link between easternmost and westernmost south-Slavic populations due to the observed lack of genetic differentiation with all other south-Slavic populations and its geographical positioning within the Balkan Peninsula. An increased heterogeneity of south Slavs, most likely mirroring turbulent demographic events within the Balkan Peninsula over time (i.e., frequent admixture and differential introgression of various gene pools), and a marked geographical stratification of Slavs to south-, east-, and west-Slavic groups, were also found. A phylogeographic analyses of 20 completely sequenced Serbian mitochondrial genomes revealed not only the presence of mtDNA lineages predominantly found within the Slavic gene pool (U4a2a*, U4a2a1, U4a2c, U4a2g, HV10), supporting a common Slavic origin, but also lineages that may have originated within the southern Europe (H5*, H5e1, H5a1v) and the Balkan Peninsula in particular (H6a2b and L2a1k). © 2014 Wiley Periodicals, Inc.

Comprehensive study of mtDNA among Southwest Asian dogs contradicts independent domestication of wolf, but implies dog–wolf hybridization

PubMed Central

Ardalan, Arman; Kluetsch, Cornelya F C; Zhang, Ai-bing; Erdogan, Metin; Uhlén, Mathias; Houshmand, Massoud; Tepeli, Cafer; Ashtiani, Seyed Reza Miraei; Savolainen, Peter

2011-01-01

Studies of mitochondrial DNA (mtDNA) diversity indicate explicitly that dogs were domesticated, probably exclusively, in southern East Asia. However, Southwest Asia (SwAsia) has had poor representation and geographical coverage in these studies. Other studies based on archaeological and genome-wide SNP data have suggested an origin of dogs in SwAsia. Hence, it has been suspected that mtDNA evidence for this scenario may have remained undetected. In the first comprehensive investigation of genetic diversity among SwAsian dogs, we analyzed 582 bp of mtDNA for 345 indigenous dogs from across SwAsia, and compared with 1556 dogs across the Old World. We show that 97.4% of SwAsian dogs carry haplotypes belonging to a universal mtDNA gene pool, but that only a subset of this pool, five of the 10 principal haplogroups, is represented in SwAsia. A high frequency of haplogroup B, potentially signifying a local origin, was not paralleled with the high genetic diversity expected for a center of origin. Meanwhile, 2.6% of the SwAsian dogs carried the rare non-universal haplogroup d2. Thus, mtDNA data give no indication that dogs originated in SwAsia through independent domestication of wolf, but dog–wolf hybridization may have formed the local haplogroup d2 within this region. Southern East Asia remains the only region with virtually full extent of genetic variation, strongly indicating it to be the primary and probably sole center of wolf domestication. An origin of dogs in southern East Asia may have been overlooked by other studies due to a substantial lack of samples from this region. PMID:22393507

Simultaneous identification and DNA barcoding of six Eimeria species infecting turkeys using PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus.

PubMed

Hafeez, Mian A; Shivaramaiah, Srichaitanya; Dorsey, Kristi Moore; Ogedengbe, Mosun E; El-Sherry, Shiem; Whale, Julia; Cobean, Julie; Barta, John R

2015-05-01

Species-specific PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus were generated that allow for the specific identification of the most common Eimeria species infecting turkeys (i.e., Eimeria adenoeides, Eimeria meleagrimitis, Eimeria gallopavonis, Eimeria meleagridis, Eimeria dispersa, and Eimeria innocua). PCR reaction chemistries were optimized with respect to divalent cation (MgCl2) and dNTP concentrations, as well as PCR cycling conditions (particularly anneal temperature for primers). Genomic DNA samples from single oocyst-derived lines of six Eimeria species were tested to establish specificity and sensitivity of these newly designed primer pairs. A mixed 60-ng total DNA sample containing 10 ng of each of the six Eimeria species was used as DNA template to demonstrate specific amplification of the correct product using each of the species-specific primer pairs. Ten nanograms of each of the five non-target Eimeria species was pooled to provide a non-target, control DNA sample suitable to test the specificity of each primer pair. The amplifications of the COI region with species-specific primer pairs from pooled samples yielded products of expected sizes (209 to 1,012 bp) and no amplification of non-target Eimeria sp. DNA was detected using the non-target, control DNA samples. These primer pairs specific for Eimeria spp. of turkeys did not amplify any of the seven Eimeria species infecting chickens. The newly developed PCR primers can be used as a diagnostic tool capable of specifically identifying six turkey Eimeria species; additionally, sequencing of the PCR amplification products yields sequence-based genotyping data suitable for identification and molecular phylogenetics.

Evaluation of single-nucleotide polymorphisms as internal controls in prenatal diagnosis of fetal blood groups.

PubMed

Doescher, Andrea; Petershofen, Eduard K; Wagner, Franz F; Schunter, Markus; Müller, Thomas H

2013-02-01

Determination of fetal blood groups in maternal plasma samples critically depends on adequate amplification of fetal DNA. We evaluated the routine inclusion of 52 single-nucleotide polymorphisms (SNPs) as internal reference in our polymerase chain reaction (PCR) settings to obtain a positive internal control for fetal DNA. DNA from 223 plasma samples of pregnant women was screened for RHD Exons 3, 4, 5, and 7 in a multiplex PCR including 52 SNPs divided into four primer pools. Amplicons were analyzed by single-base extension and the GeneScan method in a genetic analyzer. Results of D screening were compared to standard RHD genotyping of amniotic fluid or real-time PCR of fetal DNA from maternal plasma. The vast majority of all samples (97.8%) demonstrated differences in maternal and fetal SNP patterns when tested with four primer pools. These differences were not observed in less than 2.2% of the samples most probably due to an extraction failure for adequate amounts of fetal DNA. Comparison of the fetal genotypes with independent results did not reveal a single false-negative case among samples (n = 42) with positive internal control and negative fetal RHD typing. Coamplification of 52 SNPs with RHD-specific sequences for fetal blood group determination introduces a valid positive control for the amplification of fetal DNA to avoid false-negative results. This new approach does not require a paternal blood sample. It may also be applicable to other assays for fetal genotyping in maternal blood samples. © 2012 American Association of Blood Banks.

The Detection of Spotted Fever Group Rickettsia DNA in Tick Samples From Pastoral Communities in Kenya.

PubMed

Koka, Hellen; Sang, Rosemary; Kutima, Helen Lydia; Musila, Lillian

2017-05-01

In this study, ticks from pastoral communities in Kenya were tested for Rickettsia spp. infections in geographical regions where the presence of tick-borne arboviruses had previously been reported. Rickettsial and arbovirus infections have similar clinical features which makes differential diagnosis challenging when both diseases occur. The tick samples were tested for Rickettsia spp. by conventional PCR using three primer sets targeting the gltA, ompA, and ompB genes followed by amplicon sequencing. Of the tick pools screened, 25% (95/380) were positive for Rickettsia spp. DNA using the gltA primer set. Of the tick-positive pools, 60% were ticks collected from camels. Rickettsia aeschlimannii and R. africae were the main Rickettsia spp. detected in the tick pools sequenced. The findings of this study indicate that multiple Rickettsia species are circulating in ticks from pastoral communities in Kenya and could contribute to the etiology of febrile illness in these areas. Diagnosis and treatment of rickettsial infections should be a public health priority in these regions. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Reconciling evidence from ancient and contemporary genomes: a major source for the European Neolithic within Mediterranean Europe.

PubMed

Pereira, Joana B; Costa, Marta D; Vieira, Daniel; Pala, Maria; Bamford, Lisa; Harich, Nourdin; Cherni, Lotfi; Alshamali, Farida; Hatina, Jiři; Rychkov, Sergey; Stefanescu, Gheorghe; King, Turi; Torroni, Antonio; Soares, Pedro; Pereira, Luísa; Richards, Martin B

2017-03-29

Important gaps remain in our understanding of the spread of farming into Europe, due partly to apparent contradictions between studies of contemporary genetic variation and ancient DNA. It seems clear that farming was introduced into central, northern, and eastern Europe from the south by pioneer colonization. It is often argued that these dispersals originated in the Near East, where the potential source genetic pool resembles that of the early European farmers, but clear ancient DNA evidence from Mediterranean Europe is lacking, and there are suggestions that Mediterranean Europe may have resembled the Near East more than the rest of Europe in the Mesolithic. Here, we test this proposal by dating mitogenome founder lineages from the Near East in different regions of Europe. We find that whereas the lineages date mainly to the Neolithic in central Europe and Iberia, they largely date to the Late Glacial period in central/eastern Mediterranean Europe. This supports a scenario in which the genetic pool of Mediterranean Europe was partly a result of Late Glacial expansions from a Near Eastern refuge, and that this formed an important source pool for subsequent Neolithic expansions into the rest of Europe. © 2017 The Author(s).

Microbiological Analysis in Three Diverse Natural Geothermal Bathing Pools in Iceland

PubMed Central

Thorolfsdottir, Berglind Osk Th.; Marteinsson, Viggo Thor

2013-01-01

Natural thermal bathing pools contain geothermal water that is very popular to bathe in but the water is not sterilized, irradiated or treated in any way. Increasing tourism in Iceland will lead to increasing numbers of bath guests, which can in turn affect the microbial flora in the pools and therefore user safety. Today, there is no legislation that applies to natural geothermal pools in Iceland, as the water is not used for consumption and the pools are not defined as public swimming pools. In this study, we conducted a microbiological analysis on three popular but different natural pools in Iceland, located at Lýsuhóll, Hveravellir and Landmannalaugar. Total bacterial counts were performed by flow cytometry, and with plate count at 22 °C, 37 °C and 50 °C. The presence of viable coliforms, Enterococcus spp. and pseudomonads were investigated by growth experiments on selective media. All samples were screened for noroviruses by real time PCR. The results indicate higher fecal contamination in the geothermal pools where the geothermal water flow was low and bathing guest count was high during the day. The number of cultivated Pseudomonas spp. was high (13,000–40,000 cfu/100 mL) in the natural pools, and several strains were isolated and classified as opportunistic pathogens. Norovirus was not detected in the three pools. DNA was extracted from one-liter samples in each pool and analyzed by partial 16S rRNA gene sequencing. Microbial diversity analysis revealed different microbial communities between the pools and they were primarily composed of alpha-, beta- and gammaproteobacteria. PMID:23493033

78 FR 63915 - New Mailing Standards for Domestic Mailing Services Products

Federal Register 2010, 2011, 2012, 2013, 2014

2013-10-25

... locations. In addition, preparing FSS scheme pallets allows for the creation of larger pallets, which...-digit FSS scheme pools when addressed for delivery to any FSS 5-digit scheme combination per labeling... into a separate pool for each individual 5-digit FSS-scheme combination. Mailings that include 10 or...

Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens.

PubMed

Pillet, Sylvie; Verhoeven, Paul O; Epercieux, Amélie; Bourlet, Thomas; Pozzetto, Bruno

2015-06-01

A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA together with an internal control was developed on the BD Max platform combining automated DNA extraction and an open amplification procedure. Its performance was compared to those of PCR assays routinely used in the laboratory, namely, a laboratory-developed test for HSV DNA on the LightCycler instrument and a test using a commercial master mix for VZV DNA on the ABI7500fast system. Using a pool of negative cerebrospinal fluid (CSF) samples spiked with either calibrated controls for HSV-1 and VZV or dilutions of a clinical strain that was previously quantified for HSV-2, the empirical limit of detection of the BD Max assay was 195.65, 91.80, and 414.07 copies/ml for HSV-1, HSV-2, and VZV, respectively. All the samples from HSV and VZV DNA quality control panels (Quality Control for Molecular Diagnostics [QCMD], 2013, Glasgow, United Kingdom) were correctly identified by the BD Max assay. From 180 clinical specimens of various origins, 2 CSF samples were found invalid by the BD Max assay due to the absence of detection of the internal control; a concordance of 100% was observed between the BD Max assay and the corresponding routine tests. The BD Max assay detected the PCR signal 3 to 4 cycles earlier than did the routine methods. With results available within 2 h on a wide range of specimens, this sensitive and fully automated PCR assay exhibited the qualities required for detecting simultaneously HSV and VZV DNA on a routine basis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Changes of Sand Fly Populations and Leishmania infantum Infection Rates in an Irrigated Village Located in Arid Central Tunisia

PubMed Central

Barhoumi, Walid; Fares, Wasfi; Cherni, Saifedine; Derbali, Mohamed; Dachraoui, Khalil; Chelbi, Ifhem; Ramalho-Ortigao, Marcelo; Beier, John C.; Zhioua, Elyes

2016-01-01

The current spread of zoonotic visceral leishmaniasis (ZVL) throughout arid areas of Central Tunisia is a major public health concern. The main objective of this study is to investigate whether the development of irrigation in arid bio-geographical areas in Central Tunisia have led to the establishment of a stable cycle involving sand flies of the subgenus Larroussius and Leishmania infantum, and subsequently to the emergence of ZVL. Sand flies were collected from the village of Saddaguia, a highly irrigated zone located within an arid bio-geographical area of Central Tunisia by using modified Centers for Diseases Control (CDC) light traps. Morphological keys were used to identify sand flies. Collected sand flies were pooled with up to 30 specimens per pool according to date and tested by nested Polymerase Chain Reaction (PCR) DNA sequencing from positive pools was used to identify Leishmania spp. A total of 4915 sand flies (2422 females and 2493 males) were collected from Saddaguia in September and in October 2014. Morphological identification confirmed sand flies of the subgenus Larroussius to be predominant. PCR analysis followed by DNA sequencing indicated that 15 pools were infected with L. infantum yielding an overall infection rate of 0.6%. The majority of the infected pools were of sand fly species belonging to subgenus Larroussius. Intense irrigation applied to the arid bio-geographical areas in Central Tunisia is at the origin of the development of an environment capable of sustaining important populations of sand flies of the subgenus Larroussius. This has led to the establishment of stable transmission cycles of L. infantum and subsequently to the emergence of ZVL. PMID:26999176

Changes of Sand Fly Populations and Leishmania infantum Infection Rates in an Irrigated Village Located in Arid Central Tunisia.

PubMed

Barhoumi, Walid; Fares, Wasfi; Cherni, Saifedine; Derbali, Mohamed; Dachraoui, Khalil; Chelbi, Ifhem; Ramalho-Ortigao, Marcelo; Beier, John C; Zhioua, Elyes

2016-03-16

The current spread of zoonotic visceral leishmaniasis (ZVL) throughout arid areas of Central Tunisia is a major public health concern. The main objective of this study is to investigate whether the development of irrigation in arid bio-geographical areas in Central Tunisia have led to the establishment of a stable cycle involving sand flies of the subgenus Larroussius and Leishmania infantum, and subsequently to the emergence of ZVL. Sand flies were collected from the village of Saddaguia, a highly irrigated zone located within an arid bio-geographical area of Central Tunisia by using modified Centers for Diseases Control (CDC) light traps. Morphological keys were used to identify sand flies. Collected sand flies were pooled with up to 30 specimens per pool according to date and tested by nested Polymerase Chain Reaction (PCR) DNA sequencing from positive pools was used to identify Leishmania spp. A total of 4915 sand flies (2422 females and 2493 males) were collected from Saddaguia in September and in October 2014. Morphological identification confirmed sand flies of the subgenus Larroussius to be predominant. PCR analysis followed by DNA sequencing indicated that 15 pools were infected with L. infantum yielding an overall infection rate of 0.6%. The majority of the infected pools were of sand fly species belonging to subgenus Larroussius. Intense irrigation applied to the arid bio-geographical areas in Central Tunisia is at the origin of the development of an environment capable of sustaining important populations of sand flies of the subgenus Larroussius. This has led to the establishment of stable transmission cycles of L. infantum and subsequently to the emergence of ZVL.

An improved SELEX technique for selection of DNA aptamers binding to M-type 11 of Streptococcus pyogenes.

PubMed

Hamula, Camille L A; Peng, Hanyong; Wang, Zhixin; Tyrrell, Gregory J; Li, Xing-Fang; Le, X Chris

2016-03-15

Streptococcus pyogenes is a clinically important pathogen consisting of various serotypes determined by different M proteins expressed on the cell surface. The M type is therefore a useful marker to monitor the spread of invasive S. pyogenes in a population. Serotyping and nucleic acid amplification/sequencing methods for the identification of M types are laborious, inconsistent, and usually confined to reference laboratories. The primary objective of this work is to develop a technique that enables generation of aptamers binding to specific M-types of S. pyogenes. We describe here an in vitro technique that directly used live bacterial cells and the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) strategy. Live S. pyogenes cells were incubated with DNA libraries consisting of 40-nucleotides randomized sequences. Those sequences that bound to the cells were separated, amplified using polymerase chain reaction (PCR), purified using gel electrophoresis, and served as the input DNA pool for the next round of SELEX selection. A specially designed forward primer containing extended polyA20/5Sp9 facilitated gel electrophoresis purification of ssDNA after PCR amplification. A counter-selection step using non-target cells was introduced to improve selectivity. DNA libraries of different starting sequence diversity (10(16) and 10(14)) were compared. Aptamer pools from each round of selection were tested for their binding to the target and non-target cells using flow cytometry. Selected aptamer pools were then cloned and sequenced. Individual aptamer sequences were screened on the basis of their binding to the 10 M-types that were used as targets. Aptamer pools obtained from SELEX rounds 5-8 showed high affinity to the target S. pyogenes cells. Tests against non-target Streptococcus bovis, Streptococcus pneumoniae, and Enterococcus species demonstrated selectivity of these aptamers for binding to S. pyogenes. Several aptamer sequences were found to bind preferentially to the M11 M-type of S. pyogenes. Estimated binding dissociation constants (Kd) were in the low nanomolar range for the M11 specific sequences; for example, sequence E-CA20 had a Kd of 7±1 nM. These affinities are comparable to those of a monoclonal antibody. The improved bacterial cell-SELEX technique is successful in generating aptamers selective for S. pyogenes and some of its M-types. These aptamers are potentially useful for detecting S. pyogenes, achieving binding profiles of the various M-types, and developing new M-typing technologies for non-specialized laboratories or point-of-care testing. Copyright © 2015 Elsevier Inc. All rights reserved.

Transstadial Transmission of Hepatozoon canis by Rhipicephalus sanguineus (Acari: Ixodidae) in Field Conditions.

PubMed

Aktas, M; Özübek, S

2017-07-01

This study investigated possible transovarial and transstadial transmission of Hepatozoon canis by Rhipicephalus sanguineus (Latreille) ticks collected from naturally infected dogs in a municipal dog shelter and the grounds of the shelter. Four hundred sixty-five engorged nymphs were collected from 16 stray dogs that were found to be infected with H. canis by blood smear and PCR analyses and maintained in an incubator at 28 °C for moulting. Four hundred eighteen nymphs moulted to adults 14-16 d post collection. Unfed ticks from the shelter grounds comprised 1,500 larvae, 2,100 nymphs, and 85 adults; were sorted according to origin, developmental stage, and sex into 117 pools; and screened by 18S rRNA PCR for Hepatozoon infection. Of 60 adult tick pools examined, 51 were infected with H. canis. The overall maximum likelihood estimate (MLE) of infection rate was calculated as 21.0% (CI 15.80-28.21). Hepatozoon canis was detected in 31 out of 33 female pools (MLE 26.96%, CI 17.64-44.33) and 20 out of 27 male pools (MLE 14.82%, CI 20.15-46.41). Among 42 unfed nymph pools collected from the shelter, 26 were infected with H. canis, and MLE of infection was calculated as 1.9% (CI 1.25-2.77). No H. canis DNA was detected in any of the gDNA pools consisting of larva specimens. Partial sequences of the 18S rRNA gene shared 99-100% similarity with the corresponding H. canis isolates. Our results revealed the transstadial transmission of H. canis by R. sanguineus, both from larva to nymph and from nymph to adult, in field conditions. However, there were no evidence of transovarial transmission. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

PubMed Central

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

2013-01-01

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

In vitro and in vivo infectivity and pathogenicity of the lymphoid cell-derived woodchuck hepatitis virus.

PubMed

Lew, Y Y; Michalak, T I

2001-02-01

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (approximately 10(3) virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.

Isolation of candidate genes of Friedreich`s ataxia on chromosome 9q13

DOE Office of Scientific and Technical Information (OSTI.GOV)

Montermini, L.; Zara, F.; Pandolfo, M.

1994-09-01

Friedreich`s ataxia (FRDA) is an autosomal recessive degenerative disease involving the central and peripheral nervous system and the heart. The mutated gene in FRDA has recently been localized within a 450 Kb interval on chromosome 9q13 between the markers D9S202/FR1/FR8. We have been able to confirm such localization for the disease gene by analysis of extended haplotype in consanguineous families. Cases of loss of marker homozygosity, which are likely to be due to ancient recombinations, have been found to involve D9S110, D9S15, and D9S111 on the telomeric side, and FR5 on the centromeric side, while homozygosity was always found formore » a core haplotype including D9S5, FD1, and D9S202. We constructed a YAC contig spanning the region between the telomeric markers and FR5, and cosmids have been obtained from the YACs. In order to isolate transcribed sequences from the FRDA candidate region we are utilizing a combination of approaches, including hybridization of YACs and cosmids to an arrayed human heart cDNA library, cDNA direct selection, and exon amplification. A transcribed sequence near the telomeric end of the region has been isolated by cDNA direct selection using pooled cosmids as genomic template and primary human heart, muscle, brain, liver and placenta cDNAs as cDNA source. We have shown this sequence to be the human equivalent of ZO-2, a tight junction protein previously described in the dog. No mutations of this gene have been found in FRDA subjects. Additional cDNA have recently been isolated and they are currently being evaluated.« less

Gene expression deregulation in postnatal skeletal muscle of TK2 deficient mice reveals a lower pool of proliferating myogenic progenitor cells.

PubMed

Paredes, João A; Zhou, Xiaoshan; Höglund, Stefan; Karlsson, Anna

2013-01-01

Loss of thymidine kinase 2 (TK2) causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA) depletion syndrome (MDS) in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue.

The Dawn of Human Matrilineal Diversity

PubMed Central

Behar, Doron M.; Villems, Richard; Soodyall, Himla; Blue-Smith, Jason; Pereira, Luisa; Metspalu, Ene; Scozzari, Rosaria; Makkan, Heeran; Tzur, Shay; Comas, David; Bertranpetit, Jaume; Quintana-Murci, Lluis; Tyler-Smith, Chris; Wells, R. Spencer; Rosset, Saharon

2008-01-01

The quest to explain demographic history during the early part of human evolution has been limited because of the scarce paleoanthropological record from the Middle Stone Age. To shed light on the structure of the mitochondrial DNA (mtDNA) phylogeny at the dawn of Homo sapiens, we constructed a matrilineal tree composed of 624 complete mtDNA genomes from sub-Saharan Hg L lineages. We paid particular attention to the Khoi and San (Khoisan) people of South Africa because they are considered to be a unique relic of hunter-gatherer lifestyle and to carry paternal and maternal lineages belonging to the deepest clades known among modern humans. Both the tree phylogeny and coalescence calculations suggest that Khoisan matrilineal ancestry diverged from the rest of the human mtDNA pool 90,000–150,000 years before present (ybp) and that at least five additional, currently extant maternal lineages existed during this period in parallel. Furthermore, we estimate that a minimum of 40 other evolutionarily successful lineages flourished in sub-Saharan Africa during the period of modern human dispersal out of Africa approximately 60,000–70,000 ybp. Only much later, at the beginning of the Late Stone Age, about 40,000 ybp, did introgression of additional lineages occur into the Khoisan mtDNA pool. This process was further accelerated during the recent Bantu expansions. Our results suggest that the early settlement of humans in Africa was already matrilineally structured and involved small, separately evolving isolated populations. PMID:18439549

Gene Expression Deregulation in Postnatal Skeletal Muscle of TK2 Deficient Mice Reveals a Lower Pool of Proliferating Myogenic Progenitor Cells

PubMed Central

Paredes, João A.; Zhou, Xiaoshan; Höglund, Stefan; Karlsson, Anna

2013-01-01

Loss of thymidine kinase 2 (TK2) causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA) depletion syndrome (MDS) in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue. PMID:23341978

Inherited Mitochondrial Diseases of DNA Replication

PubMed Central

Copeland, William C.

2007-01-01

Mitochondrial genetic diseases can result from defects in mitochondrial DNA (mtDNA) in the form of deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These mutations may be spontaneous, maternally inherited, or a result of inherited nuclear defects in genes that maintain mtDNA. This review focuses on our current understanding of nuclear gene mutations that produce mtDNA alterations and cause mitochondrial depletion syndrome (MDS), progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). To date, all of these etiologic nuclear genes fall into one of two categories: genes whose products function directly at the mtDNA replication fork, such as POLG, POLG2, and TWINKLE, or genes whose products supply the mitochondria with deoxynucleotide triphosphate pools needed for DNA replication, such as TK2, DGUOK, TP, SUCLA2, ANT1, and possibly the newly identified MPV17. PMID:17892433

Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides.

PubMed

Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M; Högberg, Björn

2013-07-01

Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.

Hyper-Acetylation of Histone H3K56 Limits Break-Induced Replication by Inhibiting Extensive Repair Synthesis

PubMed Central

Che, Jun; Smith, Stephanie; Kim, Yoo Jung; Shim, Eun Yong; Myung, Kyungjae; Lee, Sang Eun

2015-01-01

Break-induced replication (BIR) has been implicated in restoring eroded telomeres and collapsed replication forks via single-ended invasion and extensive DNA synthesis on the recipient chromosome. Unlike other recombination subtypes, DNA synthesis in BIR likely relies heavily on mechanisms enabling efficient fork progression such as chromatin modification. Herein we report that deletion of HST3 and HST4, two redundant de-acetylases of histone H3 Lysine 56 (H3K56), inhibits BIR, sensitizes checkpoint deficient cells to deoxyribonucleotide triphosphate pool depletion, and elevates translocation-type gross chromosomal rearrangements (GCR). The basis for deficiency in BIR and gene conversion with long gap synthesis in hst3Δ hst4Δ cells can be traced to a defect in extensive DNA synthesis. Distinct from other cellular defects associated with deletion of HST3 and HST4 including thermo-sensitivity and elevated spontaneous mutagenesis, the BIR defect in hst3Δ hst4Δ cannot be offset by the deletion of RAD17 or MMS22, but rather by the loss of RTT109 or ASF1, or in combination with the H3K56R mutation, which also restores tolerance to replication stress in mrc1 mutants. Our studies suggest that acetylation of H3K56 limits extensive repair synthesis and interferes with efficient fork progression in BIR. PMID:25705897

DNA Damage and Oxidative Stress in Dyskeratosis Congenita: Analysis of Pathways and Therapeutic Stategies Using CPISPR and iPSC Model Systems

DTIC Science & Technology

2016-06-01

telomeres and characterized by a classical clinical triad of leukoplakia, skin dyspigmentation and nail dystrophy with concomitant marrow failure...DC symptomology, to a degree, corresponds to critically shortened telomeres that limits cellular replicative potential and thus prematurely exhausts...stem cell pools. Our previous findings support a hypothesis whereby shortened telomeres increase DNA damage responses within the cell leading to

Distribution and identification of sand flies naturally infected with Leishmania from the Southeastern Peruvian Amazon

PubMed Central

Zorrilla, Victor; De Los Santos, Maxy B.; Espada, Liz; Santos, Rocío del Pilar; Fernandez, Roberto; Urquia, Albino; Stoops, Craig A.; Ballard, Sarah-Blythe; Lescano, Andres G.; Vásquez, Gissella M.; Valdivia, Hugo O.

2017-01-01

Background Cutaneous leishmaniasis (CL) is an important health problem in the New World affecting civilian and military populations that are frequently exposed in endemic settings. The Peruvian region of Madre de Dios located near the border with Brazil is one of the most endemic CL regions in South America with more than 4,451 reported cases between 2010 and 2015 according to the Peruvian epidemiology directorate. However, little is known regarding the diversity and distribution of sand fly vectors in this region. In this study, we aimed to characterize the sand fly fauna in this endemic setting and identify sand fly species naturally infected with Leishmania possibly involved in pathogen transmission. Methods Sand fly collections were carried out during 2014 and 2015 in the communities of Flor de Acre, Villa Primavera, Mavila and Arca Pacahuara using CDC light traps and Shannon traps. Collected specimens were identified and non-blood-fed females were selected for Leishmania infection screening using kinetoplastid DNA-PCR (kDNA-PCR) and nested Real time PCR for species identification. Results A total of 10,897 phlebotomines belonging to the genus Lutzomyia (58 species) and Brumptomyia (2 species) were collected. Our study confirmed the widespread distribution and abundance of Lutzomyia (Trichophoromyia) spp. (24%), Lu. whitmani (19.4%) and Lu. yucumensis (15.8%) in the region. Analysis of Shannon diversity index indicates variability in sand fly composition across sites with Villa Primavera presenting the highest sand fly diversity and abundance. Leishmania screening by kDNA-PCR resulted in 45 positive pools collected from Flor de Acre (34 pools), Mavila (10 pools) and Arca Pacahuara (1 pool) and included 14 species: Lu. yucumensis, Lu. aragoi, Lu. sallesi, Lu. sherlocki, Lu. shawi, Lu. walkeri, Lu nevesi, Lu. migonei, Lu. davisi, Lu. carrerai, Lu. hirsuta, Lu. (Trichophoromyia) spp., Lu. llanosmartinsi and Lu. whitmani. Lutzomyia sherlocki, Lu. walkeri and Lu. llanosmartinsi had the highest infection rates (8%, 7% and 6%, respectively). We identified Leishmania guyanensis in two Lu. whitmani pools, and L. braziliensis in two Lu. llanosmartinsi pools and one Lu. davisi pool. Conclusions Based on our collections there is high sand fly diversity in Madre de Dios, with differences in sand fly abundance and species composition across sites. We identified 14 sand fly species naturally infected with Leishmania spp., having detected natural infection with L. (V.) guyanensis and L. (V.) braziliensis in three sand fly species. These results suggest the presence of several potential vectors that vary in their spatial and geographical distribution, which could explain the high prevalence of CL cases in this region. PMID:29107954

A Molecular Survey of Tick-Borne Pathogens from Ticks Collected in Central Queensland, Australia.

PubMed

Chalada, Melissa Judith; Stenos, John; Vincent, Gemma; Barker, Dayana; Bradbury, Richard Stewart

2018-03-01

Central Queensland (CQ) is a large and isolated, low population density, remote tropical region of Australia with a varied environment. The region has a diverse fauna and several species of ticks that feed upon that fauna. This study examined 518 individual ticks: 177 Rhipicephalus sanguineus (brown dog tick), 123 Haemaphysalis bancrofti (wallaby tick), 102 Rhipicephalus australis (Australian cattle tick), 47 Amblyomma triguttatum (ornate kangaroo tick), 57 Ixodes holocyclus (paralysis tick), 9 Bothriocroton tachyglossi (CQ short-beaked echidna tick), and 3 Ornithodoros capensis (seabird soft tick). Tick midguts were pooled by common host or environment and screened for four genera of tick-borne zoonoses by PCR and sequencing. The study examined a total of 157 midgut pools of which 3 contained DNA of Coxiella burnetii, 13 Rickettsia gravesii, 1 Rickettsia felis, and 4 other Rickettsia spp. No Borrelia spp. or Babesia spp. DNA were recovered.

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

NASA Astrophysics Data System (ADS)

Bercovici, Sivan; Geiger, Dan

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

Identification of presumed ancestral DNA sequences of phaseolin in Phaseolus vulgaris.

PubMed Central

Kami, J; Velásquez, V B; Debouck, D G; Gepts, P

1995-01-01

Common bean (Phaseolus vulgaris) consists of two major geographic gene pools, one distributed in Mexico, Central America, and Colombia and the other in the southern Andes (southern Peru, Bolivia, and Argentina). Amplification and sequencing of members of the multigene family coding for phaseolin, the major seed storage protein of the common bean, provide evidence for accumulation of tandem direct repeats in both introns and exons during evolution of the multigene family in this species. The presumed ancestral phaseolin sequences, without tandem repeats, were found in recently discovered but nearly extinct wild common bean populations of Ecuador and northern Peru that are intermediate between the two major gene pools of the species based on geographical and molecular arguments. Our results illustrate the usefulness of tandem direct repeats in establishing the polarity of DNA sequence divergence and therefore in proposing phylogenies. Images Fig. 1 Fig. 3 PMID:7862642

Unchanged thymidine triphosphate pools and thymidine metabolism in two lines of thymidine kinase 2-mutated fibroblasts.

PubMed

Frangini, Miriam; Rampazzo, Chiara; Franzolin, Elisa; Lara, Mari-Carmen; Vilà, Maya R; Martí, Ramon; Bianchi, Vera

2009-02-01

Mitochondrial thymidine kinase (TK2) catalyzes the phosphorylation of thymidine in mitochondria. Its function becomes essential for dTTP synthesis in noncycling cells, where cytosolic dTTP synthesis via R1/R2 ribonucleotide reductase and thymidine kinase 1 is turned down. Mutations in the nuclear gene for TK2 cause a fatal mtDNA depletion syndrome. Only selected cell types are affected, suggesting that the other cells compensate for the TK2 deficiency by adapting the enzyme network that regulates dTTP synthesis outside S-phase. Here we looked for such metabolic adaptation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activities of TK2, deoxycytidine kinase, thymidine phosphorylase, deoxynucleotidases and the amounts of the three ribonucleotide reductase subunits. Patient cells contained 40% or 5% TK2 activity and unchanged activities of the other enzymes. However, their mitochondrial and cytosolic dTTP pools were unchanged, and also the overall composition of the dNTP pools was normal. TK2-dependent phosphorylation of [(3)H]thymidine in intact cells and the turnover of the dTTP pool showed that even the fibroblasts with 5% residual TK2 activity synthesized dTTP at an almost normal rate. Normal fibroblasts apparently contain more TK2 than needed to maintain dTTP during quiescence, which would explain why TK2-mutated fibroblasts do not manifest mtDNA depletion despite their reduced TK2 activity.

Genomics of crop wild relatives: expanding the gene pool for crop improvement.

PubMed

Brozynska, Marta; Furtado, Agnelo; Henry, Robert J

2016-04-01

Plant breeders require access to new genetic diversity to satisfy the demands of a growing human population for more food that can be produced in a variable or changing climate and to deliver the high-quality food with nutritional and health benefits demanded by consumers. The close relatives of domesticated plants, crop wild relatives (CWRs), represent a practical gene pool for use by plant breeders. Genomics of CWR generates data that support the use of CWR to expand the genetic diversity of crop plants. Advances in DNA sequencing technology are enabling the efficient sequencing of CWR and their increased use in crop improvement. As the sequencing of genomes of major crop species is completed, attention has shifted to analysis of the wider gene pool of major crops including CWR. A combination of de novo sequencing and resequencing is required to efficiently explore useful genetic variation in CWR. Analysis of the nuclear genome, transcriptome and maternal (chloroplast and mitochondrial) genome of CWR is facilitating their use in crop improvement. Genome analysis results in discovery of useful alleles in CWR and identification of regions of the genome in which diversity has been lost in domestication bottlenecks. Targeting of high priority CWR for sequencing will maximize the contribution of genome sequencing of CWR. Coordination of global efforts to apply genomics has the potential to accelerate access to and conservation of the biodiversity essential to the sustainability of agriculture and food production. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

The influence of ethnicity in the association of WC, WHR, hypertension and PGC-1α (Gly482Ser), UCP2 -866 G/A and SIRT1 -1400 T/C polymorphisms with T2D in the population of Punjab.

PubMed

Kaul, Nabodita; Singh, Yoginder P; Bhanwer, A J S

2015-06-01

To assess the effect of ethnicity, the association of WC, WHR and hypertension along with PGC-1α (Gly482Ser), UCP2 -866 G/A and SIRT1 -1400 T/C polymorphisms in seven endogamous caste groups and pooled population of Punjab. Study was conducted on 1813 individuals (859 T2D patients and 954 healthy controls) belonging to seven endogamous groups. Waist and hip circumference, height, weight and blood pressure were recorded following standard protocol using designed performa. PGC-1α (Gly482Ser) and UCP2 -866 G/A polymorphisms were genotyped using PCR RFLP and SIRT1 -1400 T/C was genotyped by direct DNA sequencing. WHR conferred risk in Brahmins (p=0.00003), Khtaris (p=0.001) and SCs (p=0.02). Similarly, we detected that WC conferred risk in BCs (p=0.012), Brahmins (p=0.016), Jat Sikhs (0.00025), Khatris (0.005) and SCs (p=0.015). In pooled population, all three factors imparted risk (WHR (p=0.00001), hypertension (p=0.003) and WC (p=0.0000016)). With respect to gene polymorphism, PGC-1α (Gly482Ser) was associated in Banias (p=0.0003), Jat Sikhs (p=0.003) and Khatris (p=0.03). Similarly, UCP2 -866 G>A showed risk in Banias (p=0.000004), BCs (p=0.01) and SCs (p=0.01). However, SIRT1 -1400 T>C showed risk only in Khatris (p=0.004). In the pooled population of Punjab, both PGC-1α (Gly482Ser) [p=0.001] and UCP2 -866 G>A (p=0.0001) polymorphisms provided risk. Interaction analysis showed 72% of the patients had risk combination of PGC-1α XA and UCP2-866 XA genotypes. Based on the data, Khatris were found to be showing the highest susceptibility to T2D followed by SCs. Different combinations of factors provided risk in each caste group and in pooled population. Therefore, to curve the menace of T2D, detailed information about the ethnic background of the individual will be very useful for proper medical intervention. Copyright © 2015. Published by Elsevier B.V.

Identification of susceptibility genes and genetic modifiers of human diseases

NASA Astrophysics Data System (ADS)

Abel, Kenneth; Kammerer, Stefan; Hoyal, Carolyn; Reneland, Rikard; Marnellos, George; Nelson, Matthew R.; Braun, Andreas

2005-03-01

The completion of the human genome sequence enables the discovery of genes involved in common human disorders. The successful identification of these genes is dependent on the availability of informative sample sets, validated marker panels, a high-throughput scoring technology, and a strategy for combining these resources. We have developed a universal platform technology based on mass spectrometry (MassARRAY) for analyzing nucleic acids with high precision and accuracy. To fuel this technology, we generated more than 100,000 validated assays for single nucleotide polymorphisms (SNPs) covering virtually all known and predicted human genes. We also established a large DNA sample bank comprised of more than 50,000 consented healthy and diseased individuals. This combination of reagents and technology allows the execution of large-scale genome-wide association studies. Taking advantage of MassARRAY"s capability for quantitative analysis of nucleic acids, allele frequencies are estimated in sample pools containing large numbers of individual DNAs. To compare pools as a first-pass "filtering" step is a tremendous advantage in throughput and cost over individual genotyping. We employed this approach in numerous genome-wide, hypothesis-free searches to identify genes associated with common complex diseases, such as breast cancer, osteoporosis, and osteoarthritis, and genes involved in quantitative traits like high density lipoproteins cholesterol (HDL-c) levels and central fat. Access to additional well-characterized patient samples through collaborations allows us to conduct replication studies that validate true disease genes. These discoveries will expand our understanding of genetic disease predisposition, and our ability for early diagnosis and determination of specific disease subtype or progression stage.

Long Term Expression of Drosophila melanogaster Nucleoside Kinase in Thymidine Kinase 2-deficient Mice with No Lethal Effects Caused by Nucleotide Pool Imbalances*

PubMed Central

Krishnan, Shuba; Paredes, João A.; Zhou, Xiaoshan; Kuiper, Raoul V.; Hultenby, Kjell; Curbo, Sophie; Karlsson, Anna

2014-01-01

Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2−/−) mice extended the life span of Tk2−/− mice from 3 weeks to at least 20 months. The Dm-dNK+/−Tk2−/− mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK+/−Tk2−/− mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK+/− mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues. PMID:25296759

Long term expression of Drosophila melanogaster nucleoside kinase in thymidine kinase 2-deficient mice with no lethal effects caused by nucleotide pool imbalances.

PubMed

Krishnan, Shuba; Paredes, João A; Zhou, Xiaoshan; Kuiper, Raoul V; Hultenby, Kjell; Curbo, Sophie; Karlsson, Anna

2014-11-21

Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2(-/-)) mice extended the life span of Tk2(-/-) mice from 3 weeks to at least 20 months. The Dm-dNK(+/-)Tk2(-/-) mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK(+/-)Tk2(-/-) mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK(+/-) mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Circulating human papillomavirus DNA as a surveillance tool in head and neck squamous cell carcinoma: a systematic review and meta-analysis.

PubMed

Jensen, Kristina Kvist; Grønhøj, Christian; Jensen, David H; von Buchwald, Christian

2018-05-15

The incidence of human papillomavirus-induced (HPV+) head and neck squamous cell carcinoma (HNSCC) i.e. especially oropharyngeal cancers (OPSCC) is increasing and a significant proportion of patients encounter disease progression. A simple and sensitive test to identify patients with progression is an unmet need. To systematically review the literature and carry out a meta-analysis of studies, investigated circulating HPV-DNA as a biomarker for disease progression in patients with HNSCC. A systematic review and meta-analysis. PubMed, Embase and the Cochrane Library were systematically searched for articles published in English from January 1980 to November 2017. Search terms used were related to HPV, cancer sites, blood-based biomarkers and terms for specific use settings. Articles reviewed and selected by authors and data on study design, demographic variables, location, HPV status, number of pre treatment blood tests, number of post treatment blood tests, blood HPV status and number of recurrences and length of follow-up was extracted. A meta-analysis of HPV-DNA as a diagnostic test for recurrence by means of a hierarchical summary receiver operating curve (HSROC) model was performed. We identified five studies (n=600 subjects) examining circulating HPV-DNA in patients with HNSCC. In these five studies (n=411) patients had both pre and post treatment blood samples. The pooled sensitivity, in detecting a recurrence was estimated to be 54% (95% CI: 32%-74%), while the pooled specificity was 98% (95% CI: 93-99.4%). The pooled false-positive rate is 2% (95% CI: 0.6%-7%). The area under the curve (AUC) of the summary HSROC was 0.93. Positive predictive value was estimated to 93% and the negative predictive value to 94%. Plasma HPV-DNA is a promising tool for surveillance in patients with HPV-related HNSCC i.e. OPSCC and has a high specificity. By recent technical advances and by increasing follow-up blood samples the sensitivity could likely be improved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Mucopolysaccharidosis VI in cats - clarification regarding genetic testing.

PubMed

Lyons, Leslie A; Grahn, Robert A; Genova, Francesca; Beccaglia, Michela; Hopwood, John J; Longeri, Maria

2016-07-02

The release of new DNA-based diagnostic tools has increased tremendously in companion animals. Over 70 different DNA variants are now known for the cat, including DNA variants in disease-associated genes and genes causing aesthetically interesting traits. The impact genetic tests have on animal breeding and health management is significant because of the ability to control the breeding of domestic cats, especially breed cats. If used properly, genetic testing can prevent the production of diseased animals, causing the reduction of the frequency of the causal variant in the population, and, potentially, the eventual eradication of the disease. However, testing of some identified DNA variants may be unwarranted and cause undo strife within the cat breeding community and unnecessary reduction of gene pools and availability of breeding animals. Testing for mucopolysaccharidosis Type VI (MPS VI) in cats, specifically the genetic testing of the L476P (c.1427T>C) and the D520N (c.1558G>A) variants in arylsulfatase B (ARSB), has come under scrutiny. No health problems are associated with the D520N (c.1558G>A) variant, however, breeders that obtain positive results for this variant are speculating as to possible correlation with health concerns. Birman cats already have a markedly reduced gene pool and have a high frequency of the MPS VI D520N variant. Further reduction of the gene pool by eliminating cats that are heterozygous or homozygous for only the MPS VI D520N variant could lead to more inbreeding depression effects on the breed population. Herein is debated the genetic testing of the MPS VI D520N variant in cats. Surveys from different laboratories suggest the L476P (c.1427T>C) disease-associated variant should be monitored in the cat breed populations, particularly breeds with Siamese derivations and outcrosses. However, the D520N has no evidence of association with disease in cats and testing is not recommended in the absence of L476P genotyping. Selection against the D520N is not warranted in cat populations. More rigorous guidelines may be required to support the genetic testing of DNA variants in all animal species.

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

PubMed Central

2012-01-01

Background The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility. Results In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9% (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom® MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total). Conclusions The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving interval plausibly harbouring causative variants contributing to heritable variation. To our knowledge, this is the first report describing sequencing of targeted genomic regions in any livestock species using groups with divergent phenotypes for an economically important trait. PMID:22235840

Empirical Validation of Pooled Whole Genome Population Re-Sequencing in Drosophila melanogaster

PubMed Central

Zhu, Yuan; Bergland, Alan O.; González, Josefa; Petrov, Dmitri A.

2012-01-01

The sequencing of pooled non-barcoded individuals is an inexpensive and efficient means of assessing genome-wide population allele frequencies, yet its accuracy has not been thoroughly tested. We assessed the accuracy of this approach on whole, complex eukaryotic genomes by resequencing pools of largely isogenic, individually sequenced Drosophila melanogaster strains. We called SNPs in the pooled data and estimated false positive and false negative rates using the SNPs called in individual strain as a reference. We also estimated allele frequency of the SNPs using “pooled” data and compared them with “true” frequencies taken from the estimates in the individual strains. We demonstrate that pooled sequencing provides a faithful estimate of population allele frequency with the error well approximated by binomial sampling, and is a reliable means of novel SNP discovery with low false positive rates. However, a sufficient number of strains should be used in the pooling because variation in the amount of DNA derived from individual strains is a substantial source of noise when the number of pooled strains is low. Our results and analysis confirm that pooled sequencing is a very powerful and cost-effective technique for assessing of patterns of sequence variation in populations on genome-wide scales, and is applicable to any dataset where sequencing individuals or individual cells is impossible, difficult, time consuming, or expensive. PMID:22848651

Lager Yeast Comes of Age

PubMed Central

2014-01-01

Alcoholic fermentations have accompanied human civilizations throughout our history. Lager yeasts have a several-century-long tradition of providing fresh beer with clean taste. The yeast strains used for lager beer fermentation have long been recognized as hybrids between two Saccharomyces species. We summarize the initial findings on this hybrid nature, the genomics/transcriptomics of lager yeasts, and established targets of strain improvements. Next-generation sequencing has provided fast access to yeast genomes. Its use in population genomics has uncovered many more hybridization events within Saccharomyces species, so that lager yeast hybrids are no longer the exception from the rule. These findings have led us to propose network evolution within Saccharomyces species. This “web of life” recognizes the ability of closely related species to exchange DNA and thus drain from a combined gene pool rather than be limited to a gene pool restricted by speciation. Within the domesticated lager yeasts, two groups, the Saaz and Frohberg groups, can be distinguished based on fermentation characteristics. Recent evidence suggests that these groups share an evolutionary history. We thus propose to refer to the Saaz group as Saccharomyces carlsbergensis and to the Frohberg group as Saccharomyces pastorianus based on their distinct genomes. New insight into the hybrid nature of lager yeast will provide novel directions for future strain improvement. PMID:25084862

Haplotype combination of the bovine INSIG1 gene sequence variants and association with growth traits in Nanyang cattle.

PubMed

Sun, Jiajie; Gao, Yuan; Liu, Dong; Ma, Wei; Xue, Jing; Zhang, Chunlei; Lan, Xianyong; Lei, Chuzhao; Chen, Hong

2012-06-01

The insulin-induced gene 1 (INSIG1) gene encodes a protein that blocks proteolytic activation of sterol regulatory element binding proteins, which are transcription factors that activate genes that regulate cholesterol, fatty acid, and glucose metabolism. However, similar research for the bovine INSIG1 gene is lacking. Therefore, in this study, polymorphisms of the bovine INSIG1 gene were detected in 643 individuals from four cattle breeds by DNA pooling, forced PCR-RFLP, PCR-SSCP, and DNA sequencing methods. Only 10 novel SNPs were identified, which included four mutations in the coding region and the others in the introns. In Nanyang individuals, seven common haplotypes were identified based on four coding region SNPs. The haplotype GACT, with a frequency of 75.4%, was the most prevalent haplotypes and SNPs formed two linkage disequilibrium blocks with strong multi-allelic D' (D' = 1). Additionally, association analysis between mutations of the bovine INSIG1 gene and growth traits in Nanyang cattle at 6, 12, 18, and 24 months old was performed, and the results indicated that the polymorphisms were not significantly associated with body mass.

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants.

PubMed

Williams, Lindsey N; Marjavaara, Lisette; Knowels, Gary M; Schultz, Eric M; Fox, Edward J; Chabes, Andrei; Herr, Alan J

2015-05-12

Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

Routine screening of blood donations at Qingdao central blood bank, China, for hepatitis B virus (HBV) DNA with a real-time, multiplex nucleic acid test for HBV, hepatitis C virus, and human immunodeficiency virus Types 1 and 2.

PubMed

Yang, Zhongsi; Xu, Lei; Liu, Li; Feng, Qiuxia; Zhang, Longmu; Ma, Weijuan; Saldanha, John; Wang, Mingmin; Zhao, Lin

2013-10-01

The Roche cobas TaqScreen MPX test was used to evaluate the rate of hepatitis B surface antigen (HBsAg)-negative donations that were hepatitis B virus (HBV) DNA reactive from June 2010 to January 2011 in Qingdao, China. HBsAg-negative samples from 65,800 voluntary blood donors were tested with the cobas TaqScreen MPX test in pools of 6 on the Roche cobas s 201 blood screening platform. Samples positive for HBV DNA and negative for HBsAg were quantitated with the Roche COBAS AmpliPrep/COBAS TaqMan HBV test. In addition, serologic tests for HBsAg, hepatitis B surface antibody, anti-hepatitis B core antigen (anti-HBc), anti-hepatitis B e antigen (anti-HBe), and hepatitis B e antigen (HBe) were done using the Roche electrochemiluminescence immunoassay. A total of 80 nucleic acid amplification technology (NAT) test-reactive pools were identified and 59 pools (74%) resolved to a reactive sample. All samples were HBV DNA reactive and the viral load in each sample was quantitated. The viral loads of the samples ranged from less than 20 to 34,600 IU/mL; 13 samples (22%) had viral loads of more than 20 IU/mL, 27 samples (45.8%) had viral loads of less than 20 IU/mL, and 19 samples (32.2%) had undetectable viral loads. Of the 59 NAT-reactive samples, 40 (67.8%) were anti-HBc positive. Fifteen of the 59 samples could not be confirmed as NAT reactive either by an alternative NAT test or by serology. The HBV NAT yield in blood donors in Qingdao is 0.06% (38/65,800). This study confirmed the value of NAT for interdicting HBV-positive donations and preventing transfusion-transmitted HBV infections. © 2013 American Association of Blood Banks.

The color(s) of human hair--forensic hair analysis with SpectraCube.

PubMed

Birngruber, Christoph; Ramsthaler, Frank; Verhoff, Marcel A

2009-03-10

Human hair is among the most common kind of evidence secured at crime scenes. Although DNA analysis through STR-typing is possible in principle, it is not very promising for telogenic hair or single hairs. For the mixed traces frequently found in practice, composed of different hair from an unknown number of individuals, mtDNA sequencing of each individual hair seems to be the only possible, even if technically elaborate, solution. If it were possible to pool all hair belonging to an individual prior to DNA analysis, then this effort could not only be reduced, but the number of hair for an STR-approach could also be increased. Although it is possible to examine hair microscopically, this method must be considered unsuitable for pooling, since the results depend strongly on examiner experience, and the hair cannot always be correctly attributed to an individual. The goal of this study was to develop an objective non-DNA-contaminative pooling method for hair. To this end, the efficacy of spectral imaging as a method of obtaining information--beyond that obtained from a purely microscopic and morphological approach--for the identification of individuals was investigated. Three hairs each from 25 test persons (female: 18; male: 7) were examined with a SpectraCube-System and a light microscope. Six spectra were calculated for each hair, and the hairs from each individual were not only compared to each other, but also to those of the other individuals. From a forensic vantage, the examination showed, in particular, that individuals, whose hair could not be distinguished on the basis of morphology, could also not be accurately distinguished with the SpectraCube. The intra-individual differences were, in part, greater than the inter-individual differences. Altogether, the study shows that a person's hair color, as perceived, is composed of many naturally different, individual colors.

The blue man: burn from muriatic acid combined with chlorinated paint in an adult pool construction worker.

PubMed

O'Cleireachain, Marc R; Macias, Luis H; Richey, Karen J; Pressman, Melissa A; Shirah, Gina R; Caruso, Daniel M; Foster, Kevin N; Matthews, Marc R

2014-01-01

Muriatic acid (hydrochloric acid), a common cleaning and resurfacing agent for concrete pools, can cause significant burn injuries. When coating a pool with chlorinated rubber-based paint, the pool surface is initially cleansed using 31.45% muriatic acid. Here we report a 50-year-old Hispanic male pool worker who, during the process of a pool resurfacing, experienced significant contact exposure to a combination of muriatic acid and blue chlorinated rubber-based paint. Confounding the clinical situation was the inability to efficiently remove the chemical secondary to the rubber-based nature of the paint. Additionally, vigorous attempts were made to remove the rubber paint using a variety of agents, including bacitracin, chlorhexidine soap, GOOP adhesive, and Johnson's baby oil. Resultant injuries were devastating fourth-degree burns requiring an immediate operative excision and amputation. Despite aggressive operative intervention and resuscitation, he continued to have severe metabolic derangements and ultimately succumbed to his injuries. We present our attempts at debridement and the system in place to manage patients with complex chemical burns.

Deoxynucleoside stress exacerbates the phenotype of a mouse model of mitochondrial neurogastrointestinal encephalopathy

PubMed Central

Garcia-Diaz, Beatriz; Garone, Caterina; Barca, Emanuele; Mojahed, Hamed; Gutierrez, Purification; Pizzorno, Giuseppe; Tanji, Kurenai; Arias-Mendoza, Fernando; Quinzii, Caterina M.

2014-01-01

Balanced pools of deoxyribonucleoside triphosphate precursors are required for DNA replication, and alterations of this balance are relevant to human mitochondrial diseases including mitochondrial neurogastrointestinal encephalopathy. In this disease, autosomal recessive TYMP mutations cause severe reductions of thymidine phosphorylase activity; marked elevations of the pyrimidine nucleosides thymidine and deoxyuridine in plasma and tissues, and somatic multiple deletions, depletion and site-specific point mutations of mitochondrial DNA. Thymidine phosphorylase and uridine phosphorylase double knockout mice recapitulated several features of these patients including thymidine phosphorylase activity deficiency, elevated thymidine and deoxyuridine in tissues, mitochondrial DNA depletion, respiratory chain defects and white matter changes. However, in contrast to patients with this disease, mutant mice showed mitochondrial alterations only in the brain. To test the hypothesis that elevated levels of nucleotides cause unbalanced deoxyribonucleoside triphosphate pools and, in turn, pathogenic mitochondrial DNA instability, we have stressed double knockout mice with exogenous thymidine and deoxyuridine, and assessed clinical, neuroradiological, histological, molecular, and biochemical consequences. Mutant mice treated with exogenous thymidine and deoxyuridine showed reduced survival, body weight, and muscle strength, relative to untreated animals. Moreover, in treated mutants, leukoencephalopathy, a hallmark of the disease, was enhanced and the small intestine showed a reduction of smooth muscle cells and increased fibrosis. Levels of mitochondrial DNA were depleted not only in the brain but also in the small intestine, and deoxyribonucleoside triphosphate imbalance was observed in the brain. The relative proportion, rather than the absolute amount of deoxyribonucleoside triphosphate, was critical for mitochondrial DNA maintenance. Thus, our results demonstrate that stress of exogenous pyrimidine nucleosides enhances the mitochondrial phenotype of our knockout mice. Our mouse studies provide insights into the pathogenic role of thymidine and deoxyuridine imbalance in mitochondrial neurogastrointestinal encephalopathy and an excellent model to study new therapeutic approaches. PMID:24727567

Transgene expression of Drosophila melanogaster nucleoside kinase reverses mitochondrial thymidine kinase 2 deficiency.

PubMed

Krishnan, Shuba; Zhou, Xiaoshan; Paredes, João A; Kuiper, Raoul V; Curbo, Sophie; Karlsson, Anna

2013-02-15

A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK(+/-) transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK(+/-)TK2(-/-) mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK(+/-)TK2(-/-) mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency.

Recent thymic emigrants and mature naïve T cells exhibit differential DNA methylation at key cytokine loci

PubMed Central

Berkley, Amy M.; Hendricks, Deborah W.; Simmons, Kalynn B.; Fink, Pamela J.

2013-01-01

Recent thymic emigrants (RTEs) are the youngest T cells in the lymphoid periphery, and exhibit phenotypic and functional characteristics distinct from those of their more mature counterparts in the naïve peripheral T cell pool. We show here that the Il2 and Il4 promoter regions of naïve CD4+ RTEs are characterized by site-specific hypermethylation compared to those of both mature naïve (MN) T cells and the thymocyte precursors of RTEs. Thus, RTEs do not merely occupy a midpoint between the thymus and the mature T cell pool, but represent a distinct transitional T cell population. Furthermore, RTEs and MN T cells exhibit distinct CpG DNA methylation patterns both before and after activation. Compared to MN T cells, RTEs express higher levels of several enzymes that modify DNA methylation, and inhibiting methylation during culture allows RTEs to reach MN T cell levels of cytokine production. Collectively, these data suggest that the functional differences that distinguish RTEs from MN T cells are influenced by epigenetic mechanisms and provide clues to a mechanistic basis for post-thymic maturation. PMID:23686491

DNA Probe Pooling for Rapid Delineation of Chromosomal Breakpoints

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Chun-Mei; Kwan, Johnson; Baumgartner, Adolf

2009-01-30

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

Comparative genome-wide mapping versus extreme pool-genotyping and development of diagnostic SNP markers linked to QTL for adult plant resistance to stripe rust in common wheat.

PubMed

Wu, Jianhui; Huang, Shuo; Zeng, Qingdong; Liu, Shengjie; Wang, Qilin; Mu, Jingmei; Yu, Shizhou; Han, Dejun; Kang, Zhensheng

2018-06-16

A major stripe rust resistance QTL on chromosome 4BL was localized to a 4.5-Mb interval using comparative QTL mapping methods and validated in 276 wheat genotypes by haplotype analysis. CYMMIT-derived wheat line P10103 was previously identified to have adult plant resistance (APR) to stripe rust in the greenhouse and field. The conventional approach for QTL mapping in common wheat is laborious. Here, we performed QTL detection of APR using a combination of genome-wide scanning and extreme pool-genotyping. SNP-based genetic maps were constructed using the Wheat55 K SNP array to genotype a recombinant inbred line (RIL) population derived from the cross Mingxian 169 × P10103. Five stable QTL were detected across multiple environments. A fter comparing SNP profiles from contrasting, extreme DNA pools of RILs six putative QTL were located to approximate chromosome positions. A major QTL on chromosome 4B was identified in F 2:4 contrasting pools from cross Zhengmai 9023 × P10103. A consensus QTL (LOD = 26-40, PVE = 42-55%), named QYr.nwafu-4BL, was defined and localized to a 4.5-Mb interval flanked by SNP markers AX-110963704 and AX-110519862 in chromosome arm 4BL. Based on stripe rust response, marker genotypes, pedigree analysis and mapping data, QYr.nwafu-4BL is likely to be a new APR QTL. The applicability of the SNP-based markers flanking QYr.nwafu-4BL was validated on a diversity panel of 276 wheat lines. The additional minor QTL on chromosomes 4A, 5A, 5B and 6A enhanced the level of resistance conferred by QYr.nwafu-4BL. Marker-assisted pyramiding of QYr.nwafu-4BL and other favorable minor QTL in new wheat cultivars should improve the level of APR to stripe rust.

Development of the laboratory prototype "CavyPool" for assessing treatments and materials for swimming pools.

PubMed

Valeriani, F; Gianfranceschi, G; Vitali, M; Protano, C; Romano Spica, V

2017-01-01

Hygiene and surveillance in swimming pools are established by WHO Guidelines and national laws. Progress in water management and pool construction is revolutionizing the field, introducing new materials, systems, disinfection procedures or monitoring markers. Innovation advances challenge the upgrading of safety and quality in pools and the appropriate implementation of guidelines. In order to provide a device for laboratory test, a prototype was realized and applied to study and compare swimming pool materials and treatments. A pool scale-model was engineered and evaluated by computational fluid dynamics algorithms. An automated real time monitoring assured steady state. Critical control points along the water circuit were made accessible to allow the placing of different biocides or water sampling. Simulations were safely performed in a standard hood. Materials for pool surfaces and pipelines were evaluated for biofilm formation under different disinfection conditions. Adherent microorganisms were assayed by mfDNA analysis using real time PCR. The prototype reached the steady state within 5-25 hours under different conditions, showing chemical, physical and fluid-dynamic stability. A method was optimized for testing materials showing their different response to biofilm induction. Several innovative PVC samples displayed highest resistance to bacterial adhesion. A device and method was developed for testing swimming pool hygienic parameters in laboratory. It allowed to test materials for pools hygiene and maintenance, including biofilm formation. It can be applied to simulate contaminations under different water treatments or disinfection strategies. It may support technical decisions and help policymakers in acquiring evidences for comparing or validating innovative solutions.

A study of deoxyribonucleotide metabolism and its relation to DNA synthesis. Supercomputer simulation and model-system analysis.

PubMed

Heinmets, F; Leary, R H

1991-06-01

A model system (1) was established to analyze purine and pyrimidine metabolism. This system has been expanded to include macrosimulation of DNA synthesis and the study of its regulation by terminal deoxynucleoside triphosphates (dNTPs) via a complex set of interactions. Computer experiments reveal that our model exhibits adequate and reasonable sensitivity in terms of dNTP pool levels and rates of DNA synthesis when inputs to the system are varied. These simulation experiments reveal that in order to achieve maximum DNA synthesis (in terms of purine metabolism), a proper balance is required in guanine and adenine input into this metabolic system. Excessive inputs will become inhibitory to DNA synthesis. In addition, studies are carried out on rates of DNA synthesis when various parameters are changed quantitatively. The current system is formulated by 110 differential equations.

Enzymatic Production of Monoclonal Stoichiometric Single-Stranded DNA Oligonucleotides

PubMed Central

Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M.; Högberg, Björn

2013-01-01

Single-stranded oligonucleotides are important as research tools as probes for diagnostics and gene therapy. Today, production of oligonucleotides is done via solid-phase synthesis. However, the capabilities of current polymer chemistry are limited in comparison to what can be produced in biological systems. The errors in synthetic DNA increases with oligonucleotide length, and sequence diversity can often be a problem. Here, we present the Monoclonal Stoichiometric (MOSIC) method for enzymatic DNA oligonucleotide production. Using this method, we amplify oligonucleotides from clonal templates followed by digestion of a cutter-hairpin, resulting in pools of monoclonal oligonucleotides with precisely controlled relative stoichiometric ratios. We present data where MOSIC oligonucleotides, 14–378 nt long, were prepared either by in vitro rolling-circle amplification, or by amplification in Escherichia coli in the form of phagemid DNA. The formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides. PMID:23727986

Cytotoxicity and Antineoplastic Activities of Alkylamines and Their Borane Derivatives

PubMed Central

Tse, Elaine Y.; Muhammad, Rosallah A.

1996-01-01

The alkylamines and their related boron derivatives demonstrated potent cytotoxicity against the growth of murine and human tissue cultured cells. These agents did not necessarily require the boron atom to possess potent cytotoxic action in certain tumor lines. Their ability to suppress tumor cell growth was based on their inhibition of DNA and protein syntheses. DNA synthesis was reduced because purine synthesis was blocked at the enzyme site of IMP dehydrogenase by the agents. In addition ribonucleotide reductase and nucleoside kinase activities were reduced by the agents which would account for the reduced d[NTP] pools. The DNA template or molecule may be a target of the drugs with regard to binding of the drug to nucleoside bases or intercalaction of the drug between DNA base pairs. Only some Of the agents caused DNA fragmentation with reduced DNA viscosity. These effects would contribute to overall cell death afforded by the agents. PMID:18472803

Random access in large-scale DNA data storage.

PubMed

Organick, Lee; Ang, Siena Dumas; Chen, Yuan-Jyue; Lopez, Randolph; Yekhanin, Sergey; Makarychev, Konstantin; Racz, Miklos Z; Kamath, Govinda; Gopalan, Parikshit; Nguyen, Bichlien; Takahashi, Christopher N; Newman, Sharon; Parker, Hsing-Yeh; Rashtchian, Cyrus; Stewart, Kendall; Gupta, Gagan; Carlson, Robert; Mulligan, John; Carmean, Douglas; Seelig, Georg; Ceze, Luis; Strauss, Karin

2018-03-01

Synthetic DNA is durable and can encode digital data with high density, making it an attractive medium for data storage. However, recovering stored data on a large-scale currently requires all the DNA in a pool to be sequenced, even if only a subset of the information needs to be extracted. Here, we encode and store 35 distinct files (over 200 MB of data), in more than 13 million DNA oligonucleotides, and show that we can recover each file individually and with no errors, using a random access approach. We design and validate a large library of primers that enable individual recovery of all files stored within the DNA. We also develop an algorithm that greatly reduces the sequencing read coverage required for error-free decoding by maximizing information from all sequence reads. These advances demonstrate a viable, large-scale system for DNA data storage and retrieval.

A hierarchical and modular approach to the discovery of robust associations in genome-wide association studies from pooled DNA samples

PubMed Central

Sebastiani, Paola; Zhao, Zhenming; Abad-Grau, Maria M; Riva, Alberto; Hartley, Stephen W; Sedgewick, Amanda E; Doria, Alessandro; Montano, Monty; Melista, Efthymia; Terry, Dellara; Perls, Thomas T; Steinberg, Martin H; Baldwin, Clinton T

2008-01-01

Background One of the challenges of the analysis of pooling-based genome wide association studies is to identify authentic associations among potentially thousands of false positive associations. Results We present a hierarchical and modular approach to the analysis of genome wide genotype data that incorporates quality control, linkage disequilibrium, physical distance and gene ontology to identify authentic associations among those found by statistical association tests. The method is developed for the allelic association analysis of pooled DNA samples, but it can be easily generalized to the analysis of individually genotyped samples. We evaluate the approach using data sets from diverse genome wide association studies including fetal hemoglobin levels in sickle cell anemia and a sample of centenarians and show that the approach is highly reproducible and allows for discovery at different levels of synthesis. Conclusion Results from the integration of Bayesian tests and other machine learning techniques with linkage disequilibrium data suggest that we do not need to use too stringent thresholds to reduce the number of false positive associations. This method yields increased power even with relatively small samples. In fact, our evaluation shows that the method can reach almost 70% sensitivity with samples of only 100 subjects. PMID:18194558

Rolling Circle Amplification of Complete Nematode Mitochondrial Genomes

PubMed Central

Tang, Sha; Hyman, Bradley C.

2005-01-01

To enable investigation of nematode mitochondrial DNA evolution, methodology has been developed to amplify intact nematode mitochondrial genomes in preparative yields using a rolling circle replication strategy. Successful reactions were generated from whole cell template DNA prepared by alkaline lysis of the rhabditid nematode Caenorhabditis elegans and a mermithid nematode, Thaumamermis cosgrovei. These taxa, representing the two major nematode classes Chromodorea and Enoplea, maintain mitochondrial genomes of 13.8 kb and 20.0 kb, respectively. Efficient amplifications were conducted on template DNA isolated from individual or pooled nematodes that were alive or stored at -80°C. Unexpectedly, these experiments revealed that multiple T. cosgrovei mitochondrial DNA haplotypes are maintained in our local population. Rolling circle amplification products can be used as templates for standard PCR reactions with specific primers that target mitochondrial genes or for direct DNA sequencing. PMID:19262866

Chemical Biology Probes from Advanced DNA-encoded Libraries.

PubMed

Salamon, Hazem; Klika Škopić, Mateja; Jung, Kathrin; Bugain, Olivia; Brunschweiger, Andreas

2016-02-19

The identification of bioactive compounds is a crucial step toward development of probes for chemical biology studies. Screening of DNA-encoded small molecule libraries (DELs) has emerged as a validated technology to interrogate vast chemical space. DELs consist of chimeric molecules composed of a low-molecular weight compound that is conjugated to a DNA identifier tag. They are screened as pooled libraries using selection to identify "hits." Screening of DELs has identified numerous bioactive compounds. Some of these molecules were instrumental in gaining a deeper understanding of biological systems. One of the main challenges in the field is the development of synthesis methodology for DELs.

Recombination of polynucleotide sequences using random or defined primers

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Affholter, Joseph A.; Zhao, Huimin H; Giver, Lorraine J.

2000-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Metatranscriptomics of Soil Eukaryotic Communities.

PubMed

Yadav, Rajiv K; Bragalini, Claudia; Fraissinet-Tachet, Laurence; Marmeisse, Roland; Luis, Patricia

2016-01-01

Functions expressed by eukaryotic organisms in soil can be specifically studied by analyzing the pool of eukaryotic-specific polyadenylated mRNA directly extracted from environmental samples. In this chapter, we describe two alternative protocols for the extraction of high-quality RNA from soil samples. Total soil RNA or mRNA can be converted to cDNA for direct high-throughput sequencing. Polyadenylated mRNA-derived full-length cDNAs can also be cloned in expression plasmid vectors to constitute soil cDNA libraries, which can be subsequently screened for functional gene categories. Alternatively, the diversity of specific gene families can also be explored following cDNA sequence capture using exploratory oligonucleotide probes.

ECB deacylase mutants

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

2002-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Recombination of polynucleotide sequences using random or defined primers

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Affholter, Joseph A.; Zhao, Huimin; Giver, Lorraine J.

2001-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

An analysis of the relationship of seven selected variables to State Board Test Pool Examination performance of the University of Tennessee, Knoxville, College of Nursing.

PubMed

Sharp, T G

1984-02-01

The study was designed to determine whether any one of seven selected variables or a combination of the variables is predictive of performance on the State Board Test Pool Examination. The selected variables studied were: high school grade point average (HSGPA), The University of Tennessee, Knoxville, College of Nursing grade point average (GPA), and American College Test Assessment (ACT) standard scores (English, ENG; mathematics, MA; social studies, SS; natural sciences, NSC; composite, COMP). Data utilized were from graduates of the baccalaureate program of The University of Tennessee, Knoxville, College of Nursing from 1974 through 1979. The sample of 322 was selected from a total population of 572. The Statistical Analysis System (SAS) was designed to accomplish analysis of the predictive relationship of each of the seven selected variables to State Board Test Pool Examination performance (result of pass or fail), a stepwise discriminant analysis was designed for determining the predictive relationship of the strongest combination of the independent variables to overall State Board Test Pool Examination performance (result of pass or fail), and stepwise multiple regression analysis was designed to determine the strongest predictive combination of selected variables for each of the five subexams of the State Board Test Pool Examination. The selected variables were each found to be predictive of SBTPE performance (result of pass or fail). The strongest combination for predicting SBTPE performance (result of pass or fail) was found to be GPA, MA, and NSC.

Use of a pooled clone method to isolate a novel Bacillus thuringiensis Cry2A toxin with activity against Ostrinia furnacalis.

PubMed

Shu, Changlong; Zhang, Jingtao; Chen, Guihua; Liang, Gemei; He, Kanglai; Crickmore, Neil; Huang, Dafang; Zhang, Jie; Song, Fuping

2013-09-01

A pooled clone method was developed to screen for cry2A genes. This metagenomic method avoids the need to analyse isolated Bacillus thuringiensis strains by performing gene specific PCR on plasmid-enriched DNA prepared from a pooled soil sample. Using this approach the novel holotype gene cry2Ah1 was cloned and characterized. The toxin gene was over-expressed in Escherichia coli Rosetta (DE3) and the expressed toxin accumulated in both the soluble and insoluble fractions. The soluble Cry2Ah1 was found to have a weight loss activity against Ostrinia furnacalis, and a growth inhibitory activity to both Cry1Ac-susceptible and resistant Helicoverpa armigera populations. Copyright © 2013 Elsevier Inc. All rights reserved.

Exome copy number variation detection: Use of a pool of unrelated healthy tissue as reference sample.

PubMed

Wenric, Stephane; Sticca, Tiberio; Caberg, Jean-Hubert; Josse, Claire; Fasquelle, Corinne; Herens, Christian; Jamar, Mauricette; Max, Stéphanie; Gothot, André; Caers, Jo; Bours, Vincent

2017-01-01

An increasing number of bioinformatic tools designed to detect CNVs (copy number variants) in tumor samples based on paired exome data where a matched healthy tissue constitutes the reference have been published in the recent years. The idea of using a pool of unrelated healthy DNA as reference has previously been formulated but not thoroughly validated. As of today, the gold standard for CNV calling is still aCGH but there is an increasing interest in detecting CNVs by exome sequencing. We propose to design a metric allowing the comparison of two CNV profiles, independently of the technique used and assessed the validity of using a pool of unrelated healthy DNA instead of a matched healthy tissue as reference in exome-based CNV detection. We compared the CNV profiles obtained with three different approaches (aCGH, exome sequencing with a matched healthy tissue as reference, exome sequencing with a pool of eight unrelated healthy tissue as reference) on three multiple myeloma samples. We show that the usual analyses performed to compare CNV profiles (deletion/amplification ratios and CNV size distribution) lack in precision when confronted with low LRR values, as they only consider the binary status of each CNV. We show that the metric-based distance constitutes a more accurate comparison of two CNV profiles. Based on these analyses, we conclude that a reliable picture of CNV alterations in multiple myeloma samples can be obtained from whole-exome sequencing in the absence of a matched healthy sample. © 2016 WILEY PERIODICALS, INC.

Alkali metal pool boiler life tests for a 25 kWe advanced Stirling conversion system

NASA Technical Reports Server (NTRS)

Anderson, W. G.; Rosenfeld, J. H.; Noble, J.

1991-01-01

The overall operating temperature and efficiency of solar-powered Stirling engines can be improved by adding an alkali metal pool boiler heat transport system to supply heat more uniformly to the heater head tubes. One issue with liquid metal pool boilers is unstable boiling. Stable boiling is obtained with an enhanced boiling surface containing nucleation sites that promote continuous boiling. Over longer time periods, it is possible that the boiling behavior of the system will change. An 800-h life test was conducted to verify that pool boiling with the chosen fluid/surface combination remains stable as the system ages. The apparatus uses NaK boiling on a - 100 + 140 stainless steel sintered porous layer, with the addition of a small amount of xenon. Pool boiling remained stable to the end of life test. The pool boiler life test included a total of 82 cold starts, to simulate startup each morning, and 60 warm restarts, to simulate cloud cover transients. The behavior of the cold and warm starts showed no significant changes during the life test. In the experiments, the fluid/surface combination provided stable, high-performance boiling at the operating temperature of 700 C. Based on these experiments, a pool boiler was designed for a full-scale 25-kWe Stirling system.

Photolytic removal of DBPs by medium pressure UV in swimming pool water.

PubMed

Hansen, Kamilla M S; Zortea, Raissa; Piketty, Aurelia; Vega, Sergio Rodriguez; Andersen, Henrik Rasmus

2013-01-15

Medium pressure UV is used for controlling the concentration of combined chlorine (chloramines) in many public swimming pools. Little is known about the fate of other disinfection by-products (DBPs) in UV treatment. Photolysis by medium pressure UV treatment was investigated for 12 DBPs reported to be found in swimming pool water: chloroform, bromodichloromethane, dibromochloromethane, bromoform, dichloroacetonitrile, bromochloroacetonitrile, dibromoacetronitrile, trichloroacetonitrile, trichloronitromethane, dichloropropanone, trichloropropanone, and chloral hydrate. First order photolysis constants ranged 26-fold from 0.020 min(-1) for chloroform to 0.523 min(-1) for trichloronitromethane. The rate constants generally increased with bromine substitution. Using the UV removal of combined chlorine as an actinometer, the rate constants were recalculated to actual treatment doses of UV applied in a swimming pool. In an investigated public pool the UV dose was equivalent to an applied electrical energy of 1.34 kWh m(-3) d(-1) and the UV dose required to removed 90% of trichloronitromethane was 0.4 kWh m(-3) d(-1), while 2.6 kWh m(-3) d(-1) was required for chloral hydrate and the bromine containing haloacetonitriles and trihalomethanes ranged from 0.6 to 3.1 kWh m(-3) d(-1). It was predicted thus that a beneficial side-effect of applying UV for removing combined chlorine from the pool water could be a significant removal of trichloronitromethane, chloral hydrate and the bromine containing haloacetonitriles and trihalomethanes. Copyright © 2012 Elsevier B.V. All rights reserved.

DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair.

PubMed

Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

2016-08-31

DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

Single-Track Melt-Pool Measurements and Microstructures in Inconel 625

NASA Astrophysics Data System (ADS)

Ghosh, Supriyo; Ma, Li; Levine, Lyle E.; Ricker, Richard E.; Stoudt, Mark R.; Heigel, Jarred C.; Guyer, Jonathan E.

2018-06-01

We use single-track laser melting experiments and simulations on Inconel 625 to estimate the dimensions and microstructure of the resulting melt pool. Our work is based on a design-of-experiments approach which uses multiple laser power and scan speed combinations. Single-track experiments generated melt pools of certain dimensions that showed reasonable agreement with our finite-element calculations. Phase-field simulations were used to predict the size and segregation of the cellular microstructure that formed along the melt-pool boundaries for the solidification conditions that changed as a function of melt-pool dimensions.

Single-Track Melt-Pool Measurements and Microstructures in Inconel 625

NASA Astrophysics Data System (ADS)

Ghosh, Supriyo; Ma, Li; Levine, Lyle E.; Ricker, Richard E.; Stoudt, Mark R.; Heigel, Jarred C.; Guyer, Jonathan E.

2018-02-01

We use single-track laser melting experiments and simulations on Inconel 625 to estimate the dimensions and microstructure of the resulting melt pool. Our work is based on a design-of-experiments approach which uses multiple laser power and scan speed combinations. Single-track experiments generated melt pools of certain dimensions that showed reasonable agreement with our finite-element calculations. Phase-field simulations were used to predict the size and segregation of the cellular microstructure that formed along the melt-pool boundaries for the solidification conditions that changed as a function of melt-pool dimensions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Culbert, A.A.; Wallis, G.A.; Kadler, K.E.

The brittleness of bone in people with lethal (type II) osteogenesis imperfecta, a heritable disorder caused by mutations in the type I collagen genes, arises from the deposition of abnormal collagen in the bone matrix. The inability of the abnormal collagen to participate in mineralization may be caused by its failure to interact with other bone proteins. Here, we have designed a strategy to isolate the genes important for mineralization of collagen during bone formation. Cells isolated from 16-day embryonic chick calvaria and seeded post-confluence in culture deposited a mineralized matrix over a period of 2 weeks. Chick skin fibroblastsmore » seeded and cultured under the same conditions did not mineralize. Using RT-PCR, we prepared short cDNAs ({approximately}300 bp) corresponding to the 3{prime} ends of mRNA from fibroblasts and separately from the mineralizing calvarial cells. Subtractive cDNA hybridization generated a pool of cDNAs that were specific to mineralizing calvarial cells but not to fibroblasts. Screening of 100,000 plaques of a chick bone ZAP Express cDNA library with this pool of mineralizing-specific cDNAs identified ten clones which comprised full-length cDNAs for the bone proteins osteopontin (eight of the ten positives), bone sialoprotein II (one of the ten positives), and cystatin (one of the ten positives). cDNAs for type I collagen, fibronectin, alkaline phosphatase, house-keeping genes, and other genes expressed in fibroblasts were not identified in this preliminary screen. The pool of short cDNAs is likely to comprise cDNAs for further bone-specific genes and will be used to screen the entire bone cDNA library of 4.2 million clones. 30 refs., 4 figs.« less

From genomes to vaccines: Leishmania as a model.

PubMed Central

Almeida, Renata; Norrish, Alan; Levick, Mark; Vetrie, David; Freeman, Tom; Vilo, Jaak; Ivens, Alasdair; Lange, Uta; Stober, Carmel; McCann, Sharon; Blackwell, Jenefer M

2002-01-01

The 35 Mb genome of Leishmania should be sequenced by late 2002. It contains approximately 8500 genes that will probably translate into more than 10 000 proteins. In the laboratory we have been piloting strategies to try to harness the power of the genome-proteome for rapid screening of new vaccine candidate. To this end, microarray analysis of 1094 unique genes identified using an EST analysis of 2091 cDNA clones from spliced leader libraries prepared from different developmental stages of Leishmania has been employed. The plan was to identify amastigote-expressed genes that could be used in high-throughput DNA-vaccine screens to identify potential new vaccine candidates. Despite the lack of transcriptional regulation that polycistronic transcription in Leishmania dictates, the data provide evidence for a high level of post-transcriptional regulation of RNA abundance during the developmental cycle of promastigotes in culture and in lesion-derived amastigotes of Leishmania major. This has provided 147 candidates from the 1094 unique genes that are specifically upregulated in amastigotes and are being used in vaccine studies. Using DNA vaccination, it was demonstrated that pooling strategies can work to identify protective vaccines, but it was found that some potentially protective antigens are masked by other disease-exacerbatory antigens in the pool. A total of 100 new vaccine candidates are currently being tested separately and in pools to extend this analysis, and to facilitate retrospective bioinformatic analysis to develop predictive algorithms for sequences that constitute potentially protective antigens. We are also working with other members of the Leishmania Genome Network to determine whether RNA expression determined by microarray analyses parallels expression at the protein level. We believe we are making good progress in developing strategies that will allow rapid translation of the sequence of Leishmania into potential interventions for disease control in humans. PMID:11839176

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marchisio, Mario Andrea, E-mail: marchisio@hit.edu.cn

Published in 2008, Parts & Pools represents one of the first attempts to conceptualize the modular design of bacterial synthetic gene circuits with Standard Biological Parts (DNA segments) and Pools of molecules referred to as common signal carriers (e.g., RNA polymerases and ribosomes). The original framework for modeling bacterial components and designing prokaryotic circuits evolved over the last years and brought, first, to the development of an algorithm for the automatic design of Boolean gene circuits. This is a remarkable achievement since gene digital circuits have a broad range of applications that goes from biosensors for health and environment caremore » to computational devices. More recently, Parts & Pools was enabled to give a proper formal description of eukaryotic biological circuit components. This was possible by employing a rule-based modeling approach, a technique that permits a faithful calculation of all the species and reactions involved in complex systems such as eukaryotic cells and compartments. In this way, Parts & Pools is currently suitable for the visual and modular design of synthetic gene circuits in yeast and mammalian cells too.« less

Ancient and modern DNA reveal dynamics of domestication and cross-continental dispersal of the dromedary

PubMed Central

Almathen, Faisal; Charruau, Pauline; Mohandesan, Elmira; Mwacharo, Joram M.; Orozco-terWengel, Pablo; Pitt, Daniel; Abdussamad, Abdussamad M.; Uerpmann, Margarethe; Uerpmann, Hans-Peter; De Cupere, Bea; Magee, Peter; Alnaqeeb, Majed A.; Salim, Bashir; Raziq, Abdul; Dessie, Tadelle; Abdelhadi, Omer M.; Banabazi, Mohammad H.; Al-Eknah, Marzook; Walzer, Chris; Faye, Bernard; Hofreiter, Michael; Peters, Joris; Hanotte, Olivier

2016-01-01

Dromedaries have been fundamental to the development of human societies in arid landscapes and for long-distance trade across hostile hot terrains for 3,000 y. Today they continue to be an important livestock resource in marginal agro-ecological zones. However, the history of dromedary domestication and the influence of ancient trading networks on their genetic structure have remained elusive. We combined ancient DNA sequences of wild and early-domesticated dromedary samples from arid regions with nuclear microsatellite and mitochondrial genotype information from 1,083 extant animals collected across the species’ range. We observe little phylogeographic signal in the modern population, indicative of extensive gene flow and virtually affecting all regions except East Africa, where dromedary populations have remained relatively isolated. In agreement with archaeological findings, we identify wild dromedaries from the southeast Arabian Peninsula among the founders of the domestic dromedary gene pool. Approximate Bayesian computations further support the “restocking from the wild” hypothesis, with an initial domestication followed by introgression from individuals from wild, now-extinct populations. Compared with other livestock, which show a long history of gene flow with their wild ancestors, we find a high initial diversity relative to the native distribution of the wild ancestor on the Arabian Peninsula and to the brief coexistence of early-domesticated and wild individuals. This study also demonstrates the potential to retrieve ancient DNA sequences from osseous remains excavated in hot and dry desert environments. PMID:27162355

Preparation and screening of an arrayed human genomic library generated with the P1 cloning system.

PubMed Central

Shepherd, N S; Pfrogner, B D; Coulby, J N; Ackerman, S L; Vaidyanathan, G; Sauer, R H; Balkenhol, T C; Sternberg, N

1994-01-01

We describe here the construction and initial characterization of a 3-fold coverage genomic library of the human haploid genome that was prepared using the bacteriophage P1 cloning system. The cloned DNA inserts were produced by size fractionation of a Sau3AI partial digest of high molecular weight genomic DNA isolated from primary cells of human foreskin fibroblasts. The inserts were cloned into the pAd10sacBII vector and packaged in vitro into P1 phage. These were used to generate recombinant bacterial clones, each of which was picked robotically from an agar plate into a well of a 96-well microtiter dish, grown overnight, and stored at -70 degrees C. The resulting library, designated DMPC-HFF#1 series A, consists of approximately 130,000-140,000 recombinant clones that were stored in 1500 microtiter dishes. To screen the library, clones were combined in a pooling strategy and specific loci were identified by PCR analysis. On average, the library contains two or three different clones for each locus screened. To date we have identified a total of 17 clones containing the hypoxanthine-guanine phosphoribosyltransferase, human serum albumin-human alpha-fetoprotein, p53, cyclooxygenase I, human apurinic endonuclease, beta-polymerase, and DNA ligase I genes. The cloned inserts average 80 kb in size and range from 70 to 95 kb, with one 49-kb insert and one 62-kb insert. Images PMID:8146166

Policy implications for familial searching

PubMed Central

2011-01-01

In the United States, several states have made policy decisions regarding whether and how to use familial searching of the Combined DNA Index System (CODIS) database in criminal investigations. Familial searching pushes DNA typing beyond merely identifying individuals to detecting genetic relatedness, an application previously reserved for missing persons identifications and custody battles. The intentional search of CODIS for partial matches to an item of evidence offers law enforcement agencies a powerful tool for developing investigative leads, apprehending criminals, revitalizing cold cases and exonerating wrongfully convicted individuals. As familial searching involves a range of logistical, social, ethical and legal considerations, states are now grappling with policy options for implementing familial searching to balance crime fighting with its potential impact on society. When developing policies for familial searching, legislators should take into account the impact of familial searching on select populations and the need to minimize personal intrusion on relatives of individuals in the DNA database. This review describes the approaches used to narrow a suspect pool from a partial match search of CODIS and summarizes the economic, ethical, logistical and political challenges of implementing familial searching. We examine particular US state policies and the policy options adopted to address these issues. The aim of this review is to provide objective background information on the controversial approach of familial searching to inform policy decisions in this area. Herein we highlight key policy options and recommendations regarding effective utilization of familial searching that minimize harm to and afford maximum protection of US citizens. PMID:22040348

Policy implications for familial searching.

PubMed

Kim, Joyce; Mammo, Danny; Siegel, Marni B; Katsanis, Sara H

2011-11-01

In the United States, several states have made policy decisions regarding whether and how to use familial searching of the Combined DNA Index System (CODIS) database in criminal investigations. Familial searching pushes DNA typing beyond merely identifying individuals to detecting genetic relatedness, an application previously reserved for missing persons identifications and custody battles. The intentional search of CODIS for partial matches to an item of evidence offers law enforcement agencies a powerful tool for developing investigative leads, apprehending criminals, revitalizing cold cases and exonerating wrongfully convicted individuals. As familial searching involves a range of logistical, social, ethical and legal considerations, states are now grappling with policy options for implementing familial searching to balance crime fighting with its potential impact on society. When developing policies for familial searching, legislators should take into account the impact of familial searching on select populations and the need to minimize personal intrusion on relatives of individuals in the DNA database. This review describes the approaches used to narrow a suspect pool from a partial match search of CODIS and summarizes the economic, ethical, logistical and political challenges of implementing familial searching. We examine particular US state policies and the policy options adopted to address these issues. The aim of this review is to provide objective background information on the controversial approach of familial searching to inform policy decisions in this area. Herein we highlight key policy options and recommendations regarding effective utilization of familial searching that minimize harm to and afford maximum protection of US citizens.

Possible effect of biotechnology on plant gene pools in Turkey.

PubMed

Demir, Aynur

2015-01-02

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

Possible effect of biotechnology on plant gene pools in Turkey

PubMed Central

Demir, Aynur

2015-01-01

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

Extension of platelet shelf life from 4 to 5 days by implementation of a new screening strategy in Germany.

PubMed

Sireis, W; Rüster, B; Daiss, C; Hourfar, M K; Capalbo, G; Pfeiffer, H-U; Janetzko, K; Goebel, M; Kempf, V A J; Seifried, E; Schmidt, M

2011-10-01

The Paul-Ehrlich-Institute analysed all fatalities due to bacterial infections between 1997 and 2007. Thereafter, the platelet shelf life was reduced to a maximum of 4 days after blood donation because the majority of all cases of severe transfusion-transmitted bacterial infections occurred with day 5 platelets. The current study compares the analytical sensitivity and the diagnostic specificity of four rapid bacterial detection procedures. Nine transfusion-relevant bacterial strains were spiked in pooled platelets or apheresis platelets at a low concentration (10 CFU/bag). Samples were collected after day 3, day 4 and day 5 and investigated by four rapid bacterial detection methods (modified BacT/ALERT, Bactiflow, FACS method and 16s DNA PCR methods). Seven out of nine bacterial strains were adequately detected by BacT/ALERT, Bactiflow and PCR in apheresis platelets and pooled platelets after sample collection at day 3, day 4 and day 5. For three bacterial strains, analytical sensitivity was reduced for the FACS method. Two bacterial strains did not grow under the storage conditions in either pooled or apheresis platelets. A late sample collection on day 3, day 4 or day 5 after blood donation in combination with a rapid bacterial detection method offers a new opportunity to improve blood safety and reduce errors due to sampling., BacT/ALERT, Bactiflow or 16s ID-NAT are feasible for late bacterial screening in platelets may provide data which support the extension of platelet shelf life in Germany to 5 days. © 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.

Impact of phlebotomine sand flies on U.S. military operations at Tallil Air Base, Iraq: 4. Detection and identification of leishmania parasites in sand flies.

PubMed

Coleman, Russell E; Hochberg, Lisa P; Swanson, Katherine I; Lee, John S; McAvin, James C; Moulton, John K; Eddington, David O; Groebner, Jennifer L; O'Guinn, Monica L; Putnam, John L

2009-05-01

Sand flies collected between April 2003 and November 2004 at Tallil Air Base, Iraq, were evaluated for the presence of Leishmania parasites using a combination of a real-time Leishmania-generic polymerase chain reaction (PCR) assay and sequencing of a 360-bp fragment of the glucose-6-phosphate-isomerase (GPI) gene. A total of 2,505 pools containing 26,574 sand flies were tested using the real-time PCR assay. Leishmania DNA was initially detected in 536 pools; however, after extensive retesting with the real-time PCR assay, a total of 456 pools were considered positive and 80 were considered indeterminate. A total of 532 samples were evaluated for Leishmania GPI by sequencing, to include 439 PCR-positive samples, 80 PCR-indeterminate samples, and 13 PCR-negative samples. Leishmania GPI was detected in 284 samples that were sequenced, to include 281 (64%) of the PCR-positive samples and 3 (4%) of the PCR-indeterminate samples. Of the 284 sequences identified as Leishmania, 261 (91.9%) were L. tarentolae, 18 (6.3%) were L. donovani-complex parasites, 3 (1.1%) were L. tropica, and 2 were similar to both L. major and L. tropica. Minimum field infection rates were 0.09% for L. donovani-complex parasites, 0.02% for L. tropica, and 0.01% for the L. major/tropica-like parasite. Subsequent sequencing of a 600-bp region of the "Hyper" gene of 12 of the L. donovani-complex parasites showed that all 12 parasites were L. infantum. These data suggest that L. infantum was the primary leishmanial threat to U.S. military personnel deployed to Tallil Air Base. The implications of these findings are discussed.

Chromonoodles: Jump into the Gene Pool

ERIC Educational Resources Information Center

Farrar, Jennifer; Barnhart, Kelsi

2011-01-01

Chromosomes, alleles, chromatids, genotype, phenotype, mitosis, meiosis, fertilization--this vocabulary can be overwhelming, confusing, and difficult for students to tie together. However, since these terms are commonplace in the high school biology classroom, and are the basis for understanding both DNA and heredity, students must understand…

Lice outbreak in buffaloes: evidence of Anaplasma marginale transmission by sucking lice Haematopinus tuberculatus.

PubMed

Da Silva, Aleksandro Schafer; Lopes, Leandro Sâmia; Diaz, Jorge Damian Stumpfs; Tonin, Alexandre Alberto; Stefani, Lenita Moura; Araújo, Denise Nunes

2013-06-01

Lice infestations are commonly seen in buffaloes, causing damage directly to the animal, i.e., itching, skin lesions, and anemia. In addition, these insects can also be vectors for infectious diseases. The present study describes an outbreak of lice in buffaloes as well as evidence for Haematopinus tuberculatus acting as a vector of anaplasmosis. Lice and blood were collected from 4 young buffaloes (2- to 4-mo-old) and a molecular analysis for the presence of Anaplasma marginale was conducted. DNA of A. marginale was detected in the blood of all 4 animals. Twelve lice were collected and separated in 4 groups, with 3 insects each, to comprise a pool of samples. After DNA extraction and molecular analysis, a positive PCR for A. marginale was found in all pooled samples. These results identify sucking lice as potential vectors of anaplasmosis. However, additional studies are necessary to fully evaluate the vector potential of H. tuberculatus for A. maginale transmission.

In vitro selection of functional nucleic acids

NASA Technical Reports Server (NTRS)

Wilson, D. S.; Szostak, J. W.

1999-01-01

In vitro selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10(15) different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent three-dimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in vitro protein selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.

JWST Associations overview: automated generation of combined products

NASA Astrophysics Data System (ADS)

Alexov, Anastasia; Swade, Daryl; Bushouse, Howard; Diaz, Rosa; Eisenhamer, Jonathan; Hack, Warren; Kyprianou, Mark; Levay, Karen; Rahmani, Christopher; Swam, Mike; Valenti, Jeff

2018-01-01

We are presenting the design of the James Webb Space Telescope (JWST) Data Management System (DMS) automated processing of Associations. An Association captures the relationship between exposures and higher level data products, such as combined mosaics created from dithered and tiled observations. The astronomer’s intent is captured within the Proposal Planning System (PPS) and provided to DMS as candidate associations. These candidates are converted into Association Pools and Association Generator Tables that serve as input to automated processing which create the combined data products. Association Pools are generated to capture a list of exposures that could potentially form associations and provide relevant information about those exposures. The Association Generator using definitions on groupings creates one or more Association Tables from a single input Association Pool. Each Association Table defines a set of exposures to be combined and the ruleset of the combination to be performed; the calibration software creates Associated data products based on these input tables. The initial design produces automated Associations within a proposal. Additionally this JWST overall design is conducive to eventually produce Associations for observations from multiple proposals, similar to the Hubble Legacy Archive (HLA).

SNP discovery in common bean by restriction-associated DNA (RAD) sequencing for genetic diversity and population structure analysis.

PubMed

Valdisser, Paula Arielle M R; Pappas, Georgios J; de Menezes, Ivandilson P P; Müller, Bárbara S F; Pereira, Wendell J; Narciso, Marcelo G; Brondani, Claudio; Souza, Thiago L P O; Borba, Tereza C O; Vianello, Rosana P

2016-06-01

Researchers have made great advances into the development and application of genomic approaches for common beans, creating opportunities to driving more real and applicable strategies for sustainable management of the genetic resource towards plant breeding. This work provides useful polymorphic single-nucleotide polymorphisms (SNPs) for high-throughput common bean genotyping developed by RAD (restriction site-associated DNA) sequencing. The RAD tags were generated from DNA pooled from 12 common bean genotypes, including breeding lines of different gene pools and market classes. The aligned sequences identified 23,748 putative RAD-SNPs, of which 3357 were adequate for genotyping; 1032 RAD-SNPs with the highest ADT (assay design tool) score are presented in this article. The RAD-SNPs were structurally annotated in different coding (47.00 %) and non-coding (53.00 %) sequence components of genes. A subset of 384 RAD-SNPs with broad genome distribution was used to genotype a diverse panel of 95 common bean germplasms and revealed a successful amplification rate of 96.6 %, showing 73 % of polymorphic SNPs within the Andean group and 83 % in the Mesoamerican group. A slightly increased He (0.161, n = 21) value was estimated for the Andean gene pool, compared to the Mesoamerican group (0.156, n = 74). For the linkage disequilibrium (LD) analysis, from a group of 580 SNPs (289 RAD-SNPs and 291 BARC-SNPs) genotyped for the same set of genotypes, 70.2 % were in LD, decreasing to 0.10 %in the Andean group and 0.77 % in the Mesoamerican group. Haplotype patterns spanning 310 Mb of the genome (60 %) were characterized in samples from different origins. However, the haplotype frameworks were under-represented for the Andean (7.85 %) and Mesoamerican (5.55 %) gene pools separately. In conclusion, RAD sequencing allowed the discovery of hundreds of useful SNPs for broad genetic analysis of common bean germplasm. From now, this approach provides an excellent panel of molecular tools for whole genome analysis, allowing integrating and better exploring the common bean breeding practices.

The construction of cDNA library and the screening of related antigen of ascitic tumor cells of ovarian cancer.

PubMed

Hou, Q; Chen, K; Shan, Z

2015-01-01

To construct the cDNA library of the ascites tumor cells of ovarian cancer, which can be used to screen the related antigen for the early diagnosis of ovarian cancer and therapeutic targets of immune treatment. Four cases of ovarian serous cystadenocarcinoma, two cases of ovarian mucinous cystadenocarcinoma, and two cases of ovarian endometrial carcinoma in patients with ascitic tumor cells which were used to construct the cDNA library. To screen the ovarian cancer antigen gene, evaluate the enzyme, and analyze nucleotide sequence, serological analysis of recombinant tumor cDNA expression libraries (SEREX) and suppression subtractive hybridization technique (SSH) techniques were utilized. The detection method of recombinant expression-based serological mini-arrays (SMARTA) was used to detect the ovarian cancer antigen and the positive reaction of 105 cases of ovarian cancer patients and 105 normal women's autoantibodies correspondingly in serum. After two rounds of serologic screening and glycosides sequencing analysis, 59 candidates of ovarian cancer antigen gene fragments were finally identified, which corresponded to 50 genes. They were then divided into six categories: (1) the homologous genes which related to the known ovarian cancer genes, such as BARD 1 gene, etc; (2) the homologous genes which were associated with other tumors, such as TM4SFI gene, etc; (3) the genes which were expressed in a special organization, such as ILF3, FXR1 gene, etc; (4) the genes which were the same with some protein genes of special function, such as TIZ, ClD gene; (5) the homologous genes which possessed the same source with embryonic genes, such as PKHD1 gene, etc; (6) the remaining genes were the unknown genes without the homologous sequence in the gene pool, such as OV-189 genes. SEREX technology combined with SSH method is an effective research strategy which can filter tumor antigen with high specific character; the corresponding autoantibodies of TM4SFl, ClD, TIZ, BARDI, FXRI, and OV-189 gene's recombinant antigen in serum can be regarded as the biomarkers which are used to diagnose ovarian cancer. The combination of multiple antigen detection can improve diagnostic efficiency.

Solving satisfiability problems using a novel microarray-based DNA computer.

PubMed

Lin, Che-Hsin; Cheng, Hsiao-Ping; Yang, Chang-Biau; Yang, Chia-Ning

2007-01-01

An algorithm based on a modified sticker model accompanied with an advanced MEMS-based microarray technology is demonstrated to solve SAT problem, which has long served as a benchmark in DNA computing. Unlike conventional DNA computing algorithms needing an initial data pool to cover correct and incorrect answers and further executing a series of separation procedures to destroy the unwanted ones, we built solutions in parts to satisfy one clause in one step, and eventually solve the entire Boolean formula through steps. No time-consuming sample preparation procedures and delicate sample applying equipment were required for the computing process. Moreover, experimental results show the bound DNA sequences can sustain the chemical solutions during computing processes such that the proposed method shall be useful in dealing with large-scale problems.

Phylogenetic relationships of the Gomphales based on nuc-25S-rDNA, mit-12S-rDNA, and mit-atp6-DNA combined sequences

Treesearch

Admir J. Giachini; Kentaro Hosaka; Eduardo Nouhra; Joseph Spatafora; James M. Trappe

2010-01-01

Phylogenetic relationships among Geastrales, Gomphales, Hysterangiales, and Phallales were estimated via combined sequences: nuclear large subunit ribosomal DNA (nuc-25S-rDNA), mitochondrial small subunit ribosomal DNA (mit-12S-rDNA), and mitochondrial atp6 DNA (mit-atp6-DNA). Eighty-one taxa comprising 19 genera and 58 species...

cDNA Cloning, expression and characterization of an allergenic 60s ribosomal protein of almond (prunus dulcis).

PubMed

Abolhassani, Mohsen; Roux, Kenneth H

2009-06-01

Tree nuts, including almond (prunus dulcis) are a source of food allergens often associated with life-threatening allergic reactions in susceptible individuals. Although the proteins in almonds have been biochemically characterized, relatively little has been reported regarding the identity of the allergens involved in almond sensitivity. The present study was undertaken to identify the allergens of the almond by cDNA library approach. cDNA library of almond seeds was constructed in Uni-Zap XR lamda vector and expressed in E. coli XL-1 blue. Plaques were immunoscreened with pooled sera of allergic patients. The cDNA clone reacting significantly with specific IgE antibodies was selected and subcloned and subsequently expressed in E. coli. The amino acids deducted from PCR product of clone showed homology to 60s acidic ribosomal protein of almond. The expressed protein was 11,450 Dalton without leader sequence. Immunoreactivity of the recombinant 60s ribosomal protein (r60sRP) was evaluated with dot blot analysis using pooled and individual sera of allergic patients. The data showed that r60sRP and almond extract (as positive control) possess the ability to bind the IgE antibodies. The results showed that expressed protein is an almond allergen.Whether this r60sRP represents a major allergen of almond needs to be further studied which requires a large number of sera from the almond atopic patients and also need to determine the IgE-reactive frequencies of each individual allergen.

Sequence analysis of malacoherpesvirus proteins: Pan-herpesvirus capsid module and replication enzymes with an ancient connection to "Megavirales".

PubMed

Mushegian, Arcady; Karin, Eli Levy; Pupko, Tal

2018-01-01

The order Herpesvirales includes animal viruses with large double-strand DNA genomes replicating in the nucleus. The main capsid protein in the best-studied family Herpesviridae contains a domain with HK97-like fold related to bacteriophage head proteins, and several virion maturation factors are also homologous between phages and herpesviruses. The origin of herpesvirus DNA replication proteins is less well understood. While analyzing the genomes of herpesviruses in the family Malacohepresviridae, we identified nearly 30 families of proteins conserved in other herpesviruses, including several phage-related domains in morphogenetic proteins. Herpesvirus DNA replication factors have complex evolutionary history: some are related to cellular proteins, but others are closer to homologs from large nucleocytoplasmic DNA viruses. Phylogenetic analyses suggest that the core replication machinery of herpesviruses may have been recruited from the same pool as in the case of other large DNA viruses of eukaryotes. Published by Elsevier Inc.

Transgene Expression of Drosophila melanogaster Nucleoside Kinase Reverses Mitochondrial Thymidine Kinase 2 Deficiency*♦

PubMed Central

Krishnan, Shuba; Zhou, Xiaoshan; Paredes, João A.; Kuiper, Raoul V.; Curbo, Sophie; Karlsson, Anna

2013-01-01

A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK+/− transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK+/−TK2−/− mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK+/−TK2−/− mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency. PMID:23288848

Real-time polymerase chain reaction for diagnosing infectious mononucleosis in pediatric patients: A systematic review and meta-analysis.

PubMed

Jiang, Sha-Yi; Yang, Jing-Wei; Shao, Jing-Bo; Liao, Xue-Lian; Lu, Zheng-Hua; Jiang, Hui

2016-05-01

In this meta-analysis, we evaluated the diagnostic role of Epstein-Barr virus deoxyribonucleic acid detection and quantitation in the serum of pediatric and young adult patients with infectious mononucleosis. The primary outcome of this meta-analysis was the sensitivity and specificity of Epstein-Barr virus (EBV) deoxyribonucleic acid (DNA) detection and quantitation using polymerase chain reaction (PCR). A systematic review and meta-analysis was performed by searching for articles that were published through September 24, 2014 in the following databases: Medline, Cochrane, EMBASE, and Google Scholar. The following keywords were used for the search: "Epstein-Barr virus," "infectious mononucleosis," "children/young adults/infant/pediatric," and "polymerase chain reaction or PCR." Three were included in this analysis. We found that for detection by PCR, the pooled sensitivity for detecting EBV DNA was 77% (95%CI, 66-86%) and the pooled specificity for was 98% (95%CI, 93-100%). Our findings indicate that this PCR-based assay has high specificity and good sensitivity for detecting of EBV DNA, indicating it may useful for identifying patients with infectious mononucleosis. This assay may also be helpful to identify young athletic patients or highly physically active pediatric patients who are at risk for a splenic rupture due to acute infectious mononucleosis. © 2015 Wiley Periodicals, Inc.

The use of (GTG)5 oligonucleotide as an RAPD primer to type Campylobacter concisus.

PubMed

Matsheka, M I; Lastovica, A J; Zappe, H; Elisha, B G

2006-06-01

DNA fingerprinting using (GTG)(5) oligonucleotide as a primer in a random amplified polymorphic DNA (RAPD) assay was assessed by typing isolates of Campylobacter concisus strains, collected over a period of 8 years. RAPD analysis using the (GTG)(5) oligonucleotide as a primer was used to type 100 isolates of C. concisus comprising mostly isolates from children with diarrhoea. Using this method, 86% of the isolates were found to be genotypically diverse. Of these heterogeneous isolates, 25 of the strains were also shown to be genetically distinct in a previous study using pulsed field gel electrophoresis. The remaining isolates (14) could be classified into five profile groups based on the DNA fingerprinting patterns. The assay successfully identified epidemiologically linked strains from the unrelated genetically diverse pool of strains. Laboratory RADP typing using the (GTG)(5) primer proved to be useful in distinguishing related strains of C. concisus from a large pool of unrelated strains of this organism. RAPD typing using (GTG)(5) is a simple method that could be used to investigate the epidemiology of C. concisus. The results suggest that homologous lineages of C. concisus may exist within an otherwise heterogeneous species complex. However, these data need to be confirmed using a more robust typing method.

Analysis of medieval mtDNA from Napole cemetery provides new insights into the early history of Polish state.

PubMed

Płoszaj, Tomasz; Jędrychowska-Dańska, Krystyna; Masłowska, Alicja; Kozłowski, Tomasz; Chudziak, Wojciech; Bojarski, Jacek; Robaszkiewicz, Agnieszka; Witas, Henryk W

2017-02-01

Contemporary historical anthropology and classical archaeology are concerned not only with such fundamental issues as the origins of ancient human populations and migration routes, but also with the formation and development of inter-population relations and the mixing of gene pools as a result of inter-breeding between individuals representing different cultural units. The contribution of immigrants to the analysed autochthonous population and their effect on the gene pool of that population has proven difficult to evaluate with classical morphological methods. The burial of one individual in the studied Napole cemetery located in central Poland had the form of a chamber grave, which is typical of Scandinavian culture from that period. However, this fact cannot be interpreted as absolute proof that the individual (in the biological sense) was allochtonous. This gives rise to the question as to who was actually buried in that cemetery. The ancient DNA results indicate that one of the individuals had an mtDNA haplotype typical of Iron Age northern Europe, which suggests that he could have arrived from that area at a later period. This seems to indirectly confirm the claims of many anthropologists that the development of the early medieval Polish state was significantly and directly influenced by the Scandinavians.

Sources of PCR-induced distortions in high-throughput sequencing data sets

PubMed Central

Kebschull, Justus M.; Zador, Anthony M.

2015-01-01

PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error—bias, stochasticity, template switches and polymerase errors—on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules. PMID:26187991

Antiretroviral therapy-induced mitochondrial toxicity: potential mechanisms beyond polymerase-γ inhibition.

PubMed

Selvaraj, S; Ghebremichael, M; Li, M; Foli, Y; Langs-Barlow, A; Ogbuagu, A; Barakat, L; Tubridy, E; Edifor, R; Lam, W; Cheng, Y-C; Paintsil, E

2014-07-01

We hypothesized that competition between nucleotide reverse-transcriptase inhibitor triphosphate and endogenous deoxyribonucleotide triphosphate (dNTP) may lead to depletion of dNTP pools and mitochondrial dysfunction independent of polymerase-γ (pol-γ) inhibition. We collected peripheral blood mononuclear cells from 75 adults (25 cases: HIV-infected patients with mitochondrial toxicity, 25 HIV-infected positive controls, and 25 HIV-negative controls). We observed statistically significant individual and group differences in ribonucleotide (RN) and deoxyribonucleotide (dRN) pools. The median values for the RN pools were 10,062 (interquartile range (IQR): 7,090-12,590), 4,360 (IQR: 3,058-6,838), and 2,968 (IQR: 2,538-4,436) pmol/10(6) cells for negative controls, positive controls, and cases, respectively. Cases had significantly higher absolute mitochondrial DNA copy number as compared with negative controls (P < 0.05). Moreover, cases had significantly higher expression levels of pol-γ, nucleotide transporters, cellular kinases, and adenosine triphosphate (ATP)-binding cassette (ABC) proteins as compared with controls. Antiretroviral therapy (ART) perturbs RN and dRN pools. Depletion of RN and dRN pools may be associated with ART-induced mitochondrial toxicity independent of pol-γ inhibition.

Nuclear and chloroplast DNA differentiation in Andean potatoes.

PubMed

Sukhotu, Thitaporn; Kamijima, Osamu; Hosaka, Kazuyoshi

2004-02-01

Over 3500 accessions of Andean landraces have been known in potato, classified into 7 cultivated species ranging from 2x to 5x (Hawkes 1990). Chloroplast DNA (ctDNA), distinguished into T, W, C, S, and A types, showed extensive overlaps in their frequencies among cultivated species and between cultivated and putative ancestral wild species. In this study, 76 accessions of cultivated and 19 accessions of wild species were evaluated for ctDNA types and examined by ctDNA high-resolution markers (ctDNA microsatellites and H3 marker) and nuclear DNA restriction fragment length polymorphisms (RFLPs). ctDNA high-resolution markers identified 25 different ctDNA haplotypes. The S- and A-type ctDNAs were discriminated as unique haplotypes from 12 haplotypes having C-type ctDNA and T-type ctDNA from 10 haplotypes having W-type ctDNA. Differences among ctDNA types were strongly correlated with those of ctDNA high-resolution markers (r = 0.822). Differentiation between W-type ctDNA and C-, S-, and A-type ctDNAs was supported by nDNA RFLPs in most species except for those of recent or immediate hybrid origin. However, differentiation among C-, S-, and A-type ctDNAs was not clearly supported by nDNA RFLPs, suggesting that frequent genetic exchange occurred among them and (or) they shared the same gene pool owing to common ancestry.

Complex interactions of the Eastern and Western Slavic populations with other European groups as revealed by mitochondrial DNA analysis.

PubMed

Grzybowski, Tomasz; Malyarchuk, Boris A; Derenko, Miroslava V; Perkova, Maria A; Bednarek, Jarosław; Woźniak, Marcin

2007-06-01

Mitochondrial DNA sequence variation was examined by the control region sequencing (HVS I and HVS II) and RFLP analysis of haplogroup-diagnostic coding region sites in 570 individuals from four regional populations of Poles and two Russian groups from northwestern part of the country. Additionally, sequences of complete mitochondrial genomes representing K1a1b1a subclade in Polish and Polish Roma populations have been determined. Haplogroup frequency patterns revealed in Poles and Russians are similar to those characteristic of other Europeans. However, there are several features of Slavic mtDNA pools seen on the level of regional populations which are helpful in the understanding of complex interactions of the Eastern and Western Slavic populations with other European groups. One of the most important is the presence of subhaplogroups U5b1b1, D5, Z1 and U8a with simultaneous scarcity of haplogroup K in populations of northwestern Russia suggesting the participation of Finno-Ugrian tribes in the formation of mtDNA pools of Russians from this region. The results of genetic structure analyses suggest that Russians from Velikii Novgorod area (northwestern Russia) and Poles from Suwalszczyzna (northeastern Poland) differ from all remaining Polish and Russian samples. Simultaneously, northwestern Russians and northeastern Poles bear some similarities to Baltic (Latvians) and Finno-Ugrian groups (Estonians) of northeastern Europe, especially on the level of U5 haplogroup frequencies. The occurrence of K1a1b1a subcluster in Poles and Polish Roma is one of the first direct proofs of the presence of Ashkenazi-specific mtDNA lineages in non-Jewish European populations.

Ionization in liquids. Progress report, September 1, 1977-April 30, 1981

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bakale, G.

1980-12-19

Quasifree electrons simulate the behavior of unsolvated or dry electrons in aqueous media including the special case of biological systems. A model of direct radiosensitization was developed based on dry charge-carriers having an extended lifetime in the sheath of structured water that surrounds polar biomolecules. In this model, the pre-solvation lifetimes of dry electrons increased with an increase in the rotational times of solvent molecules. During the development of this model, an increasing number of radiosensitizers were found to be carcinogenic. Measurement of the k/sub e/'s of known carcinogens and noncarcinogens revealed that carcinogens attached quasifree electrons at diffusion-controlled rates,more » whereas the k/sub e/'s of noncarcinogens were significantly less. To explore the k/sub e/-carcinogenicity correlation further, a study of quasifree electron attachment to the water pools of reversed micelles was conducted. The degree of structuredness of the water pools which determines the k/sub e/ of the reversed micellar systems was also controlled. Another approach to controlling the microenvironment of quasifree electrons in biological systems was done in studies of radiation-induced damage to DNA in concentrated DNA solutions. The high concentration of DNA introduces more structure into the solutions than that occurring in typical in vitro experiments. The structural enhancement by DNA extends the lifetime of unsolvated charge-carriers. The DNA-damaging effects of radiolyticaly produced charge-carriers were also determined in studies of synergistic mutagenesis in bacteria simultaneously exposed to ionizing radiation and electrophilic chemical carcinogens. The attachment-detachment equilibrium of nicotine in hexane solutions was also studied. Both the kinetics and the thermodynamics of electron reactions were studied. (ERB)« less

Linking the sub-Saharan and West Eurasian gene pools: maternal and paternal heritage of the Tuareg nomads from the African Sahel

PubMed Central

Pereira, Luísa; Černý, Viktor; Cerezo, María; Silva, Nuno M; Hájek, Martin; Vašíková, Alžběta; Kujanová, Martina; Brdička, Radim; Salas, Antonio

2010-01-01

The Tuareg presently live in the Sahara and the Sahel. Their ancestors are commonly believed to be the Garamantes of the Libyan Fezzan, ever since it was suggested by authors of antiquity. Biological evidence, based on classical genetic markers, however, indicates kinship with the Beja of Eastern Sudan. Our study of mitochondrial DNA (mtDNA) sequences and Y chromosome SNPs of three different southern Tuareg groups from Mali, Burkina Faso and the Republic of Niger reveals a West Eurasian-North African composition of their gene pool. The data show that certain genetic lineages could not have been introduced into this population earlier than ∼9000 years ago whereas local expansions establish a minimal date at around 3000 years ago. Some of the mtDNA haplogroups observed in the Tuareg population were involved in the post-Last Glacial Maximum human expansion from Iberian refugia towards both Europe and North Africa. Interestingly, no Near Eastern mtDNA lineages connected with the Neolithic expansion have been observed in our population sample. On the other hand, the Y chromosome SNPs data show that the paternal lineages can very probably be traced to the Near Eastern Neolithic demic expansion towards North Africa, a period that is otherwise concordant with the above-mentioned mtDNA expansion. The time frame for the migration of the Tuareg towards the African Sahel belt overlaps that of early Holocene climatic changes across the Sahara (from the optimal greening ∼10 000 YBP to the extant aridity beginning at ∼6000 YBP) and the migrations of other African nomadic peoples in the area. PMID:20234393

Linking the sub-Saharan and West Eurasian gene pools: maternal and paternal heritage of the Tuareg nomads from the African Sahel.

PubMed

Pereira, Luísa; Cerný, Viktor; Cerezo, María; Silva, Nuno M; Hájek, Martin; Vasíková, Alzbeta; Kujanová, Martina; Brdicka, Radim; Salas, Antonio

2010-08-01

The Tuareg presently live in the Sahara and the Sahel. Their ancestors are commonly believed to be the Garamantes of the Libyan Fezzan, ever since it was suggested by authors of antiquity. Biological evidence, based on classical genetic markers, however, indicates kinship with the Beja of Eastern Sudan. Our study of mitochondrial DNA (mtDNA) sequences and Y chromosome SNPs of three different southern Tuareg groups from Mali, Burkina Faso and the Republic of Niger reveals a West Eurasian-North African composition of their gene pool. The data show that certain genetic lineages could not have been introduced into this population earlier than approximately 9000 years ago whereas local expansions establish a minimal date at around 3000 years ago. Some of the mtDNA haplogroups observed in the Tuareg population were involved in the post-Last Glacial Maximum human expansion from Iberian refugia towards both Europe and North Africa. Interestingly, no Near Eastern mtDNA lineages connected with the Neolithic expansion have been observed in our population sample. On the other hand, the Y chromosome SNPs data show that the paternal lineages can very probably be traced to the Near Eastern Neolithic demic expansion towards North Africa, a period that is otherwise concordant with the above-mentioned mtDNA expansion. The time frame for the migration of the Tuareg towards the African Sahel belt overlaps that of early Holocene climatic changes across the Sahara (from the optimal greening approximately 10 000 YBP to the extant aridity beginning at approximately 6000 YBP) and the migrations of other African nomadic peoples in the area.

Design of 240,000 orthogonal 25mer DNA barcode probes.

PubMed

Xu, Qikai; Schlabach, Michael R; Hannon, Gregory J; Elledge, Stephen J

2009-02-17

DNA barcodes linked to genetic features greatly facilitate screening these features in pooled formats using microarray hybridization, and new tools are needed to design large sets of barcodes to allow construction of large barcoded mammalian libraries such as shRNA libraries. Here we report a framework for designing large sets of orthogonal barcode probes. We demonstrate the utility of this framework by designing 240,000 barcode probes and testing their performance by hybridization. From the test hybridizations, we also discovered new probe design rules that significantly reduce cross-hybridization after their introduction into the framework of the algorithm. These rules should improve the performance of DNA microarray probe designs for many applications.

Design of 240,000 orthogonal 25mer DNA barcode probes

PubMed Central

Xu, Qikai; Schlabach, Michael R.; Hannon, Gregory J.; Elledge, Stephen J.

2009-01-01

DNA barcodes linked to genetic features greatly facilitate screening these features in pooled formats using microarray hybridization, and new tools are needed to design large sets of barcodes to allow construction of large barcoded mammalian libraries such as shRNA libraries. Here we report a framework for designing large sets of orthogonal barcode probes. We demonstrate the utility of this framework by designing 240,000 barcode probes and testing their performance by hybridization. From the test hybridizations, we also discovered new probe design rules that significantly reduce cross-hybridization after their introduction into the framework of the algorithm. These rules should improve the performance of DNA microarray probe designs for many applications. PMID:19171886

Improving Estimates and Forecasts of Lake Carbon Pools and Fluxes Using Data Assimilation

NASA Astrophysics Data System (ADS)

Zwart, J. A.; Hararuk, O.; Prairie, Y.; Solomon, C.; Jones, S.

2017-12-01

Lakes are biogeochemical hotspots on the landscape, contributing significantly to the global carbon cycle despite their small areal coverage. Observations and models of lake carbon pools and fluxes are rarely explicitly combined through data assimilation despite significant use of this technique in other fields with great success. Data assimilation adds value to both observations and models by constraining models with observations of the system and by leveraging knowledge of the system formalized by the model to objectively fill information gaps. In this analysis, we highlight the utility of data assimilation in lake carbon cycling research by using the Ensemble Kalman Filter to combine simple lake carbon models with observations of lake carbon pools. We demonstrate the use of data assimilation to improve a model's representation of lake carbon dynamics, to reduce uncertainty in estimates of lake carbon pools and fluxes, and to improve the accuracy of carbon pool size estimates relative to estimates derived from observations alone. Data assimilation techniques should be embraced as valuable tools for lake biogeochemists interested in learning about ecosystem dynamics and forecasting ecosystem states and processes.

Screening Yield of HIV Antigen/Antibody Combination and Pooled HIV RNA Testing for Acute HIV Infection in a High-Prevalence Population.

PubMed

Peters, Philip J; Westheimer, Emily; Cohen, Stephanie; Hightow-Weidman, Lisa B; Moss, Nicholas; Tsoi, Benjamin; Hall, Laura; Fann, Charles; Daskalakis, Demetre C; Beagle, Steve; Patel, Pragna; Radix, Asa; Foust, Evelyn; Kohn, Robert P; Marmorino, Jenni; Pandori, Mark; Fu, Jie; Samandari, Taraz; Gay, Cynthia L

2016-02-16

Although acute HIV infection contributes disproportionately to onward HIV transmission, HIV testing has not routinely included screening for acute HIV infection. To evaluate the performance of an HIV antigen/antibody (Ag/Ab) combination assay to detect acute HIV infection compared with pooled HIV RNA testing. Multisite, prospective, within-individual comparison study conducted between September 2011 and October 2013 in 7 sexually transmitted infection clinics and 5 community-based programs in New York, California, and North Carolina. Participants were 12 years or older and seeking HIV testing, without known HIV infection. All participants with a negative rapid HIV test result were screened for acute HIV infection with an HIV Ag/Ab combination assay (index test) and pooled human immunodeficiency virus 1 (HIV-1) RNA testing. HIV RNA testing was the reference standard, with positive reference standard result defined as detectable HIV-1 RNA on an individual RNA test. Number and proportion with acute HIV infections detected. Among 86,836 participants with complete test results (median age, 29 years; 75.0% men; 51.8% men who have sex with men), established HIV infection was diagnosed in 1158 participants (1.33%) and acute HIV infection was diagnosed in 168 participants (0.19%). Acute HIV infection was detected in 134 participants with HIV Ag/Ab combination testing (0.15% [95% CI, 0.13%-0.18%]; sensitivity, 79.8% [95% CI, 72.9%-85.6%]; specificity, 99.9% [95% CI, 99.9%-99.9%]; positive predictive value, 59.0% [95% CI, 52.3%-65.5%]) and in 164 participants with pooled HIV RNA testing (0.19% [95% CI, 0.16%-0.22%]; sensitivity, 97.6% [95% CI, 94.0%-99.4%]; specificity, 100% [95% CI, 100%-100%]; positive predictive value, 96.5% [95% CI, 92.5%-98.7%]; sensitivity comparison, P < .001). Overall HIV Ag/Ab combination testing detected 82% of acute HIV infections detectable by pooled HIV RNA testing. Compared with rapid HIV testing alone, HIV Ag/Ab combination testing increased the relative HIV diagnostic yield (both established and acute HIV infections) by 10.4% (95% CI, 8.8%-12.2%) and pooled HIV RNA testing increased the relative HIV diagnostic yield by 12.4% (95% CI, 10.7%-14.3%). In a high-prevalence population, HIV screening using an HIV Ag/Ab combination assay following a negative rapid test detected 82% of acute HIV infections detectable by pooled HIV RNA testing, with a positive predictive value of 59%. Further research is needed to evaluate this strategy in lower-prevalence populations and in persons using preexposure prophylaxis for HIV prevention.

Accuracy of combined dynamic contrast-enhanced magnetic resonance imaging and diffusion-weighted imaging for breast cancer detection: a meta-analysis.

PubMed

Zhang, Li; Tang, Min; Min, Zhiqian; Lu, Jun; Lei, Xiaoyan; Zhang, Xiaoling

2016-06-01

Magnetic resonance imaging (MRI) is increasingly being used to examine patients with suspected breast cancer. To determine the diagnostic performance of combined dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and diffusion-weighted imaging (DWI) for breast cancer detection. A comprehensive search of the PUBMED, EMBASE, Web of Science, and Cochrane Library databases was performed up to September 2014. Statistical analysis included pooling of sensitivity and specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and diagnostic accuracy using the summary receiver operating characteristic (SROC). All analyses were conducted using STATA (version 12.0), RevMan (version 5.2), and Meta-Disc 1.4 software programs. Fourteen studies were analyzed, which included a total of 1140 patients with 1276 breast lesions. The pooled sensitivity and specificity of combined DCE-MRI and DWI were 91.6% and 85.5%, respectively. The pooled sensitivity and specificity of DWI-MRI were 86.0% and 75.6%, respectively. The pooled sensitivity and specificity of DCE-MRI were 93.2% and 71.1%. The area under the SROC curve (AUC-SROC) of combined DCE-MRI and DWI was 0.94, the DCE-MRI of 0.85. Deeks testing confirmed no significant publication bias in all studies. Combined DCE-MRI and DWI had superior diagnostic accuracy than either DCE-MRI or DWI alone for the diagnosis of breast cancer. © The Foundation Acta Radiologica 2015.

A defect in the thymidine kinase 2 gene causing isolated mitochondrial myopathy without mtDNA depletion.

PubMed

Leshinsky-Silver, E; Michelson, M; Cohen, S; Ginsberg, M; Sadeh, M; Barash, V; Lerman-Sagie, T; Lev, D

2008-07-01

Isolated mitochondrial myopathies (IMM) are either due to primary defects in mtDNA, in nuclear genes that control mtDNA abundance and structure such as thymidine kinase 2 (TK2), or due to CoQ deficiency. Defects in the TK2 gene have been found to be associated with mtDNA depletion attributed to a depleted mitochondrial dNTP pool in non-dividing cells. We report an unusual case of IMM, homozygous for the H90N mutation in the TK2 gene but unlike other cases with the same mutation, does not demonstrate mtDNA depletion. The patient's clinical course is relatively mild and a muscle biopsy showed ragged red muscle fibers with a mild decrease in complexes I and an increase in complexes IV and II activities. This report extends the phenotypic expression of TK2 defects and suggests that all patients who present with an IMM even with normal quantities of mtDNA should be screened for TK2 mutations.

The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage

PubMed Central

Fu, Haiqing; Martin, Melvenia M.; Regairaz, Marie; Huang, Liang; You, Yang; Lin, Chi-Mei; Ryan, Michael; Kim, RyangGuk; Shimura, Tsutomu; Pommier, Yves; Aladjem, Mirit I.

2015-01-01

The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81 deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81 deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins. PMID:25879486

Assessment of circulating copy number variant detection for cancer screening.

PubMed

Molparia, Bhuvan; Nichani, Eshaan; Torkamani, Ali

2017-01-01

Current high-sensitivity cancer screening methods, largely utilizing correlative biomarkers, suffer from false positive rates that lead to unnecessary medical procedures and debatable public health benefit overall. Detection of circulating tumor DNA (ctDNA), a causal biomarker, has the potential to revolutionize cancer screening. Thus far, the majority of ctDNA studies have focused on detection of tumor-specific point mutations after cancer diagnosis for the purpose of post-treatment surveillance. However, ctDNA point mutation detection methods developed to date likely lack either the scope or analytical sensitivity necessary to be useful for cancer screening, due to the low (<1%) ctDNA fraction derived from early stage tumors. On the other hand, tumor-derived copy number variant (CNV) detection is hypothetically a superior means of ctDNA-based cancer screening for many tumor types, given that, relative to point mutations, each individual tumor CNV contributes a much larger number of ctDNA fragments to the overall pool of circulating free DNA (cfDNA). A small number of studies have demonstrated the potential of ctDNA CNV-based screening in select cancer types. Here we perform an in silico assessment of the potential for ctDNA CNV-based cancer screening across many common cancers, and suggest ctDNA CNV detection shows promise as a broad cancer screening methodology.

Structure and Function of the Splice Variants of TMPRSS2-ERG, a Prevalent Genomic Alteration in Prostate Cancer

DTIC Science & Technology

2009-09-01

binding ETS domain) and five type II (without ETS domain). Fusion-positive type I– and type II–containing phages were amplified with T3 and T7 primers...will be performed to identify the authentic 3’ UTRs from the mRNA pool from CaP patient specimens. Using phage excision strategy, we will use to... phage DNA sequences plasmids (cDNA) clones were generated by using phage excision strategy. Figure 1. ERG splice variants in prostate cancer

Genome-wide association of porcine lung lesions using DNA pooling

USDA-ARS?s Scientific Manuscript database

Respiratory disease in swine is a primary health concern for producers. Pleuropneumonia and enzootic pneumonia occur at a high incidence on most farms and have a negative effect on feed efficiency, growth, and animal welfare. Mycoplasma pneumonia is one of the most important because it increases sus...

[35S]-LABELING OF THE SALMONELLA TYPHIMURIUM GLUTATHIONE POOL TO ASSESS GLUTATHIONE-MEDIATED DNA BINDING BY 1,2-DIBROMOETHANE

EPA Science Inventory

Biotransformation of drugs and environmental chemicals to reactive intermediates is often studied with the use of radiolabeled compounds that are synthesized by expensive and technically difficult procedures. In general, glutathione (GSH) conjugation serves as a detoxification m...

Molecular Characterization of Cultivated Pawpaw (Asimina triloba) Using RAPD Markers

Treesearch

Hongwen Huang; Desmond R. Layne; Thomas L. Kubisiak

2003-01-01

Thirty-four extant pawpaw [Asimina triloba (L.) Dunal] cultivars and advanced selections representing a large portion of the gene pool of cultivated pawpaws were investigated using 71 randomly amplified polymorphic DNA (RAPD) markers to establish genetic identities and evaluate genetic relatedness. All 34 cultivated pawpaws were uniquely...

Species From Feces: Order-Wide Identification of Chiroptera From Guano and Other Non-Invasive Genetic Samples

PubMed Central

Williamson, Charles H. D.; Sanchez, Daniel E.; Sobek, Colin J.; Chambers, Carol L.

2016-01-01

Bat guano is a relatively untapped reservoir of information, having great utility as a DNA source because it is often available at roosts even when bats are not and is an easy type of sample to collect from a difficult-to-study mammalian order. Recent advances from microbial community studies in primer design, sequencing, and analysis enable fast, accurate, and cost-effective species identification. Here, we borrow from this discipline to develop an order-wide DNA mini-barcode assay (Species from Feces) based on a segment of the mitochondrial gene cytochrome c oxidase I (COI). The assay works effectively with fecal DNA and is conveniently transferable to low-cost, high-throughput Illumina MiSeq technology that also allows simultaneous pairing with other markers. Our PCR primers target a region of COI that is highly discriminatory among Chiroptera (92% species-level identification of barcoded species), and are sufficiently degenerate to allow hybridization across diverse bat taxa. We successfully validated our system with 54 bat species across both suborders. Despite abundant arthropod prey DNA in guano, our primers were highly specific to bats; no arthropod DNA was detected in thousands of feces run on Sanger and Illumina platforms. The assay is extendable to fecal pellets of unknown age as well as individual and pooled guano, to allow for individual (using singular fecal pellets) and community (using combined pellets collected from across long-term roost sites) analyses. We developed a searchable database (http://nau.edu/CEFNS/Forestry/Research/Bats/Search-Tool/) that allows users to determine the discriminatory capability of our markers for bat species of interest. Our assay has applications worldwide for examining disease impacts on vulnerable species, determining species assemblages within roosts, and assessing the presence of bat species that are vulnerable or facing extinction. The development and analytical pathways are rapid, reliable, and inexpensive, and can be applied to ecology and conservation studies of other taxa. PMID:27654850

Species From Feces: Order-Wide Identification of Chiroptera From Guano and Other Non-Invasive Genetic Samples.

PubMed

Walker, Faith M; Williamson, Charles H D; Sanchez, Daniel E; Sobek, Colin J; Chambers, Carol L

Bat guano is a relatively untapped reservoir of information, having great utility as a DNA source because it is often available at roosts even when bats are not and is an easy type of sample to collect from a difficult-to-study mammalian order. Recent advances from microbial community studies in primer design, sequencing, and analysis enable fast, accurate, and cost-effective species identification. Here, we borrow from this discipline to develop an order-wide DNA mini-barcode assay (Species from Feces) based on a segment of the mitochondrial gene cytochrome c oxidase I (COI). The assay works effectively with fecal DNA and is conveniently transferable to low-cost, high-throughput Illumina MiSeq technology that also allows simultaneous pairing with other markers. Our PCR primers target a region of COI that is highly discriminatory among Chiroptera (92% species-level identification of barcoded species), and are sufficiently degenerate to allow hybridization across diverse bat taxa. We successfully validated our system with 54 bat species across both suborders. Despite abundant arthropod prey DNA in guano, our primers were highly specific to bats; no arthropod DNA was detected in thousands of feces run on Sanger and Illumina platforms. The assay is extendable to fecal pellets of unknown age as well as individual and pooled guano, to allow for individual (using singular fecal pellets) and community (using combined pellets collected from across long-term roost sites) analyses. We developed a searchable database (http://nau.edu/CEFNS/Forestry/Research/Bats/Search-Tool/) that allows users to determine the discriminatory capability of our markers for bat species of interest. Our assay has applications worldwide for examining disease impacts on vulnerable species, determining species assemblages within roosts, and assessing the presence of bat species that are vulnerable or facing extinction. The development and analytical pathways are rapid, reliable, and inexpensive, and can be applied to ecology and conservation studies of other taxa.

Phylogenetic relationships among Lactuca (Asteraceae) species and related genera based on ITS-1 DNA sequences.

PubMed

Koopman, W J; Guetta, E; van de Wiel, C C; Vosman, B; van den Berg, R G

1998-11-01

Internal transcribed spacer (ITS-1) sequences from 97 accessions representing 23 species of Lactuca and related genera were determined and used to evaluate species relationships of Lactuca sensu lato (s.l.). The ITS-1 phylogenies, calculated using PAUP and PHYLIP, correspond better to the classification of Feráková than to other classifications evaluated, although the inclusion of sect. Lactuca subsect. Cyanicae is not supported. Therefore, exclusion of subsect. Cyanicae from Lactuca sensu Feráková is proposed. The amended genus contains the entire gene pool (sensu Harlan and De Wet) of cultivated lettuce (Lactuca sativa). The position of the species in the amended classification corresponds to their position in the lettuce gene pool. In the ITS-1 phylogenies, a clade with L. sativa, L. serriola, L. dregeana, L. altaica, and L. aculeata represents the primary gene pool. L. virosa and L. saligna, branching off closest to this clade, encompass the secondary gene pool. L. virosa is possibly of hybrid origin. The primary and secondary gene pool species are classified in sect. Lactuca subsect. Lactuca. The species L. quercina, L. viminea, L. sibirica, and L. tatarica, branching off next, represent the tertiary gene pool. They are classified in Lactuca sect. Lactucopsis, sect. Phaenixopus, and sect. Mulgedium, respectively. L. perennis and L. tenerrima, classified in sect. Lactuca subsect. Cyanicae, form clades with species from related genera and are not part of the lettuce gene pool.

In search of the genetic footprints of Sumerians: a survey of Y-chromosome and mtDNA variation in the Marsh Arabs of Iraq

PubMed Central

2011-01-01

Background For millennia, the southern part of the Mesopotamia has been a wetland region generated by the Tigris and Euphrates rivers before flowing into the Gulf. This area has been occupied by human communities since ancient times and the present-day inhabitants, the Marsh Arabs, are considered the population with the strongest link to ancient Sumerians. Popular tradition, however, considers the Marsh Arabs as a foreign group, of unknown origin, which arrived in the marshlands when the rearing of water buffalo was introduced to the region. Results To shed some light on the paternal and maternal origin of this population, Y chromosome and mitochondrial DNA (mtDNA) variation was surveyed in 143 Marsh Arabs and in a large sample of Iraqi controls. Analyses of the haplogroups and sub-haplogroups observed in the Marsh Arabs revealed a prevalent autochthonous Middle Eastern component for both male and female gene pools, with weak South-West Asian and African contributions, more evident in mtDNA. A higher male than female homogeneity is characteristic of the Marsh Arab gene pool, likely due to a strong male genetic drift determined by socio-cultural factors (patrilocality, polygamy, unequal male and female migration rates). Conclusions Evidence of genetic stratification ascribable to the Sumerian development was provided by the Y-chromosome data where the J1-Page08 branch reveals a local expansion, almost contemporary with the Sumerian City State period that characterized Southern Mesopotamia. On the other hand, a more ancient background shared with Northern Mesopotamia is revealed by the less represented Y-chromosome lineage J1-M267*. Overall our results indicate that the introduction of water buffalo breeding and rice farming, most likely from the Indian sub-continent, only marginally affected the gene pool of autochthonous people of the region. Furthermore, a prevalent Middle Eastern ancestry of the modern population of the marshes of southern Iraq implies that if the Marsh Arabs are descendants of the ancient Sumerians, also the Sumerians were most likely autochthonous and not of Indian or South Asian ancestry. PMID:21970613

In search of the genetic footprints of Sumerians: a survey of Y-chromosome and mtDNA variation in the Marsh Arabs of Iraq.

PubMed

Al-Zahery, Nadia; Pala, Maria; Battaglia, Vincenza; Grugni, Viola; Hamod, Mohammed A; Hooshiar Kashani, Baharak; Olivieri, Anna; Torroni, Antonio; Santachiara-Benerecetti, Augusta S; Semino, Ornella

2011-10-04

For millennia, the southern part of the Mesopotamia has been a wetland region generated by the Tigris and Euphrates rivers before flowing into the Gulf. This area has been occupied by human communities since ancient times and the present-day inhabitants, the Marsh Arabs, are considered the population with the strongest link to ancient Sumerians. Popular tradition, however, considers the Marsh Arabs as a foreign group, of unknown origin, which arrived in the marshlands when the rearing of water buffalo was introduced to the region. To shed some light on the paternal and maternal origin of this population, Y chromosome and mitochondrial DNA (mtDNA) variation was surveyed in 143 Marsh Arabs and in a large sample of Iraqi controls. Analyses of the haplogroups and sub-haplogroups observed in the Marsh Arabs revealed a prevalent autochthonous Middle Eastern component for both male and female gene pools, with weak South-West Asian and African contributions, more evident in mtDNA. A higher male than female homogeneity is characteristic of the Marsh Arab gene pool, likely due to a strong male genetic drift determined by socio-cultural factors (patrilocality, polygamy, unequal male and female migration rates). Evidence of genetic stratification ascribable to the Sumerian development was provided by the Y-chromosome data where the J1-Page08 branch reveals a local expansion, almost contemporary with the Sumerian City State period that characterized Southern Mesopotamia. On the other hand, a more ancient background shared with Northern Mesopotamia is revealed by the less represented Y-chromosome lineage J1-M267*. Overall our results indicate that the introduction of water buffalo breeding and rice farming, most likely from the Indian sub-continent, only marginally affected the gene pool of autochthonous people of the region. Furthermore, a prevalent Middle Eastern ancestry of the modern population of the marshes of southern Iraq implies that if the Marsh Arabs are descendants of the ancient Sumerians, also the Sumerians were most likely autochthonous and not of Indian or South Asian ancestry.

Building a Better Applicant Pool--A Case Study of the Use of Predictive Modeling and Market Segmentation to Build and Enroll Better Pools of Students

ERIC Educational Resources Information Center

Herridge, Bart; Heil, Robert

2003-01-01

Predictive modeling has been a popular topic in higher education for the last few years. This case study shows an example of an effective use of modeling combined with market segmentation to strategically divide large, unmanageable prospect and inquiry pools and convert them into applicants, and eventually, enrolled students. (Contains 6 tables.)

Screening and Characterization of RAPD Markers in Viscerotropic Leishmania Parasites

PubMed Central

Mkada–Driss, Imen; Talbi, Chiraz; Guerbouj, Souheila; Driss, Mehdi; Elamine, Elwaleed M.; Cupolillo, Elisa; Mukhtar, Moawia M.; Guizani, Ikram

2014-01-01

Visceral leishmaniasis (VL) is mainly due to the Leishmania donovani complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL Leishmania parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 L. donovani, L. archibaldi and L. infantum strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5′ end transversions, and presence of inter– and intra– taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between L. infantum and L. donovani. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents. PMID:25313833

Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology.

PubMed Central

Siehnel, R J; Worobec, E A; Hancock, R E

1988-01-01

The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes. Images PMID:2834340

A new source of cytoplasmic male sterility in pearl millet: RFLP analysis of mitochondrial DNA.

PubMed

Sujata, V; Sivaramakrishnan, S; Rai, K N; Seetha, K

1994-06-01

A new source of cytoplasmic male sterility (cms) in pearl millet (Pennisetum glaucum (L.) R.Br.) derived from a half-sib progeny of the Early Gene Pool (EGP 261) and used in a male-sterile line, ICMA 90111, was compared with other known cms sources for RFLP of mitochondrial (mt) DNA. Southern blot hybridization of mtDNA from ICMA 90111 digested with several restriction enzymes and probed with homologous mtDNA clones from pearl millet and heterologous gene clones from maize and wheat revealed the RFLP patterns of ICMA 90111 distinct from others studied so far. The dendrogram of male-sterile lines constructed from the Southern blot hybridization patterns indicated that ICMA 90111 represents a separate group. Our results suggest that this source of cms is unique in several respects.

DNA-based cryptographic methods for data hiding in DNA media.

PubMed

Marwan, Samiha; Shawish, Ahmed; Nagaty, Khaled

2016-12-01

Information security can be achieved using cryptography, steganography or a combination of them, where data is firstly encrypted using any of the available cryptography techniques and then hid into any hiding medium. Recently, the famous genomic DNA has been introduced as a hiding medium, known as DNA steganography, due to its notable ability to hide huge data sets with a high level of randomness and hence security. Despite the numerous cryptography techniques, to our knowledge only the vigenere cipher and the DNA-based playfair cipher have been combined with the DNA steganography, which keeps space for investigation of other techniques and coming up with new improvements. This paper presents a comprehensive analysis between the DNA-based playfair, vigenere, RSA and the AES ciphers, each combined with a DNA hiding technique. The conducted analysis reports the performance diversity of each combined technique in terms of security, speed, hiding capacity in addition to both key size and data size. Moreover, this paper proposes a modification of the current combined DNA-based playfair cipher technique, which makes it not only simple and fast but also provides a significantly higher hiding capacity and security. The conducted extensive experimental studies confirm such outstanding performance in comparison with all the discussed combined techniques. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Analysis of plastome and chondriome genome types in potato somatic hybrids from Solanum tuberosum × Solanum etuberosum.

PubMed

Tiwari, Jagesh K; Chandel, Poonam; Singh, Bir Pal; Bhardwaj, Vinay

2014-01-01

Cytoplasm types of the potato somatic hybrids from Solanum tuberosum × Solanum etuberosum were analysed using chloroplast (cp) and mitochondrial (mt) organelle genomes-specific markers. Of the 29 markers (15 cpDNA and 14 mtDNA) amplified in the 26 genotypes, 5 cpDNA (H3, NTCP4, NTCP8, NTCP9, and ALC1/ALC3) and 13 mtDNA markers showed polymorphism. The cluster analysis based on the mtDNA markers detected higher diversity compared with the cpDNA markers. Presence of new mtDNA fragments of the markers, namely, T11-2, Nsm1, pumD, Nsm3, and Nsm4, were observed, while monomorphic loci revealed highly conserved genomic regions in the somatic hybrids. The study revealed that the somatic hybrids had diverse cytoplasm types consisting predominantly of T-, W-, and C-, with a few A- and S-type cp genomes; and α-, β-, and γ-type mt genomes. Somatic hybridization has unique potential to widen the cytoplasm types of the cultivated gene pools from wild species through introgression by breeding methods.

77 FR 3470 - Combined Notice of Filings

Federal Register 2010, 2011, 2012, 2013, 2014

2012-01-24

... that the Commission has received the following Natural Gas Pipeline Rate and Refund Report filings... LLC, Missouri Joint Municipal Electric Util., City of Independence, Missouri, Kansas Power Pool... Municipal Electric Utility Commission, the City of Independence, Missouri, Kansas Power Pool and Kansas City...

Analysis of Combined Data from Heterogeneous Study Designs: A Methodological Proposal from the Patient Navigation Research program

PubMed Central

Roetzheim, Richard G.; Freund, Karen M.; Corle, Don K.; Murray, David M.; Snyder, Frederick R.; Kronman, Andrea C.; Jean-Pierre, Pascal; Raich, Peter C.; Holden, Alan E. C.; Darnell, Julie S.; Warren-Mears, Victoria; Patierno, Steven; Design, PNRP; Committee, Analysis

2013-01-01

Background The Patient Navigation Research Program (PNRP) is a cooperative effort of nine research projects, each employing its own unique study design. To evaluate projects such as PNRP, it is desirable to perform a pooled analysis to increase power relative to the individual projects. There is no agreed upon prospective methodology, however, for analyzing combined data arising from different study designs. Expert opinions were thus solicited from members of the PNRP Design and Analysis Committee Purpose To review possible methodologies for analyzing combined data arising from heterogeneous study designs. Methods The Design and Analysis Committee critically reviewed the pros and cons of five potential methods for analyzing combined PNRP project data. Conclusions were based on simple consensus. The five approaches reviewed included: 1) Analyzing and reporting each project separately, 2) Combining data from all projects and performing an individual-level analysis, 3) Pooling data from projects having similar study designs, 4) Analyzing pooled data using a prospective meta analytic technique, 5) Analyzing pooled data utilizing a novel simulated group randomized design. Results Methodologies varied in their ability to incorporate data from all PNRP projects, to appropriately account for differing study designs, and in their impact from differing project sample sizes. Limitations The conclusions reached were based on expert opinion and not derived from actual analyses performed. Conclusions The ability to analyze pooled data arising from differing study designs may provide pertinent information to inform programmatic, budgetary, and policy perspectives. Multi-site community-based research may not lend itself well to the more stringent explanatory and pragmatic standards of a randomized controlled trial design. Given our growing interest in community-based population research, the challenges inherent in the analysis of heterogeneous study design are likely to become more salient. Discussion of the analytic issues faced by the PNRP and the methodological approaches we considered may be of value to other prospective community-based research programs. PMID:22273587

Analysis of combined data from heterogeneous study designs: an applied example from the patient navigation research program.

PubMed

Roetzheim, Richard G; Freund, Karen M; Corle, Don K; Murray, David M; Snyder, Frederick R; Kronman, Andrea C; Jean-Pierre, Pascal; Raich, Peter C; Holden, Alan Ec; Darnell, Julie S; Warren-Mears, Victoria; Patierno, Steven

2012-04-01

The Patient Navigation Research Program (PNRP) is a cooperative effort of nine research projects, with similar clinical criteria but with different study designs. To evaluate projects such as PNRP, it is desirable to perform a pooled analysis to increase power relative to the individual projects. There is no agreed-upon prospective methodology, however, for analyzing combined data arising from different study designs. Expert opinions were thus solicited from the members of the PNRP Design and Analysis Committee. To review possible methodologies for analyzing combined data arising from heterogeneous study designs. The Design and Analysis Committee critically reviewed the pros and cons of five potential methods for analyzing combined PNRP project data. The conclusions were based on simple consensus. The five approaches reviewed included the following: (1) analyzing and reporting each project separately, (2) combining data from all projects and performing an individual-level analysis, (3) pooling data from projects having similar study designs, (4) analyzing pooled data using a prospective meta-analytic technique, and (5) analyzing pooled data utilizing a novel simulated group-randomized design. Methodologies varied in their ability to incorporate data from all PNRP projects, to appropriately account for differing study designs, and to accommodate differing project sample sizes. The conclusions reached were based on expert opinion and not derived from actual analyses performed. The ability to analyze pooled data arising from differing study designs may provide pertinent information to inform programmatic, budgetary, and policy perspectives. Multisite community-based research may not lend itself well to the more stringent explanatory and pragmatic standards of a randomized controlled trial design. Given our growing interest in community-based population research, the challenges inherent in the analysis of heterogeneous study design are likely to become more salient. Discussion of the analytic issues faced by the PNRP and the methodological approaches we considered may be of value to other prospective community-based research programs.

Prevalence of anxiety and depressive disorders among youth with intellectual disabilities: A systematic review and meta-analysis.

PubMed

Maïano, Christophe; Coutu, Sylvain; Tracey, Danielle; Bouchard, Stéphane; Lepage, Geneviève; Morin, Alexandre J S; Moullec, Grégory

2018-04-06

The purpose of this meta-analytic study was to determine the pooled prevalence estimates of anxiety and depressive disorders among children and adolescents with intellectual disabilities (ID) and to assess the extent to which these pooled prevalence rates differed according to studies' characteristics. A systematic literature search was performed in nine databases and 21 studies, published between 1975 and 2015, met the inclusion criteria. The resulting pooled prevalence estimates of combined subtypes of anxiety and depressive disorders were respectively (a) 5.4% and 2.8% across samples; (b) 1.2% and 0.03% among children; and (c) 7.9% and 1.4% among adolescents. Pooled prevalence estimates for specific subtypes of anxiety disorders ranged from (a) 0.2% to 11.5% across samples; (b) 0.7% to 17.6% among children; and (c) 0.6% to 19.8% among adolescents. Pooled prevalence estimates of dysthymic disorder and major depressive disorder were respectively (a) 3.4% and 2.5% across samples; (b) 2.1% and 3.2% among children; and (c) 6.9% and 5.7% among adolescents. Finally, subgroup analyses showed significant variations in the pooled prevalence estimates of combined subtypes of anxiety disorders, obsessive-compulsive disorder, and generalized anxiety disorder; and combined subtypes of depressive disorders. The present findings of this meta-analysis should be interpreted with caution given several limitations related to the characteristics of the populations, diagnostic method and sampling method. Findings provide recommendations for future studies investigating psychological disorders among youth with ID, as well as how clinicians and policy makers can improve diagnostic practices and support for youth with ID. Copyright © 2018 Elsevier B.V. All rights reserved.

[The Russian gene pool: gene geography of Alu-insertions (ACE, APOA1, B65, PV92 TPA25)].

PubMed

Solov'eva, D S; Balanovskaia, E V; Kuznetsova, M A; Vasinskaia, O A; Frolova, S A; Pocheshkhova, E A; Evseeva, I V; Boldyreva, M N; Balanovskiĭ, O P

2010-01-01

The analysis of five Alu insertion loci (ACE, AP4OA1, B65, PV92, TPA25) has been carried out for the first time in 10 Russian populations (1088 individuals), covered all parts of historical area of the Russian ethnos. Depending on locus, Russian populations exhibit similarity with their western (European populations) or with the eastern (populations of the Ural region) neighbors. Considering frequencies of the studied Alu-insertions, Russian gene pool exhibits low variation: average difference between populations is d = 0.007, whereas on classical markers, mtDNA and Y chromosome heterogeneity of Russian gene pool is essentially higher (0.013, 0.033 and 0.142 respectively). Therefore, this set of five Alu insertions has lower variability on the intra-ethnic level. However in inter-ethnic comparisons the clear pattern was obtained: 13 Eastern European ethnic groups formed three clusters, according with their historical and geographical position--East Slavic, Caucasian and South Ural clusters. The obtained data confirms efficiency of using Alu insertions for studying genetic differentiation and history of a gene pool of the Eastern European populations.

Disinfection by-products and microbial contamination in the treatment of pool water with granular activated carbon.

PubMed

Uhl, W; Hartmann, C

2005-01-01

For swimming pools, it is generally agreed that free chlorine levels have to be maintained to guarantee adequate disinfection. Recommended free chlorine levels can vary between 0.3 and 0.6 mg/L in Germany and up to 3 mg/L in other countries. Bathers introduce considerable amounts of organic matter, mainly in the form of such as urine and sweat, into the pool water. As a consequence, disinfection byproducts (DBPs) are formed. Regulations in Germany recommend levels of combined chlorine of less than 0.2 mg/L and levels of trihalomethanes (THMs) of less than 20 microg/L. Haloacetic acids (HAAs), haloacetonitriles (HANs), chloropicrin and chloral hydrate are also detected in considerable amounts. However, these compounds are not regulated yet. Swimming pool staff and swimmers, especially athletes, are primarily exposed to these byproducts by inhalation and/or dermal uptake. In Germany, new regulations for swimming pool water treatment generally require the use of activated carbon. In this project, three different types of granular activated carbon (GAC) (one standard GAC, two catalytic GACs) are compared for their long time behaviour in pool water treatment. In a pilot plant operated with real swimming pool water, production and removal of disinfection byproducts (THMs, HAAs, AOXs), of biodegradable substances (AOC), of bacteria (Pseudomonas aeruginosa, Legionella, coliforms, HPC) as well as the removal of chlorine and chloramines are monitored as function of GAC bed depth. Combined chlorine penetrates deeper in the filter bed than free chlorine does. However, both, free and combined chlorine removal efficiencies decrease over the time of filter operation. The decreases of removal efficiencies are also observed for parameters such as dissolved organic carbon, spectral absorption coefficient, adsorbable organic carbon and most of the disinfection byproducts. However, THMs, especially chloroform are produced in the filter bed. The GAC beds were contaminated microbially, especially with P. aeruginosa. The contamination was not removable by backwashing with chlorine concentrations up to 2 mg/l free chlorine.

Achievements, Challenges, and Opportunities in DNA-Encoded Library Research: An Academic Point of View.

PubMed

Yuen, Lik Hang; Franzini, Raphael M

2017-05-04

DNA-encoded chemical libraries (DECLs) are pools of DNA-tagged small molecules that enable facile screening and identification of bio-macromolecule binders. The successful development of DECLs has led to their increasingly important role in drug development, and screening hits have entered clinical trials. In this review, we summarize the development and currently active research areas of DECLs with a focus on contributions from groups at academic institutes. We further look at opportunities and future directions of DECL research in medicinal chemistry and chemical biology based on the symbiotic relationship between academia and industry. Challenges associated with the application of DECLs in academic drug discovery are further discussed. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interdecadal heterogeneity in mitochondrial DNA of Atlantic haddock (Melanogrammus aeglefinus) from Georges Bank.

PubMed

Purcell, M K; Kornfield, I; Fogarty, M; Parker, A

1996-09-01

Atlantic haddock of Georges Bank are characterized by large fluctuations in population size and a recent collapse of the commercial fishery. DNA extracted from dried scales of Georges Bank haddock, archived by the U.S. National Marine Fisheries Service (NMFS), reveals significant heterogeneity in frequencies of four mitochondrial DNA control region haplotypes between 1975 and 1985 cohorts. Several processes may be responsible for this temporal variation, the most attractive hypothesis being that haddock from other geographic regions episodically contribute to the Georges Bank gene pool. Thus, the population of haddock spawning on Georges Bank may not be genetically discrete and, with respect to Atlantic haddock, Georges Bank may not be viewed as a closed system.

76 FR 49467 - Combined Notice of Filings #1

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-10

.... Applicants: Wolverine Power Supply Cooperative, Inc., Midwest Independent Transmission System Operator, Inc. Description: Wolverine Power Supply Cooperative, Inc. submits tariff filing per 35.13(a)(2)(iii: Wolverine...: Southwest Power Pool, Inc. Description: Southwest Power Pool, Inc. submits tariff filing per 35.17(b...

Molecular identification of Plasmodium spp. and blood meal sources of anophelines in environmental reserves on São Luís Island, state of Maranhão, Brazil.

PubMed

Figueiredo, Mayra Araguaia Pereira; Di Santi, Silvia Maria; Manrique, Wilson Gómez; Gonçalves, Luiz Ricardo; André, Marcos Rogério; Machado, Rosangela Zacarias

2017-04-26

Considering the diversity of feeding habits that females of some species of anophelines present, it is important to understand which vertebrates are part of blood food sources and how important is the role of each in the ecoepidemiology of malaria. There are many vector species for Plasmodium spp. in the State of Maranhão, Brazil. In São Luís Island, Anopheles aquasalis is the main vector for human malaria; this species is abundant in areas with primates that are positive for Plasmodium. Anopheles aquasalis has natural exophilic and zoophilic feeding behavior, but in cases of high density and absence of animals, presents quite varied behavior, and feeds on human blood. In this context, the objective of the present study was to identify Plasmodium spp. and the blood meal sources of anophelines in two environmental reserves on São Luís Island, state of Maranhão, using molecular methods. Between June and July 2013, female anophelines were collected in the Sítio Aguahy Private Reserve, in the municipality of São José de Ribamar, and in the Sítio Mangalho Reserve, located within the Maracanã Environmental Protection Area, in the municipality of São Luís. CDC-type light traps, Shannon traps and protected human bait were used during three consecutive hours in peridomestic and wooded areas. Pools of anophelines were formed using mosquitoes of the same species that had been caught at the same site on the same date. A genus-specific amplification protocol based on the 18S rRNA gene was used for qPCR and cPCR. A total of 416 anophelines were collected, of the following species: An. aquasalis (399), An. mediopunctatus (3), An. shannoni (1), An. nuneztovari (sensu lato) (1), An. goeldii (1), An. evansae (2) and An. (Nyssorhynchus) sp. (9), comprising 54 pools. Two pools were positive for Plasmodium (2/54) based on the 18S rRNA gene. In the phylogenetic analysis using the maximum likelihood method, based on a 240 bp fragment of the 18S rRNA gene, it was found that the sequences of Plasmodium sp. amplified from pools of An. aquasalis (pool 2) and An. nuneztovari (s.l.) (pool 10) were phylogenetically related to a clade of P. falciparum isolates from India, and to a clade of Plasmodium sp. isolates from psittacines in Brazil, respectively. Cat, dog and human DNA were identified in the blood meals of the anophelines sampled. The species An. aquasalis was the most abundant anopheline species in São Luís Island. Plasmodium spp. DNA was detected, thus confirming the importance of this species as the main vector on São Luís Island, Brazil. In addition, the presence of An. nuneztovari (s.l.) with DNA positive for Plasmodium spp. confirms its importance as a secondary vector.

Specificity and Catalytic Mechanism in Family 5 Uracil DNA Glycosylase*

PubMed Central

Xia, Bo; Liu, Yinling; Li, Wei; Brice, Allyn R.; Dominy, Brian N.; Cao, Weiguo

2014-01-01

UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that family 5 UDGb from Thermus thermophilus HB8 is not only a uracil DNA glycosyase acting on G/U, T/U, C/U, and A/U base pairs, but also a hypoxanthine DNA glycosylase acting on G/I, T/I, and A/I base pairs and a xanthine DNA glycosylase acting on all double-stranded and single-stranded xanthine-containing DNA. Analysis of potentials of mean force indicates that the tendency of hypoxanthine base flipping follows the order of G/I > T/I, A/I > C/I, matching the trend of hypoxanthine DNA glycosylase activity observed in vitro. Genetic analysis indicates that family 5 UDGb can also act as an enzyme to remove uracil incorporated into DNA through the existence of dUTP in the nucleotide pool. Mutational analysis coupled with molecular modeling and molecular dynamics analysis reveals that although hydrogen bonding to O2 of uracil underlies the UDG activity in a dissociative fashion, Tth UDGb relies on multiple catalytic residues to facilitate its excision of hypoxanthine and xanthine. This study underscores the structural and functional diversity in the UDG superfamily. PMID:24838246

Optimization and evaluation of single-cell whole-genome multiple displacement amplification.

PubMed

Spits, C; Le Caignec, C; De Rycke, M; Van Haute, L; Van Steirteghem, A; Liebaers, I; Sermon, K

2006-05-01

The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research. (c) 2006 Wiley-Liss, Inc.

Genetic diversity and differentiation in Prunus species (Rosaceae) using chloroplast and mitochondrial DNA CAPS markers.

PubMed

Ben Mustapha, S; Ben Tamarzizt, H; Baraket, G; Abdallah, D; Salhi Hannachi, A

2015-04-27

Chloroplast (cpDNA) and mitochondrial DNA (mtDNA) were analyzed to establish genetic relationships among Tunisian plum cultivars using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique. Two mtDNA regions (nad 1 b/c and nad 4 1/2) and a cpDNA region (trnL-trnF) were amplified and digested using restriction enzymes. Seventy and six polymorphic sites were revealed in cpDNA and mtDNA, respectively. As a consequence, cpDNA appears to be more polymorphic than mtDNA. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram showed that accessions were distributed independently of their geographical origin, and introduced and local cultivars appear to be closely related. Both UPGMA and principal component analysis grouped Tunisian plum accessions into similar clusters. The analysis of the pooled sequences allowed the detection of 17 chlorotypes and 12 mitotypes. The unique haplotypes detected for cultivars are valuable for management and preservation of the plum local resources. From this study, PCR-RFLP analysis appears to be a useful approach to detect and identify cytoplasmic variation in plum trees. Our results also provide useful information for the management of genetic resources and to establish a program to improve the genetic resources available for plums.

Hydrology of the Floral City Pool of Tsala Apopka Lake, west-central Florida

USGS Publications Warehouse

Bradner, L.A.

1988-01-01

Tsala Apopka Lake, in west-central Florida, has an area of about 19,000 acres and is divided into three water-management pools, with the Floral City Pool, the most upgradient. The Floral City Pool, which has a surface area of approximately 4,750 acres, contains an extensive combination of lakes, wetlands, and connecting canals. The Pool receives inflow from the Withlacoochee River through two canals. Outflow is through one manmade canal and one natural slough. Canal flow is partially controlled by manmade structures. A cumulative deficit of 19.4 inches of rainfall from August 1984 through May 1985 reduced surface-water inflow to the Floral City Pool to about 0.5 cu ft/sec by May 1985. During May 1985, pool levels declined approximately 0.04 ft/day. By the end of May, there was no observable outflow. From June 1985 through September 1985, 39.8 inches of rainfall caused above-average inflow to the Floral City Pool and a pool-level increase of 6.2 ft. The inflow of 340 CFS nearly equaled the outflow of 338 CFS by the end of September. (USGS)

Poly(adenylic acid) complementary DNA real-time polymerase chain reaction in pancreatic ductal juice in patients undergoing pancreaticoduodenectomy.

PubMed

Oliveira-Cunha, Melissa; Byers, Richard J; Siriwardena, Ajith K

2010-03-01

There is a need to develop methods of early diagnosis for pancreatic cancer. Pancreatic juice is easily collected by endoscopic retrograde cholangiopancreatography and may facilitate diagnosis using molecular markers. The aim of this work was to explore the feasibility of measurement of gene expression in RNA isolated from ductal juice. Intraoperative sampling of pancreatic juice was undertaken in 27 patients undergoing pancreaticoduodenectomy for suspected tumor. Total RNA was extracted and used as template for poly(adenylic acid) (poly[A]) polymerase chain reaction (PCR) to generate a globally amplified complementary DNA pool representative of all expressed messenger RNAs. Real-time PCR was performed for trefoil factor 2 (TFF2), carboxypeptidase B1 (CPB1), and kallikrein-related peptidase 3 (KLK3) in a subset of samples; all samples were normalized for 3 reference genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], PSMB6, and beta-2-microglobulin [B2M]). The median volume of the pancreatic juice obtained was 1245 microL (range, 50-5000 microL). The RNA integrity number ranged from 1.9 to 10. Reverse transcriptase PCR was positive for pancreas-specific genes (TFF2 and CPB1) and negative for prostatic-specific antigen in all samples. These results demonstrate that RNA analysis of pancreatic juice is feasible using a combination of poly(A) PCR and real-time PCR. In addition, the poly(A) complementary DNA generated can be probed for multiple genes and is indefinitely renewable, thereby representing a molecular block of importance for future research.

Environmental controls of C, N and P biogeochemistry in peatland pools.

PubMed

Arsenault, Julien; Talbot, Julie; Moore, Tim R

2018-08-01

Pools are common in northern peatlands but studies have seldom focused on their nutrient biogeochemistry, especially in relation to their morphological characteristics and through seasons. We determined the environmental characteristics controlling carbon (C), nitrogen (N) and phosphorus (P) biogeochemistry in pools and assessed their evolution over the course of the 2016 growing season in a subboreal ombrotrophic peatland of eastern Canada. We showed that water chemistry variations in 62 pools were significantly explained by depth (81.9%) and the surrounding vegetation type (14.8%), but not by pool area or shape. Shallow pools had larger dissolved organic carbon (DOC) and total nitrogen (TN) concentrations and lower pH than deep pools, while pools surrounded by coniferous trees had more recalcitrant DOC than pools where vegetation was dominated by mosses. The influence of depth on pool biogeochemistry was confirmed by the seasonal survey of pools of different sizes with 47.1% of the variation in pool water chemistry over time significantly explained. Of this, 67.3% was explained by the interaction between time and pool size and 32.7% by pool size alone. P concentrations were small in all pools all summer long and combined with high N:P ratios, are indicative of P-limitation. Our results show that pool biogeochemistry is influenced by internal processes and highlight the spatial and temporal heterogeneity of nutrient biogeochemistry in ombrotrophic peatlands. Copyright © 2018 Elsevier B.V. All rights reserved.

Vaccination with Combination DNA and Virus-Like Particles Enhances Humoral and Cellular Immune Responses upon Boost with Recombinant Modified Vaccinia Virus Ankara Expressing Human Immunodeficiency Virus Envelope Proteins.

PubMed

Gangadhara, Sailaja; Kwon, Young-Man; Jeeva, Subbiah; Quan, Fu-Shi; Wang, Baozhong; Moss, Bernard; Compans, Richard W; Amara, Rama Rao; Jabbar, M Abdul; Kang, Sang-Moo

2017-12-19

Heterologous prime boost with DNA and recombinant modified vaccinia virus Ankara (rMVA) vaccines is considered as a promising vaccination approach against human immunodeficiency virus (HIV-1). To further enhance the efficacy of DNA-rMVA vaccination, we investigated humoral and cellular immune responses in mice after three sequential immunizations with DNA, a combination of DNA and virus-like particles (VLP), and rMVA expressing HIV-1 89.6 gp120 envelope proteins (Env). DNA prime and boost with a combination of VLP and DNA vaccines followed by an rMVA boost induced over a 100-fold increase in Env-specific IgG antibody titers compared to three sequential immunizations with DNA and rMVA. Cellular immune responses were induced by VLP-DNA and rMVA vaccinations at high levels in CD8 T cells, CD4 T cells, and peripheral blood mononuclear cells secreting interferon (IFN)-γ, and spleen cells producing interleukin (IL)-2, 4, 5 cytokines. This study suggests that a DNA and VLP combination vaccine with MVA is a promising strategy in enhancing the efficacy of DNA-rMVA vaccination against HIV-1.

Exploring DNA variant segregation types in pooled genome sequencing enables effective mapping of weeping trait in Malus

USDA-ARS?s Scientific Manuscript database

In recent years, next generation sequencing (NGS) based bulked segregant analysis (BSA) has become a powerful approach for allele discovery in non-model plant species. However, challenges remain, particular for out-crossing species with complex genomes. Here, the genetic control of a weeping bran...

Use of Molecular Methods for the Rapid Mass Detection of Schistosoma mansoni (Platyhelminthes: Trematoda) in Biomphalaria spp. (Gastropoda: Planorbidae)

PubMed Central

Jannotti-Passos, Liana Konovaloffi; Dos Santos Carvalho, Omar

2017-01-01

The low stringency-polymerase chain reaction (LS-PCR) and loop-mediated isothermal amplification (LAMP) assays were used to detect the presence of S. mansoni DNA in (1) Brazilian intermediate hosts (Biomphalaria glabrata, B. straminea, and B. tenagophila) with patent S. mansoni infections, (2) B. glabrata snails with prepatent S. mansoni infections, (3) various mixtures of infected and noninfected snails; and (4) snails infected with other trematode species. The assays showed high sensitivity and specificity and could detect S. mansoni DNA when one positive snail was included in a pool of 1,000 negative specimens of Biomphalaria. These molecular approaches can provide a low-cost, effective, and rapid method for detecting the presence of S. mansoni in pooled samples of field-collected Biomphalaria. These assays should aid mapping of transmission sites in endemic areas, especially in low prevalence regions and improve schistosomiasis surveillance. It will be a useful tool to monitor low infection rates of snails in areas where control interventions are leading towards the elimination of schistosomiasis. PMID:28246533

Statistical aspects of genetic association testing in small samples, based on selective DNA pooling data in the arctic fox.

PubMed

Szyda, Joanna; Liu, Zengting; Zatoń-Dobrowolska, Magdalena; Wierzbicki, Heliodor; Rzasa, Anna

2008-01-01

We analysed data from a selective DNA pooling experiment with 130 individuals of the arctic fox (Alopex lagopus), which originated from 2 different types regarding body size. The association between alleles of 6 selected unlinked molecular markers and body size was tested by using univariate and multinomial logistic regression models, applying odds ratio and test statistics from the power divergence family. Due to the small sample size and the resulting sparseness of the data table, in hypothesis testing we could not rely on the asymptotic distributions of the tests. Instead, we tried to account for data sparseness by (i) modifying confidence intervals of odds ratio; (ii) using a normal approximation of the asymptotic distribution of the power divergence tests with different approaches for calculating moments of the statistics; and (iii) assessing P values empirically, based on bootstrap samples. As a result, a significant association was observed for 3 markers. Furthermore, we used simulations to assess the validity of the normal approximation of the asymptotic distribution of the test statistics under the conditions of small and sparse samples.

GPFrontend and GPGraphics: graphical analysis tools for genetic association studies.

PubMed

Uebe, Steffen; Pasutto, Francesca; Krumbiegel, Mandy; Schanze, Denny; Ekici, Arif B; Reis, André

2010-09-21

Most software packages for whole genome association studies are non-graphical, purely text based programs originally designed to run with UNIX-like operating systems. Graphical output is often not intended or supposed to be performed with other command line tools, e.g. gnuplot. Using the Microsoft .NET 2.0 platform and Visual Studio 2005, we have created a graphical software package to analyze data from microarray whole genome association studies, both for a DNA-pooling based approach as well as regular single sample data. Part of this package was made to integrate with GenePool 0.8.2, a previously existing software suite for GNU/Linux systems, which we have modified to run in a Microsoft Windows environment. Further modifications cause it to generate some additional data. This enables GenePool to interact with the .NET parts created by us. The programs we developed are GPFrontend, a graphical user interface and frontend to use GenePool and create metadata files for it, and GPGraphics, a program to further analyze and graphically evaluate output of different WGA analysis programs, among them also GenePool. Our programs enable regular MS Windows users without much experience in bioinformatics to easily visualize whole genome data from a variety of sources.

Trypanosomatid parasites in Austrian mosquitoes

PubMed Central

Schoener, Ellen; Uebleis, Sarah Susanne; Cuk, Claudia; Nawratil, Michaela; Obwaller, Adelheid G.; Zechmeister, Thomas; Rádrová, Jana; Zittra, Carina

2018-01-01

Trypanosomatid flagellates have not been studied in Austria in any detail. In this study, specific nested PCR, targeted on the ribosomal small subunit, was used to determine the occurrence and diversity of trypanosomatids in wild-caught mosquitoes sampled across Eastern Austria in the years 2014−2015. We collected a total of 29,975 mosquitoes of 19 species divided in 1680 pools. Of these, 298 (17.7%), representing 12 different mosquito species, were positive for trypanosomatid DNA. In total, seven trypanosomatid spp. were identified (three Trypanosoma, three Crithidia and one Herpetomonas species), with the highest parasite species diversity found in the mosquito host Coquillettidia richiardii. The most frequent parasite species belonged to the mammalian Trypanosoma theileri/cervi species complex (found in 105 pools; 6.3%). The avian species T. culicavium (found in 69 pools; 4.1%) was only detected in mosquitoes of the genus Culex, which corresponds to their preference for avian hosts. Monoxenous trypanosomatids of the genus Crithidia and Herpetomonas were found in 20 (1.3%) mosquito pools. One third (n = 98) of the trypanosomatid positive mosquito pools carried more than one parasite species. This is the first large scale study of trypanosomatid parasites in Austrian mosquitoes and our results are valuable in providing an overview of the diversity of these parasites in Austria. PMID:29672618

Exposure to UV radiation and risk of Hodgkin lymphoma: a pooled analysis

PubMed Central

Glaser, Sally L.; Schupp, Clayton W.; Ekström Smedby, Karin; de Sanjosé, Silvia; Kane, Eleanor; Melbye, Mads; Forétova, Lenka; Maynadié, Marc; Staines, Anthony; Becker, Nikolaus; Nieters, Alexandra; Brennan, Paul; Boffetta, Paolo; Cocco, Pierluigi; Glimelius, Ingrid; Clavel, Jacqueline; Hjalgrim, Henrik; Chang, Ellen T.

2013-01-01

Ultraviolet radiation (UVR) exposure has been inversely associated with Hodgkin lymphoma (HL) risk, but only inconsistently, only in a few studies, and without attention to HL heterogeneity. We conducted a pooled analysis of HL risk focusing on type and timing of UVR exposure and on disease subtypes by age, histology, and tumor-cell Epstein-Barr virus (EBV) status. Four case-control studies contributed 1320 HL cases and 6381 controls. We estimated lifetime, adulthood, and childhood UVR exposure and history of sunburn and sunlamp use. We used 2-stage estimation with mixed-effects models and weighted pooled effect estimates by inverse marginal variances. We observed statistically significant inverse associations with HL risk for UVR exposures during childhood and adulthood, sunburn history, and sunlamp use, but we found no significant dose-response relationships. Risks were significant only for EBV-positive HL (pooled odds ratio, 0.56; 95% confidence interval, 0.35 to 0.91 for the highest overall UVR exposure category), with a significant linear trend for overall exposure (P = .03). Pooled relative risk estimates were not heterogeneous across studies. Increased UVR exposure may protect against HL, particularly EBV-positive HL. Plausible mechanisms involving UVR induction of regulatory T cells or the cellular DNA damage response suggest opportunities for new prevention targets. PMID:24016459

Experimental characterization of the weld pool flow in a TIG configuration

NASA Astrophysics Data System (ADS)

Stadler, M.; Masquère, M.; Freton, P.; Franceries, X.; Gonzalez, J. J.

2014-11-01

Tungsten Inert Gas (TIG) welding process relies on heat transfer between plasma and work piece leading to a metallic weld pool. Combination of different forces produces movements on the molten pool surface. One of our aims is to determine the velocity on the weld pool surface. This provides a set of data that leads to a deeper comprehension of the flow behavior and allows us to validate numerical models used to study TIG parameters. In this paper, two diagnostic methods developed with high speed imaging for the determination of velocity of an AISI 304L stainless steel molten pool are presented. Application of the two methods to a metallic weld pool under helium with a current intensity of 100 A provides velocity values around 0.70 m/s which are in good agreement with literature works.

Illuminating the Prevalence of Trypanosoma brucei s.l. in Glossina Using LAMP as a Tool for Xenomonitoring

PubMed Central

Cunningham, Lucas J.; Lingley, Jessica K.; Haines, Lee R.; Ndung’u, Joseph M.; Torr, Stephen J.; Adams, Emily R.

2016-01-01

Background As the reality of eliminating human African trypanosomiasis (HAT) by 2020 draws closer, the need to detect and identify the remaining areas of transmission increases. Here, we have explored the feasibility of using commercially available LAMP kits, designed to detect the Trypanozoon group of trypanosomes, as a xenomonitoring tool to screen tsetse flies for trypanosomes to be used in future epidemiological surveys. Methods and Findings The DNA extraction method was simplified and worked with the LAMP kits to detect a single positive fly when pooled with 19 negative flies, and the absolute lowest limit of detection that the kits were able to work at was the equivalent of 0.1 trypanosome per ml. The DNA from Trypanosoma brucei brucei could be detected six days after the fly had taken a blood meal containing dead trypanosomes, and when confronted with a range of non-target species, from both laboratory-reared flies and wild-caught flies, the kits showed no evidence of cross-reacting. Conclusion We have shown that it is possible to use a simplified DNA extraction method in conjunction with the pooling of tsetse flies to decrease the time it would take to screen large numbers of flies for the presence of Trypanozoon trypanosomes. The use of commercially-available LAMP kits provides a reliable and highly sensitive tool for xenomonitoring and identifying potential sleeping sickness transmission sites. PMID:26890882

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

PubMed Central

Barakat, Tahsin Stefan; Gribnau, Joost

2014-01-01

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

HIV-1 DNA predicts disease progression and post-treatment virological control

PubMed Central

Williams, James P; Hurst, Jacob; Stöhr, Wolfgang; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Cooper, David; Schechter, Mauro; Tambussi, Giuseppe; Fidler, Sarah; Carrington, Mary; Babiker, Abdel; Weber, Jonathan

2014-01-01

In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials. Clinical trial registration: ISRCTN76742797 and EudraCT2004-000446-20 DOI: http://dx.doi.org/10.7554/eLife.03821.001 PMID:25217531

Mapping of the Gynoecy in Bitter Gourd (Momordica charantia) Using RAD-Seq Analysis

PubMed Central

Matsumura, Hideo; Miyagi, Norimichi; Taniai, Naoki; Fukushima, Mai; Tarora, Kazuhiko; Shudo, Ayano; Urasaki, Naoya

2014-01-01

Momordica charantia is a monoecious plant of the Cucurbitaceae family that has both male and female unisexual flowers. Its unique gynoecious line, OHB61-5, is essential as a maternal parent in the production of F1 cultivars. To identify the DNA markers for this gynoecy, a RAD-seq (restriction-associated DNA tag sequencing) analysis was employed to reveal genome-wide DNA polymorphisms and to genotype the F2 progeny from a cross between OHB61-5 and a monoecious line. Based on a RAD-seq analysis of F2 individuals, a linkage map was constructed using 552 co-dominant markers. In addition, after analyzing the pooled genomic DNA from monoecious or gynoecious F2 plants, several SNP loci that are genetically linked to gynoecy were identified. GTFL-1, the closest SNP locus to the putative gynoecious locus, was converted to a conventional DNA marker using invader assay technology, which is applicable to the marker-assisted selection of gynoecy in M. charantia breeding. PMID:24498029

Seepage investigation using geophysical techniques at Coursier Lake Dam, B.C., Canada

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sirles, P.

1997-10-01

Subsurface seepage flow at Coursier Lake Dam was identified by onshore and offshore self-potential surveys, and electrical resistivity profiles and soundings during a Deficiency Investigation by BChydro. For typical seepage investigations baseline geophysical data are collected at {open_quotes}low-pool{close_quotes} level and the measurements are repeated when high hydraulic gradient conditions exist. At Coursier Lake Dam a rather unanticipated outcome of the low-pool surveys was that significant seepage beneath the structure was detected. The low-pool results were conclusive enough that, when combined with visual inspection and observation of sinkholes on the embankment, an immediate restriction was placed on the pool elevation. Thus,more » because of the identified potential hazard, the remaining geophysical investigations were conducted under a {open_quotes}minimum-pool{close_quotes} reservoir level in order to complete the comparative study. Therefore, the dam was studied under low- and minimum-pool reservoir conditions in the spring and fall of 1993, respectively. Low-pool data indicated very high resistivities (3000 to 5000 ohm-m) throughout the embankment indicating a coarse-average grain size, probably unsaturated sands and gravels. Higher resistivities (>5000 ohm-m) were obtained within the foundation deposits along the downstream toe indicating a combination of lower moisture content, coarser average grain size and higher porosity than the embankment. These electrical data indicate the subsurface conditions in the embankment and the foundation to be conducive to seepage. Results from low-pool SP surveys, performed both on-shore and offshore, indicate a dispersed or sheet flow seepage occurring nearly 1100 feet upstream of the intake. Therefore, apparently the seepage source begins far upstream of the embankment within the foundation deposits.« less

Eighty routes to a ribonucleotide world; dispersion and stringency in the decisive selection.

PubMed

Yarus, Michael

2018-05-21

We examine the initial emergence of genetics; that is, of an inherited chemical capability. The crucial actors are ribonucleotides, occasionally meeting in a prebiotic landscape. Previous work identified six influential variables during such random ribonucleotide pooling. Geochemical pools can be in periodic danger (e.g., from tides) or constant danger (e.g., from unfavorable weather). Such pools receive Gaussian nucleotide amounts sporadically, at random times, or get varying substrates simultaneously. Pools use cross-templated RNA synthesis (5'-5' product from 5'-3' template) or para-templated (5'-5' product from 5'-5' template) synthesis. Pools can undergo mild or strong selection, and be recently initiated (early) or late in age. Considering > 80 combinations of these variables, selection calculations identify a superior route. Most likely, an early, sporadically fed, cross-templating pool in constant danger, receiving ≥ 1 mM nucleotides while under strong selection for a coenzyme-like product will host selection of the first encoded biochemical functions. Predominantly templated products emerge from a critical event, the starting bloc selection, which exploits inevitable differences among early pools. Favorable selection has a simple rationale; it is increased by product dispersion (sd/mean), by selection intensity (mild or strong), or by combining these factors as stringency, reciprocal fraction of pools selected (1/sfsel). To summarize: chance utility, acting via a preference for disperse, templated coenzyme-like dinucleotides, uses stringent starting bloc selection to quickly establish majority encoded/genetic expression. Despite its computational origin, starting bloc selection is largely independent of specialized assumptions. This ribodinucleotide route to inheritance may also have facilitated 5'-3' chemical RNA replication. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Declining populations of the fingernail clam Musculium transversum in the upper Mississippi River

USGS Publications Warehouse

Wilson, D.M.; Naimo, T.J.; Weiner, J.G.; Anderson, R.V.; Sandheinrich, M.B.; Sparks, Richard E.

1995-01-01

We examined recent temporal trends in the abundance of fingernail clams Musculium transversum (formerly Sphaerium transversum) in the upper Mississippi River. Historical data on densities of fingernail clams were obtained from regional scientists and published literature. We also sampled benthos in six navigation pools in summer 1991, finding very few fingernail clams. The combined data set, including historical data and sampling results, extended from 1973 to 1992 and was sufficient to statistically evaluate trends in densities of fingernail clams in eight pools. Populations of fingernail clams declined significantly in five of the eight pools examined (Pools 2, 5, 7, 9, and 19), which spanned a 700-km reach of river from St. Paul, Minnesota, to Keokuk, Iowa. Densities in Pool 19, which had the longest historical record on fingernail clam abundance, averaged 30 000 m super(-2) in 1985 and progressively declined to zero in 1990. Combined data from all eight pools showed a significant decline in abundance of fingernail clams. An evaluation of potential causal factors led us to hypothesize that the population declines in Pools 2 to 9 were linked to point-source pollution rather than to dredging activity or commercial navigation traffic. In Pool 19, the declines of fingernail clams may have resulted from low-flow conditions during drought periods, but the causal mechanisms by which low flow influences fingernail clam abundance are unclear. The decrease in fingernail clam populations may adversely affect certain fish and wildlife, such as migrating lesser scaup Aythya affinis, which feed heavily on the small mollusk. Moreover, the decreases in populations of this pollution-sensitive mollusk may signal a large-scale deterioration in the health of this riverine ecosystem.

Selection and characterization of a DNA aptamer to crystal violet.

PubMed

Chen, Yang; Wang, Jine; Zhang, Yajie; Xu, Lijun; Gao, Tian; Wang, Bing; Pei, Renjun

2018-06-13

Aptamers are short single-stranded DNA or RNA, which can be selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). In order to develop novel light-up probes to substitute G-quadruplex (G4), we selected a DNA aptamer for crystal violet (CV), a triphenylmethane light-up dye, by a modified affinity chromatography-based SELEX. The ssDNA pool was first coupled on streptavidin-coated agarose beads through a biotin labeled complementary oligonucleotide, and then the aptamer sequences would be released from agarose beads by CV affinity. This method is simple, straightforward and effective. The aptamer sequence with a low micromolar dissociation constant (Kd) and good specificity was achieved after 11 rounds of selection. The light-up properties of the CV-aptamer were also investigated, and the CV showed dramatic fluorescence enhancement. The CV-aptamer pair could be further used as a novel light-up fluorescent probe to design biosensors.

SynTrack: DNA Assembly Workflow Management (SynTrack) v2.0.1

DOE Office of Scientific and Technical Information (OSTI.GOV)

MENG, XIANWEI; SIMIRENKO, LISA

2016-12-01

SynTrack is a dynamic, workflow-driven data management system that tracks the DNA build process: Management of the hierarchical relationships of the DNA fragments; Monitoring of process tasks for the assembly of multiple DNA fragments into final constructs; Creations of vendor order forms with selectable building blocks. Organizing plate layouts barcodes for vendor/pcr/fusion/chewback/bioassay/glycerol/master plate maps (default/condensed); Creating or updating Pre-Assembly/Assembly process workflows with selected building blocks; Generating Echo pooling instructions based on plate maps; Tracking of building block orders, received and final assembled for delivering; Bulk updating of colony or PCR amplification information, fusion PCR and chewback results; Updating with QA/QCmore » outcome with .csv & .xlsx template files; Re-work assembly workflow enabled before and after sequencing validation; and Tracking of plate/well data changes and status updates and reporting of master plate status with QC outcomes.« less

Applying plant DNA barcodes to identify species of Parnassia (Parnassiaceae).

PubMed

Yang, Jun-Bo; Wang, Yi-Ping; Möller, Michael; Gao, Lian-Ming; Wu, Ding

2012-03-01

DNA barcoding is a technique to identify species by using standardized DNA sequences. In this study, a total of 105 samples, representing 30 Parnassia species, were collected to test the effectiveness of four proposed DNA barcodes (rbcL, matK, trnH-psbA and ITS) for species identification. Our results demonstrated that all four candidate DNA markers have a maximum level of primer universality and sequencing success. As a single DNA marker, the ITS region provided the highest species resolution with 86.7%, followed by trnH-psbA with 73.3%. The combination of the core barcode regions, matK+rbcL, gave the lowest species identification success (63.3%) among any combination of multiple markers and was found unsuitable as DNA barcode for Parnassia. The combination of ITS+trnH-psbA achieved the highest species discrimination with 90.0% resolution (27 of 30 sampled species), equal to the four-marker combination and higher than any two or three marker combination including rbcL or matK. Therefore, matK and rbcL should not be used as DNA barcodes for the species identification of Parnassia. Based on the overall performance, the combination of ITS+trnH-psbA is proposed as the most suitable DNA barcode for identifying Parnassia species. DNA barcoding is a useful technique and provides a reliable and effective mean for the discrimination of Parnassia species, and in combination with morphology-based taxonomy, will be a robust approach for tackling taxonomically complex groups. In the light of our findings, we found among the three species not identified a possible cryptic speciation event in Parnassia. © 2011 Blackwell Publishing Ltd.

Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

DOEpatents

Gardner, Shea N; Mariella, Jr., Raymond P; Christian, Allen T; Young, Jennifer A; Clague, David S

2013-06-25

A method of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths.

Extracellular DNA in single- and multiple-species unsaturated biofilms.

PubMed

Steinberger, R E; Holden, P A

2005-09-01

The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.

Mapping RNA Structure In Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq).

PubMed

Watters, Kyle E; Lucks, Julius B

2016-01-01

Mapping RNA structure with selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry has proven to be a versatile method for characterizing RNA structure in a variety of contexts. SHAPE reagents covalently modify RNAs in a structure-dependent manner to create adducts at the 2'-OH group of the ribose backbone at nucleotides that are structurally flexible. The positions of these adducts are detected using reverse transcriptase (RT) primer extension, which stops one nucleotide before the modification, to create a pool of cDNAs whose lengths reflect the location of SHAPE modification. Quantification of the cDNA pools is used to estimate the "reactivity" of each nucleotide in an RNA molecule to the SHAPE reagent. High reactivities indicate nucleotides that are structurally flexible, while low reactivities indicate nucleotides that are inflexible. These SHAPE reactivities can then be used to infer RNA structures by restraining RNA structure prediction algorithms. Here, we provide a state-of-the-art protocol describing how to perform in vitro RNA structure probing with SHAPE chemistry using next-generation sequencing to quantify cDNA pools and estimate reactivities (SHAPE-Seq). The use of next-generation sequencing allows for higher throughput, more consistent data analysis, and multiplexing capabilities. The technique described herein, SHAPE-Seq v2.0, uses a universal reverse transcription priming site that is ligated to the RNA after SHAPE modification. The introduced priming site allows for the structural analysis of an RNA independent of its sequence.

Mechanisms to account for maintenance of the soluble methionine pool in transgenic Arabidopsis plants expressing antisense cystathionine gamma-synthase cDNA.

PubMed

Gakière, B; Ravanel, S; Droux, M; Douce, R; Job, D

2000-10-01

To investigate the role of cystathionine gamma-synthase (CGS) in the regulation of methionine synthesis Arabidopsis plants were transformed with a full-length antisense CGS cDNA and transformants analysed. Plants that were heterozygous for the transgene showed a 20-fold reduction of CGS activity that was accompanied by severe growth retardation and morphological abnormalities, from germination to flowering. Application of exogenous methionine to the transgenic lines restored normal growth. Surprisingly, transformed Arabidopsis plants exhibited a modest decrease in methionine content (35% reduction of the wild-type level) but a seven-fold decrease in the soluble pool of S-methylmethionine (SMM), a compound that plays a major role in storage and transport of reduced sulphur and labile methyl moieties. Several mechanisms can account for the maintenance of the soluble pool of methionine. First, the observed 20-fold increase in O-phosphohomoserine, a substrate of CGS, could compensate for the depressed level of CGS polypeptide by increasing the net rate of catalysis supported by the remaining enzyme. Second, the transgenic plants exhibited a two-fold increased level of cystathionine beta-lyase, the second enzyme in the methionine biosynthetic pathway. This indicates that enzymes other than CGS are subjected to a regulatory control by methionine or one of its metabolites. In addition to these mechanisms affecting de novo methionine synthesis, the recruitment of SMM to produce methionine may account for the small change of methionine levels in transgenic lines.

Accuracy of non-invasive prenatal testing using cell-free DNA for detection of Down, Edwards and Patau syndromes: a systematic review and meta-analysis

PubMed Central

Taylor-Phillips, Sian; Freeman, Karoline; Geppert, Julia; Agbebiyi, Adeola; Uthman, Olalekan A; Madan, Jason; Clarke, Angus; Quenby, Siobhan; Clarke, Aileen

2016-01-01

Objective To measure test accuracy of non-invasive prenatal testing (NIPT) for Down, Edwards and Patau syndromes using cell-free fetal DNA and identify factors affecting accuracy. Design Systematic review and meta-analysis of published studies. Data sources PubMed, Ovid Medline, Ovid Embase and the Cochrane Library published from 1997 to 9 February 2015, followed by weekly autoalerts until 1 April 2015. Eligibility criteria for selecting studies English language journal articles describing case–control studies with ≥15 trisomy cases or cohort studies with ≥50 pregnant women who had been given NIPT and a reference standard. Results 41, 37 and 30 studies of 2012 publications retrieved were included in the review for Down, Edwards and Patau syndromes. Quality appraisal identified high risk of bias in included studies, funnel plots showed evidence of publication bias. Pooled sensitivity was 99.3% (95% CI 98.9% to 99.6%) for Down, 97.4% (95.8% to 98.4%) for Edwards, and 97.4% (86.1% to 99.6%) for Patau syndrome. The pooled specificity was 99.9% (99.9% to 100%) for all three trisomies. In 100 000 pregnancies in the general obstetric population we would expect 417, 89 and 40 cases of Downs, Edwards and Patau syndromes to be detected by NIPT, with 94, 154 and 42 false positive results. Sensitivity was lower in twin than singleton pregnancies, reduced by 9% for Down, 28% for Edwards and 22% for Patau syndrome. Pooled sensitivity was also lower in the first trimester of pregnancy, in studies in the general obstetric population, and in cohort studies with consecutive enrolment. Conclusions NIPT using cell-free fetal DNA has very high sensitivity and specificity for Down syndrome, with slightly lower sensitivity for Edwards and Patau syndrome. However, it is not 100% accurate and should not be used as a final diagnosis for positive cases. Trial registration number CRD42014014947. PMID:26781507

Accuracy of urinary human papillomavirus testing for presence of cervical HPV: systematic review and meta-analysis

PubMed Central

Pathak, Neha; Dodds, Julie; Khan, Khalid

2014-01-01

Objective To determine the accuracy of testing for human papillomavirus (HPV) DNA in urine in detecting cervical HPV in sexually active women. Design Systematic review and meta-analysis. Data sources Searches of electronic databases from inception until December 2013, checks of reference lists, manual searches of recent issues of relevant journals, and contact with experts. Eligibility criteria Test accuracy studies in sexually active women that compared detection of urine HPV DNA with detection of cervical HPV DNA. Data extraction and synthesis Data relating to patient characteristics, study context, risk of bias, and test accuracy. 2×2 tables were constructed and synthesised by bivariate mixed effects meta-analysis. Results 16 articles reporting on 14 studies (1443 women) were eligible for meta-analysis. Most used commercial polymerase chain reaction methods on first void urine samples. Urine detection of any HPV had a pooled sensitivity of 87% (95% confidence interval 78% to 92%) and specificity of 94% (95% confidence interval 82% to 98%). Urine detection of high risk HPV had a pooled sensitivity of 77% (68% to 84%) and specificity of 88% (58% to 97%). Urine detection of HPV 16 and 18 had a pooled sensitivity of 73% (56% to 86%) and specificity of 98% (91% to 100%). Metaregression revealed an increase in sensitivity when urine samples were collected as first void compared with random or midstream (P=0.004). Limitations The major limitations of this review are the lack of a strictly uniform method for the detection of HPV in urine and the variation in accuracy between individual studies. Conclusions Testing urine for HPV seems to have good accuracy for the detection of cervical HPV, and testing first void urine samples is more accurate than random or midstream sampling. When cervical HPV detection is considered difficult in particular subgroups, urine testing should be regarded as an acceptable alternative. PMID:25232064

Supercomputer analysis of purine and pyrimidine metabolism leading to DNA synthesis.

PubMed

Heinmets, F

1989-06-01

A model-system is established to analyze purine and pyrimidine metabolism leading to DNA synthesis. The principal aim is to explore the flow and regulation of terminal deoxynucleoside triophosphates (dNTPs) in various input and parametric conditions. A series of flow equations are established, which are subsequently converted to differential equations. These are programmed (Fortran) and analyzed on a Cray chi-MP/48 supercomputer. The pool concentrations are presented as a function of time in conditions in which various pertinent parameters of the system are modified. The system is formulated by 100 differential equations.

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

NASA Astrophysics Data System (ADS)

Scheingross, Joel S.; Lamb, Michael P.

2016-05-01

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

The scaffold protein Nde1 safeguards the brain genome during S phase of early neural progenitor differentiation

PubMed Central

Houlihan, Shauna L; Feng, Yuanyi

2014-01-01

Successfully completing the S phase of each cell cycle ensures genome integrity. Impediment of DNA replication can lead to DNA damage and genomic disorders. In this study, we show a novel function for NDE1, whose mutations cause brain developmental disorders, in safeguarding the genome through S phase during early steps of neural progenitor fate restrictive differentiation. Nde1 mutant neural progenitors showed catastrophic DNA double strand breaks concurrent with the DNA replication. This evoked DNA damage responses, led to the activation of p53-dependent apoptosis, and resulted in the reduction of neurons in cortical layer II/III. We discovered a nuclear pool of Nde1, identified the interaction of Nde1 with cohesin and its associated chromatin remodeler, and showed that stalled DNA replication in Nde1 mutants specifically occurred in mid-late S phase at heterochromatin domains. These findings suggest that NDE1-mediated heterochromatin replication is indispensible for neuronal differentiation, and that the loss of NDE1 function may lead to genomic neurological disorders. DOI: http://dx.doi.org/10.7554/eLife.03297.001 PMID:25245017

A preamplification approach to GMO detection in processed foods.

PubMed

Del Gaudio, S; Cirillo, A; Di Bernardo, G; Galderisi, U; Cipollaro, M

2010-03-01

DNA is widely used as a target for GMO analysis because of its stability and high detectability. Real-time PCR is the method routinely used in most analytical laboratories due to its quantitative performance and great sensitivity. Accurate DNA detection and quantification is dependent on the specificity and sensitivity of the amplification protocol as well as on the quality and quantity of the DNA used in the PCR reaction. In order to enhance the sensitivity of real-time PCR and consequently expand the number of analyzable target genes, we applied a preamplification technique to processed foods where DNA can be present in low amounts and/or in degraded forms thereby affecting the reliability of qualitative and quantitative results. The preamplification procedure utilizes a pool of primers targeting genes of interest and is followed by real-time PCR reactions specific for each gene. An improvement of Ct values was found comparing preamplified vs. non-preamplified DNA. The strategy reported in the present study will be also applicable to other fields requiring quantitative DNA testing by real-time PCR.

Development of a Medium Density Combined-Species SNP Array for Pacific and European Oysters (Crassostrea gigas and Ostrea edulis).

PubMed

Gutierrez, Alejandro P; Turner, Frances; Gharbi, Karim; Talbot, Richard; Lowe, Natalie R; Peñaloza, Carolina; McCullough, Mark; Prodöhl, Paulo A; Bean, Tim P; Houston, Ross D

2017-07-05

SNP arrays are enabling tools for high-resolution studies of the genetic basis of complex traits in farmed and wild animals. Oysters are of critical importance in many regions from both an ecological and economic perspective, and oyster aquaculture forms a key component of global food security. The aim of our study was to design a combined-species, medium density SNP array for Pacific oyster ( Crassostrea gigas ) and European flat oyster ( Ostrea edulis ), and to test the performance of this array on farmed and wild populations from multiple locations, with a focus on European populations. SNP discovery was carried out by whole-genome sequencing (WGS) of pooled genomic DNA samples from eight C. gigas populations, and restriction site-associated DNA sequencing (RAD-Seq) of 11 geographically diverse O. edulis populations. Nearly 12 million candidate SNPs were discovered and filtered based on several criteria, including preference for SNPs segregating in multiple populations and SNPs with monomorphic flanking regions. An Affymetrix Axiom Custom Array was created and tested on a diverse set of samples ( n = 219) showing ∼27 K high quality SNPs for C. gigas and ∼11 K high quality SNPs for O. edulis segregating in these populations. A high proportion of SNPs were segregating in each of the populations, and the array was used to detect population structure and levels of linkage disequilibrium (LD). Further testing of the array on three C. gigas nuclear families ( n = 165) revealed that the array can be used to clearly distinguish between both families based on identity-by-state (IBS) clustering parental assignment software. This medium density, combined-species array will be publicly available through Affymetrix, and will be applied for genome-wide association and evolutionary genetic studies, and for genomic selection in oyster breeding programs. Copyright © 2017 Gutierrez et al.

Bacteria community study of combined periodontal-endodontic lesions using denaturing gradient gel electrophoresis and sequencing analysis.

PubMed

Li, Hong; Guan, Rui; Sun, Jinghua; Hou, Benxiang

2014-10-01

The entire microbial population and predominant microflora of root canals (RCs) and adjacent periodontal pockets (PPs) from teeth with combined periodontal-endodontic lesions were determined and compared. Pooled RC and PP samples were collected from the molars of 20 patients diagnosed with combined periodontal-endodontic lesions. DNA was extracted for polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE), cloning, and sequence analysis. A coefficient of similarity (Cs) was used to determine the similarity of the bacterial profiles from RCs and PPs. Significantly fewer bands were produced by PCR-DGGE from RCs (5.9 ± 1.7) than from PPs (8.0 ± 1.8) (P <0.001). The average Cs of the RC and PP samples was 93.81% ± 10.26%. Overall, 60 genera/species were identified by sequencing. Of these, the predominant genera in RCs were Porphyromonas sp. (13.9%), Filifactor sp. (12.5%), and Parvimonas sp. (11.1%), similar to the genera obtained from PP samples. In total, 43 genera/species were common to the RC and PP samples. The most prevalent bacteria in both the RC and PP samples were (in descending order) Filifactor alocis, Parvimonas micra, Porphyromonas gingivalis, and Tannerella forsythia. The high similarity in the sets of organisms present in both RC and PP samples in this study suggests that the pocket could be a source of RC infection. The data also demonstrate that combined periodontal-endodontic lesions consist of a diverse and complex microbial community.

Controls on Filling and Evacuation of Sediment in Waterfall Plunge Pools

NASA Astrophysics Data System (ADS)

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

2014-12-01

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

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

ERIC Educational Resources Information Center

Millard, Julie T

2011-01-01

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

Nasopharyngeal carcinoma heterogeneity of DNA content identified on cytologic preparations.

PubMed

Maohuai, C; Chang, A R; Lo, D

2001-06-01

To evaluate tumor heterogeneity of DNA content in nasopharyngeal carcinoma (NPC) performed on cytologic specimens. Image cytometric analysis of DNA ploidy status of 40 NPCs was performed on nasopharyngeal brushing smears stained with the Feulgen method after hematoxylin eosin staining. If the DNA distribution pattern from the same tumor exhibited diploid, aneuploid or/and tetraploid peaks or some combination of these patterns, the presence of tumor heterogeneity of DNA content was identified. Thirty-four cases (85%) had a nondiploid DNA pattern among the 40 NPCs. Twenty-eight cases exhibited tumor heterogeneity of DNA content (70%). Of the 28 tumors, 13 (46%) had a combination of diploid and tetraploid patterns, 10 (37%) had a combination of diploid and aneuploid patterns, 3 cases (11%) had a combination of tetraploid and aneuploid patterns, and 2 cases had two aneuploid stem lines. The relationship between DNA ploidy pattern and tumor histologic and cytologic morphology was also examined. There is a high incidence of DNA content heterogeneity in NPC. The relevance of tumor heterogeneity to the biologic behavior of NPC awaits further study. DNA quantification with image cytometry on destained cytologic preparations is feasible and reliable.

Origin and evolution of Andigena potatoes revealed by chloroplast and nuclear DNA markers.

PubMed

Sukhotu, Thitaporn; Hosaka, Kazuyoshi

2006-06-01

Andigena potatoes (Solanum tuberosum L. subsp. andigena Hawkes) (2n = 4x = 48) are important, native-farmer-selected cultivars in the Andes, which form a primary gene pool for improving a worldwide grown potato (S. tuberosum subsp. tuberosum). To elucidate the origin of Andigena, 196 Andigena accessions were compared with 301 accessions of 33 closely related cultivated and wild species using several types of chloroplast DNA (ctDNA) markers and nuclear DNA (nDNA) restriction fragment length polymorphism (RFLP) markers. Fourteen ctDNA types (haplotypes) and 115 RFLP bands were detected in Andigena, of which the main haplotypes and frequent RFLP bands were mostly shared with a cultivated diploid species, S. stenotomum Juz. et Buk. Principal component analysis of nDNA polymorphisms revealed a progressive and continuous variation from Peruvian wild species with C-type ctDNA to a group of wild species having S-type ctDNA in its variation range (S. bukasovii, S. canasense, S. candolleanum, and S. multidissectum), to cultivated diploid potatoes (S. phureja and S. stenotomum), and to cultivated tetraploid potatoes (Andigena and Chilean S. tuberosum subsp. tuberosum). These results suggest that the initial Andigena population arose with multiple origins exclusively from S. stenotomum. The overall evolutionary process toward the present-day Andigena was discussed.

GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells.

PubMed

Kostyuk, Svetlana; Smirnova, Tatiana; Kameneva, Larisa; Porokhovnik, Lev; Speranskij, Anatolij; Ershova, Elizaveta; Stukalov, Sergey; Izevskaya, Vera; Veiko, Natalia

2015-01-01

Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA) and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci). As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR), PCNA (FACS)) and antiapoptotic genes (BCL2 (RT-PCR and FACS), BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR)). Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs). Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR), in the level of fatty acid binding protein FABP4 (FACS analysis) and in the level of fat (Oil Red O). GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose-derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis.

GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

PubMed Central

Smirnova, Tatiana; Kameneva, Larisa; Porokhovnik, Lev; Speranskij, Anatolij; Ershova, Elizaveta; Stukalov, Sergey; Izevskaya, Vera; Veiko, Natalia

2015-01-01

Background. Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Principal Findings. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA) and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci). As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR), PCNA (FACS)) and antiapoptotic genes (BCL2 (RT-PCR and FACS), BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR)). Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs). Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR), in the level of fatty acid binding protein FABP4 (FACS analysis) and in the level of fat (Oil Red O). Conclusions. GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose—derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis. PMID:26273425

High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.

PubMed

Inagaki, Soichi; Henry, Isabelle M; Lieberman, Meric C; Comai, Luca

2015-01-01

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.

Combination Testing Using a Single MSH5 Variant alongside HLA Haplotypes Improves the Sensitivity of Predicting Coeliac Disease Risk in the Polish Population.

PubMed

Paziewska, Agnieszka; Cukrowska, Bozena; Dabrowska, Michalina; Goryca, Krzysztof; Piatkowska, Magdalena; Kluska, Anna; Mikula, Michal; Karczmarski, Jakub; Oralewska, Beata; Rybak, Anna; Socha, Jerzy; Balabas, Aneta; Zeber-Lubecka, Natalia; Ambrozkiewicz, Filip; Konopka, Ewa; Trojanowska, Ilona; Zagroba, Malgorzata; Szperl, Malgorzata; Ostrowski, Jerzy

2015-01-01

Assessment of non-HLA variants alongside standard HLA testing was previously shown to improve the identification of potential coeliac disease (CD) patients. We intended to identify new genetic variants associated with CD in the Polish population that would improve CD risk prediction when used alongside HLA haplotype analysis. DNA samples of 336 CD and 264 unrelated healthy controls were used to create DNA pools for a genome wide association study (GWAS). GWAS findings were validated with individual HLA tag single nucleotide polymorphism (SNP) typing of 473 patients and 714 healthy controls. Association analysis using four HLA-tagging SNPs showed that, as was found in other populations, positive predicting genotypes (HLA-DQ2.5/DQ2.5, HLA-DQ2.5/DQ2.2, and HLA-DQ2.5/DQ8) were found at higher frequencies in CD patients than in healthy control individuals in the Polish population. Both CD-associated SNPs discovered by GWAS were found in the CD susceptibility region, confirming the previously-determined association of the major histocompatibility (MHC) region with CD pathogenesis. The two most significant SNPs from the GWAS were rs9272346 (HLA-dependent; localized within 1 Kb of DQA1) and rs3130484 (HLA-independent; mapped to MSH5). Specificity of CD prediction using the four HLA-tagging SNPs achieved 92.9%, but sensitivity was only 45.5%. However, when a testing combination of the HLA-tagging SNPs and the MSH5 SNP was used, specificity decreased to 80%, and sensitivity increased to 74%. This study confirmed that improvement of CD risk prediction sensitivity could be achieved by including non-HLA SNPs alongside HLA SNPs in genetic testing.

Crowd density estimation based on convolutional neural networks with mixed pooling

NASA Astrophysics Data System (ADS)

Zhang, Li; Zheng, Hong; Zhang, Ying; Zhang, Dongming

2017-09-01

Crowd density estimation is an important topic in the fields of machine learning and video surveillance. Existing methods do not provide satisfactory classification accuracy; moreover, they have difficulty in adapting to complex scenes. Therefore, we propose a method based on convolutional neural networks (CNNs). The proposed method improves performance of crowd density estimation in two key ways. First, we propose a feature pooling method named mixed pooling to regularize the CNNs. It replaces deterministic pooling operations with a parameter that, by studying the algorithm, could combine the conventional max pooling with average pooling methods. Second, we present a classification strategy, in which an image is divided into two cells and respectively categorized. The proposed approach was evaluated on three datasets: two ground truth image sequences and the University of California, San Diego, anomaly detection dataset. The results demonstrate that the proposed approach performs more effectively and easily than other methods.

DNA-binding regulates site-specific ubiquitination of IRF-1.

PubMed

Landré, Vivien; Pion, Emmanuelle; Narayan, Vikram; Xirodimas, Dimitris P; Ball, Kathryn L

2013-02-01

Understanding the determinants for site-specific ubiquitination by E3 ligase components of the ubiquitin machinery is proving to be a challenge. In the present study we investigate the role of an E3 ligase docking site (Mf2 domain) in an intrinsically disordered domain of IRF-1 [IFN (interferon) regulatory factor-1], a short-lived IFNγ-regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3 ligases such as CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and MDM2 (murine double minute 2), which dock to the Mf2 domain, was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3 docking site was not available when IRF-1 was in its DNA-bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination, highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA-binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.

3D finite element simulation of TIG weld pool

NASA Astrophysics Data System (ADS)

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

2012-07-01

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

Sample pooling for real-time PCR detection and virulence determination of the footrot pathogen Dichelobacter nodosus.

PubMed

Frosth, Sara; König, Ulrika; Nyman, Ann-Kristin; Aspán, Anna

2017-09-01

Dichelobacter nodosus is the principal cause of ovine footrot and strain virulence is an important factor in disease severity. Therefore, detection and virulence determination of D. nodosus is important for proper diagnosis of the disease. Today this is possible by real-time PCR analysis. Analysis of large numbers of samples is costly and laborious; therefore, pooling of individual samples is common in surveillance programs. However, pooling can reduce the sensitivity of the method. The aim of this study was to develop a pooling method for real-time PCR analysis that would allow sensitive detection and simultaneous virulence determination of D. nodosus. A total of 225 sheep from 17 flocks were sampled using ESwabs within the Swedish Footrot Control Program in 2014. Samples were first analysed individually and then in pools of five by real-time PCR assays targeting the 16S rRNA and aprV2/B2 genes of D. nodosus. Each pool consisted of four negative and one positive D. nodosus samples with varying amounts of the bacterium. In the individual analysis, 61 (27.1%) samples were positive in the 16S rRNA and the aprV2/B2 PCR assays and 164 (72.9%) samples were negative. All samples positive in the aprV2/B2 PCR-assay were of aprB2 variant. The pooled analysis showed that all 41 pools were also positive for D. nodosus 16S rRNA and the aprB2 variant. The diagnostic sensitivity for pooled and individual samples was therefore similar. Our method includes concentration of the bacteria before DNA-extraction. This may account for the maintenance of diagnostic sensitivity. Diagnostic sensitivity in the real-time PCR assays of the pooled samples were comparable to the sensitivity obtained for individually analysed samples. Even sub-clinical infections were able to be detected in the pooled PCR samples which is important for control of the disease. This method may therefore be implemented in footrot control programs where it can replace analysis of individual samples.

A computational proposal for designing structured RNA pools for in vitro selection of RNAs.

PubMed

Kim, Namhee; Gan, Hin Hark; Schlick, Tamar

2007-04-01

Although in vitro selection technology is a versatile experimental tool for discovering novel synthetic RNA molecules, finding complex RNA molecules is difficult because most RNAs identified from random sequence pools are simple motifs, consistent with recent computational analysis of such sequence pools. Thus, enriching in vitro selection pools with complex structures could increase the probability of discovering novel RNAs. Here we develop an approach for engineering sequence pools that links RNA sequence space regions with corresponding structural distributions via a "mixing matrix" approach combined with a graph theory analysis. We define five classes of mixing matrices motivated by covariance mutations in RNA; these constructs define nucleotide transition rates and are applied to chosen starting sequences to yield specific nonrandom pools. We examine the coverage of sequence space as a function of the mixing matrix and starting sequence via clustering analysis. We show that, in contrast to random sequences, which are associated only with a local region of sequence space, our designed pools, including a structured pool for GTP aptamers, can target specific motifs. It follows that experimental synthesis of designed pools can benefit from using optimized starting sequences, mixing matrices, and pool fractions associated with each of our constructed pools as a guide. Automation of our approach could provide practical tools for pool design applications for in vitro selection of RNAs and related problems.

Study on Dynamic Development of Three-dimensional Weld Pool Surface in Stationary GTAW

NASA Astrophysics Data System (ADS)

Huang, Jiankang; He, Jing; He, Xiaoying; Shi, Yu; Fan, Ding

2018-04-01

The weld pool contains abundant information about the welding process. In particular, the type of the weld pool surface shape, i. e., convex or concave, is determined by the weld penetration. To detect it, an innovative laser-vision-based sensing method is employed to observe the weld pool surface of the gas tungsten arc welding (GTAW). A low-power laser dots pattern is projected onto the entire weld pool surface. Its reflection is intercepted by a screen and captured by a camera. Then the dynamic development process of the weld pool surface can be detected. By observing and analyzing, the change of the reflected laser dots reflection pattern, for shape of the weld pool surface shape, was found to closely correlate to the penetration of weld pool in the welding process. A mathematical model was proposed to correlate the incident ray, reflected ray, screen and surface of weld pool based on structured laser specular reflection. The dynamic variation of the weld pool surface and its corresponding dots laser pattern were simulated and analyzed. By combining the experimental data and the mathematical analysis, the results show that the pattern of the reflected laser dots pattern is closely correlated to the development of weld pool, such as the weld penetration. The concavity of the pool surface was found to increase rapidly after the surface shape was changed from convex to concave during the stationary GTAW process.

Zinc as a second messenger of mitogenic induction. Effects on diadenosine tetraphosphate (Ap4A) and DNA synthesis.

PubMed

Grummt, F; Weinmann-Dorsch, C; Schneider-Schaulies, J; Lux, A

1986-03-01

DNA synthesis and adenosine(5')tetraphosphate(5')adenosine (Ap4A) levels decrease in cells treated with EDTA. The inhibitory effect of EDTA can be reversed with micromolar amounts of ZnCl2. ZnCl2 in micromolar concentrations also inhibits Ap4A hydrolase and stimulates amino acid-dependent Ap4A synthesis, suggesting that Zn2+ is modulating intracellular Ap4A pools. Serum addition to G1-arrested cells enhances uptake of Zn, whereas serum depletion leads to a fivefold decrease of the rates of zinc uptake. These results are discussed by regarding Zn2+ as a putative 'second messenger' of mitogenic induction and Ap4A as a possible 'third messenger' and trigger of DNA synthesis.

Post-glacial evolution of Panicum virgatum: centers of diversity and gene pools revealed by SSR markers and cpDNA sequences

USDA-ARS?s Scientific Manuscript database

Switchgrass (Panicum virgatum) is native to the tallgrass prairie and associated ecosystems of the central and eastern USA. It is highly valued as a component in tallgrass prairie and savanna restoration and conservation projects and a potential bioenergy feedstock. The purpose of this study was to ...

77 FR 68133 - Guidance for Industry: Use of Nucleic Acid Tests on Pooled and Individual Samples From Donors of...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-15

... Blood and Blood Components, Including Source Plasma, To Reduce the Risk of Transmission of Hepatitis B... Components, including Source Plasma, to Reduce the Risk of Transmission of Hepatitis B Virus,'' dated October... (NAT) to screen blood donors for hepatitis B virus (HBV) deoxyribonucleic acid (DNA) and...

Identification of ssDNA aptamers specific to clinical isolates of Streptococcus mutans strains with different cariogenicity.

PubMed

Cui, Wei; Liu, Jiaojiao; Su, Donghua; Hu, Danyang; Hou, Shuai; Hu, Tongnan; Yang, Jiyong; Luo, Yanping; Xi, Qing; Chu, Bingfeng; Wang, Chenglong

2016-06-01

Streptococcus mutans, a Gram-positive facultative anaerobic bacterium, is considered to be a major etiological factor for dental caries. In this study, plaques from dental enamel surfaces of caries-active and caries-free individuals were obtained and cultivated for S. mutans isolation. Morphology examination, biochemical characterization, and polymerase chain reaction were performed to identify S. mutans The cariogenicity of S. mutans strains isolated from clinical specimens was evaluated by testing the acidogenicity, aciduricity, extracellular polysaccharide production, and adhesion ability of the bacteria. Finally, subtractive SELEX (systematic evolution of ligands by exponential enrichment) technology targeting whole intact cells was used to screen for ssDNA aptamers specific to the strains with high cariogenicity. After nine rounds of subtractive SELEX, sufficient pool enrichment was achieved as shown by radioactive isotope analysis. The enriched pool was cloned and sequenced randomly, followed by MEME online and RNA structure software analysis of the sequences. Results from the flow cytometry indicated that aptamers H1, H16, H4, L1, L10, and H19 could discriminate highly cariogenic S. mutans strains from poorly cariogenic strains. Among these, Aptamer H19 had the strongest binding capacity with cariogenic S. mutans strains with a dissociation constant of 69.45 ± 38.53 nM. In conclusion, ssDNA aptamers specific to highly cariogenic clinical S. mutans strains were successfully obtained. These ssDNA aptamers might be used for the early diagnosis and treatment of dental caries. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

PAX1 and SOX1 methylation as an initial screening method for cervical cancer: a meta-analysis of individual studies in Asians

PubMed Central

Cui, Zhaolei; Xiao, Zhenzhou; Hu, Minhua; Jiang, Chuanhui; Lin, Yingying; Chen, Yansong

2016-01-01

Background Epigenetic alterations of gene or DNA methylation have been highlighted as promising biomarkers for early cervical cancer screening. Herein, we evaluated the diagnostic performance of paired boxed gene 1 (PAX1) and sex determining region Y-box 1 (SOX1) methylation for cervical cancer detection. Methods Eligible studies were retrieved by searching the electronic databases. Study quality was assessed according to the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) checklist. The bivariate meta-analysis model was employed to plot the summary receiver operator characteristic (SROC) curve using Stata 12.0 software. Results The pooled sensitivity of PAX1 methylation was estimated to be 0.73 [95% confidence interval (CI): 0.70–0.75] in differentiating patients with HSIL (high-grade squamous intraepithelial lesion) or CIN3+ (cervical intraepithelial neoplasia type III/worse) or cervical cancer from normal individuals, corresponding to a specificity of 0.87 (95% CI: 0.85–0.89) and area under the curve (AUC) of 0.91. The SOX1 methylation test yielded an AUC of 0.82, under which, the pooled sensitivity was 0.71 (95% CI: 0.67–0.74) and specificity was 0.64 (95% CI: 0.61–0.67). Notably, the stratified analysis suggested that combing parallel testing of PAX1 methylation and human papillomavirus (HPV) DNA (AUC, sensitivity, and specificity of 0.89, 0.75, and 0.81, respectively) achieved higher accuracy than single HPV DNA testing (AUC, sensitivity, and specificity of 0.77, 0.81, and 0.70, respectively). Conclusions PAX1 or SOX1 methylation has a prospect to be an auxiliary biomarker for cervical cancer screening, and parallel testing of PAX1 methylation and HPV DNA in cervical swabs confers an improved diagnostic accuracy than single HPV DNA testing. PMID:27826568

75 FR 29750 - Combined Notice of Filings #1

Federal Register 2010, 2011, 2012, 2013, 2014

2010-05-27

.... submits joint application for approval under section 203 of the Federal Power Act. Filed Date: 05/18/2010.... Eastern Time on Monday, June 7, 2010. Docket Numbers: ER09-1397-002. Applicants: Southwest Power Pool, Inc. Description: Southwest Power Pool, Inc submits clean copy of the Revised Agreements and redlined copies of the...

Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis

PubMed Central

Ng, Chun Kiat; Miller, Dana; Cao, Bin

2015-01-01

Introduction As bioaerosol research attracts increasing attention, there is a need for additional efforts that focus on method development to deal with different environmental samples. Bioaerosol environmental samples typically have very low biomass concentrations in the air, which often leaves researchers with limited options in choosing the downstream analysis steps, especially when culture-independent methods are intended. Objectives This study investigates the impacts of three important factors that can influence the performance of culture-independent DNA-based analysis in dealing with bioaerosol environmental samples engaged in this study. The factors are: 1) enhanced high temperature sonication during DNA extraction; 2) effect of sampling duration on DNA recoverability; and 3) an alternative method for concentrating composite samples. In this study, DNA extracted from samples was analysed using the Qubit fluorometer (for direct total DNA measurement) and quantitative polymerase chain reaction (qPCR). Results and Findings The findings suggest that additional lysis from high temperature sonication is crucial: DNA yields from both high and low biomass samples increased up to 600% when the protocol included 30-min sonication at 65°C. Long air sampling duration on a filter media was shown to have a negative impact on DNA recoverability with up to 98% of DNA lost over a 20-h sampling period. Pooling DNA from separate samples during extraction was proven to be feasible with margins of error below 30%. PMID:26619279

[Estimation of Genetic Diversity of Romanov Sheep by the Coefficient of Genetic Originality Based on ISSR-Fingerprinting Data].

PubMed

Nesteruk, L V; Makarova, N N; Svishcheva, G R; Stolpovsky, Yu A

2015-07-01

Estimation of the state of the genetic diversity and the originality of the breed structure is required for the conservation and management of domestic breeds of agricultural animals. The Romanov breed of sheep from the leading breeding and gene pool farms in Yaroslavl oblast (Russia) is the object of our study. ISS R fingerprinting was used as a molecular method of the study of sheep gene pools. Forty-three DNA fragments were detected (25 and 18, respectively) by two primers ((AG)9C and (GA)9C). Of the discovered ISSR markers, 81% were polymorphic. The coefficient of genetic originality was for the first time used for the study of the specificity and originality of the Romanov-breed gene pool. Based on its values, the studied individuals were divided into five classes depending on the frequency of the ISSR fragment. The most original or the rarest, as well as typical genotypes, were singled out in the Romanov sheep gene pool. Use the obtained data on genetic originality was proposed as a means to increase the efficiency of selection and breeding during the breeding of autochthonous breeds of domesticated animal species.

Chloroplast DNA analysis of Tunisian cork oak populations (Quercus suber L.): sequence variations and molecular evolution of the trnL (UAA)-trnF (GAA) region.

PubMed

Abdessamad, A; Baraket, G; Sakka, H; Ammari, Y; Ksontini, M; Hannachi, A Salhi

2016-10-24

Sequences of the trnL-trnF spacer and combined trnL-trnF region in chloroplast DNA of cork oak (Quercus suber L.) were analyzed to detect polymorphisms and to elucidate molecular evolution and demographic history. The aligned sequences varied in length and nucleotide composition. The overall ratio of transition/transversion (ti/tv) of 0.724 for the intergenic spacer and 0.258 for the pooled sequences were estimated, and indicated that transversions are more frequent than transitions. The molecular evolution and demographic history of Q. suber were investigated. Neutrality tests (Tajima's D and Fu and Li) ruled out the null hypothesis of a strictly neutral model, and Fu's Fs and Ramos-Onsins and Rozas' R2 confirmed the recent expansion of cork oak trees, validating its persistency in North Africa since the last glaciation during the Quaternary. The observed uni-modal mismatch distribution and the Harpending's raggedness index confirmed the demographic history model for cork oak. A phylogenetic dendrogram showed that the distribution of Q. suber trees occurs independently of geographical origin, the relief of the population site, and the bioclimatic stages. The molecular history and cytoplasmic diversity suggest that in situ and ex situ conservation strategies can be recommended for preserving landscape value and facing predictable future climatic changes.

Extinct New Zealand megafauna were not in decline before human colonization

PubMed Central

Allentoft, Morten Erik; Heller, Rasmus; Oskam, Charlotte L.; Lorenzen, Eline D.; Hale, Marie L.; Gilbert, M. Thomas P.; Jacomb, Christopher; Holdaway, Richard N.; Bunce, Michael

2014-01-01

The extinction of New Zealand's moa (Aves: Dinornithiformes) followed the arrival of humans in the late 13th century and was the final event of the prehistoric Late Quaternary megafauna extinctions. Determining the state of the moa populations in the pre-extinction period is fundamental to understanding the causes of the event. We sampled 281 moa individuals and combined radiocarbon dating with ancient DNA analyses to help resolve the extinction debate and gain insights into moa biology. The samples, which were predominantly from the last 4,000 years preceding the extinction, represent four sympatric moa species excavated from five adjacent fossil deposits. We characterized the moa assemblage using mitochondrial DNA and nuclear microsatellite markers developed specifically for moa. Although genetic diversity differed significantly among the four species, we found that the millennia preceding the extinction were characterized by a remarkable degree of genetic stability in all species, with no loss of heterozygosity and no shifts in allele frequencies over time. The extinction event itself was too rapid to be manifested in the moa gene pools. Contradicting previous claims of a decline in moa before Polynesian settlement in New Zealand, our findings indicate that the populations were large and stable before suddenly disappearing. This interpretation is supported by approximate Bayesian computation analyses. Our analyses consolidate the disappearance of moa as the most rapid, human-facilitated megafauna extinction documented to date. PMID:24639531

Effects of late quaternary climate change on Palearctic shrews.

PubMed

Prost, Stefan; Klietmann, Johannes; van Kolfschoten, Thijs; Guralnick, Robert P; Waltari, Eric; Vrieling, Klaas; Stiller, Mathias; Nagel, Doris; Rabeder, Gernot; Hofreiter, Michael; Sommer, Robert S

2013-06-01

The Late Quaternary was a time of rapid climatic oscillations and drastic environmental changes. In general, species can respond to such changes by behavioral accommodation, distributional shifts, ecophenotypic modifications (nongenetic), evolution (genetic) or ultimately face local extinction. How those responses manifested in the past is essential for properly predicting future ones especially as the current warm phase is further intensified by rising levels of atmospheric carbon dioxide. Here, we use ancient DNA (aDNA) and morphological features in combination with ecological niche modeling (ENM) to investigate genetic and nongenetic responses of Central European Palearctic shrews to past climatic change. We show that a giant form of shrew, previously described as an extinct Pleistocene Sorex species, represents a large ecomorph of the common shrew (Sorex araneus), which was replaced by populations from a different gene-pool and with different morphology after the Pleistocene Holocene transition. We also report the presence of the cold-adapted tundra shrew (S. tundrensis) in Central Europe. This species is currently restricted to Siberia and was hitherto unknown as an element of the Pleistocene fauna of Europe. Finally, we show that there is no clear correlation between climatic oscillations within the last 50 000 years and body size in shrews and conclude that a special nonanalogous situation with regard to biodiversity and food supply in the Late Glacial may have caused the observed large body size. © 2013 Blackwell Publishing Ltd.

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

PubMed

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

2015-08-01

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

Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins

PubMed Central

2018-01-01

ABSTRACT African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs. IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The responses in immunized pigs to these individual antigens were compared to identify the most immunogenic. Lethal challenge of pigs immunized with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to those in pigs immunized with control vectors. Novel immunogenic ASFV proteins have been identified for further testing as vaccine candidates. PMID:29386289

Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins.

PubMed

Jancovich, James K; Chapman, Dave; Hansen, Debra T; Robida, Mark D; Loskutov, Andrey; Craciunescu, Felicia; Borovkov, Alex; Kibler, Karen; Goatley, Lynnette; King, Katherine; Netherton, Christopher L; Taylor, Geraldine; Jacobs, Bertram; Sykes, Kathryn; Dixon, Linda K

2018-04-15

African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs. IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The responses in immunized pigs to these individual antigens were compared to identify the most immunogenic. Lethal challenge of pigs immunized with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to those in pigs immunized with control vectors. Novel immunogenic ASFV proteins have been identified for further testing as vaccine candidates. Copyright © 2018 Jancovich et al.

First Detection of Leishmania tropica DNA and Trypanosoma Species in Sergentomyia Sand Flies (Diptera: Psychodidae) from an Outbreak Area of Cutaneous Leishmaniasis in Ghana

PubMed Central

Nzelu, Chukwunonso O.; Kato, Hirotomo; Puplampu, Naiki; Desewu, Kwame; Odoom, Shirley; Wilson, Michael D.; Sakurai, Tatsuya; Katakura, Ken; Boakye, Daniel A.

2014-01-01

Background Leishmania major and an uncharacterized species have been reported from human patients in a cutaneous leishmaniasis (CL) outbreak area in Ghana. Reports from the area indicate the presence of anthropophilic Sergentomyia species that were found with Leishmania DNA. Methodology/Principal Findings In this study, we analyzed the Leishmania DNA positive sand fly pools by PCR-RFLP and ITS1 gene sequencing. The trypanosome was determined using the SSU rRNA gene sequence. We observed DNA of L. major, L. tropica and Trypanosoma species to be associated with the sand fly infections. This study provides the first detection of L. tropica DNA and Trypanosoma species as well as the confirmation of L. major DNA within Sergentomyia sand flies in Ghana and suggests that S. ingrami and S. hamoni are possible vectors of CL in the study area. Conclusions/Significance The detection of L. tropica DNA in this CL focus is a novel finding in Ghana as well as West Africa. In addition, the unexpected infection of Trypanosoma DNA within S. africana africana indicates that more attention is necessary when identifying parasitic organisms by PCR within sand fly vectors in Ghana and other areas where leishmaniasis is endemic. PMID:24516676

Dynamic changes to survivin subcellular localization are initiated by DNA damage

PubMed Central

Asumen, Maritess Gay; Ifeacho, Tochukwu V; Cockerham, Luke; Pfandl, Christina; Wall, Nathan R

2010-01-01

Subcellular distribution of the apoptosis inhibitor survivin and its ability to relocalize as a result of cell cycle phase or therapeutic insult has led to the hypothesis that these subcellular pools may coincide with different survivin functions. The PIK kinases (ATM, ATR and DNA-PK) phosphorylate a variety of effector substrates that propagate DNA damage signals, resulting in various biological outputs. Here we demonstrate that subcellular repartitioning of survivin in MCF-7 cells as a result of UV light-mediated DNA damage is dependent upon DNA damage-sensing proteins as treatment with the pan PIK kinase inhibitor wortmannin repartitioned survivin in the mitochondria and diminished it from the cytosol and nucleus. Mitochondrial redistribution of survivin, such as was recorded after wortmannin treatment, occurred in cells lacking any one of the three DNA damage sensing protein kinases: DNA-PK, ATM or ATR. However, failed survivin redistribution from the mitochondria in response to low-dose UV occurred only in the cells lacking ATM, implying that ATM may be the primary kinase involved in this process. Taken together, this data implicates survivian’s subcellular distribution is a dynamic physiological process that appears responsive to UV light-initiated DNA damage and that its distribution may be responsible for its multifunctionality. PMID:20856848

Electron microscopic visualization of sites of nascent DNA synthesis by streptavidin-gold binding to biotinylated nucleotides incorporated in vivo

PubMed Central

1988-01-01

Biotinylated nucleotides (bio-11-dCTP, bio-11-dUTP, and bio-7-dATP) were microinjected into unfertilized and fertilized Xenopus laevis eggs. The amounts introduced were comparable to in vivo deoxy- nucleoside triphosphate pools. At various times after microinjection, DNA was extracted from eggs or embryos and subjected to electrophoresis on agarose gels. Newly synthesized biotinylated DNA was analyzed by Southern transfer and visualized using either the BluGENE or Detek-hrp streptavidin-based nucleic acid detection systems. Quantitation of the amount of biotinylated DNA observed at various times showed that the microinjected biotinylated nucleotides were efficiently incorporated in vivo, both into replicating endogenous chromosomal DNA and into replicating microinjected exogenous plasmid DNA. At least one biotinylated nucleotide could be incorporated in vivo for every eight nucleotides of DNA synthesized. Control experiments also showed that heavily biotinylated DNA was not subjected to detectable DNA repair during early embryogenesis (for at least 5 h after activation of the eggs). The incorporated biotinylated nucleotides were visualized by electron microscopy by using streptavidin-colloidal gold or streptavidin-ferritin conjugates to bind specifically to the biotin groups projecting from the newly replicated DNA. The incorporated biotinylated nucleotides were thus made visible as electron-dense spots on the underlying DNA molecules. Biotinylated nucleotides separated by 20-50 bases could be resolved. We conclude that nascent DNA synthesized in vivo in Xenopus laevis eggs can be visualized efficiently and specifically using the techniques described. PMID:3392102

Molecular survey of Coxiella burnetii in wildlife and ticks at wildlife-livestock interfaces in Kenya.

PubMed

Ndeereh, David; Muchemi, Gerald; Thaiyah, Andrew; Otiende, Moses; Angelone-Alasaad, Samer; Jowers, Michael J

2017-07-01

Coxiella burnetii is the causative agent of Q fever, a zoonotic disease of public health importance. The role of wildlife and their ticks in the epidemiology of C. burnetii in Kenya is unknown. This study analysed the occurrence and prevalence of the pathogen in wildlife and their ticks at two unique wildlife-livestock interfaces of Laikipia and Maasai Mara National Reserve (MMNR) with the aim to determine the potential risk of transmission to livestock and humans. Blood from 79 and 73 animals in Laikipia and MMNR, respectively, and 756 and 95 ixodid ticks in each of the areas, respectively, was analysed. Ticks were pooled before analyses into 137 and 29 samples in Laikipia and MMNR, respectively, of one to eight non-engorged ticks according to species and animal host. Real-time PCR amplifying the repetitive insertion element IS1111a of the transposase gene was used to detect C. burnetii DNA. Although none of the animals and ticks from MMNR tested positive, ticks from Laikipia had an overall pooled prevalence of 2.92% resulting in a maximum-likelihood estimate of prevalence of 0.54%, 95% CI 0.17-1.24. Ticks positive for C. burnetii DNA belonged to the genus Rhipicephalus at a pooled prevalence of 2.96% (maximum-likelihood estimate of prevalence of 0.54%, 95% CI 0.17-1.26). These ticks were Rhipicephalus appendiculatus, R. pulchellus and R. evertsi at pooled prevalence of 3.77, 3.03 and 2.04%, respectively. The presence of C. burnetii in ticks suggests circulation of the pathogen in Laikipia and demonstrates they may play a potential role in the epidemiology of Q fever in this ecosystem. The findings warrant further studies to understand the presence of C. burnetii in domestic animals and their ticks within both study areas.

Targeting DNA double strand break repair with hyperthermia and DNA-PKcs inhibition to enhance the effect of radiation treatment.

PubMed

van Oorschot, Bregje; Granata, Giovanna; Di Franco, Simone; Ten Cate, Rosemarie; Rodermond, Hans M; Todaro, Matilde; Medema, Jan Paul; Franken, Nicolaas A P

2016-10-04

Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation.The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 μM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found.In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.

Formation and processing of DNA damage substrates for the hNEIL enzymes.

PubMed

Fleming, Aaron M; Burrows, Cynthia J

2017-06-01

Reactive oxygen species (ROS) are harnessed by the cell for signaling at the same time as being detrimental to cellular components such as DNA. The genome and transcriptome contain instructions that can alter cellular processes when oxidized. The guanine (G) heterocycle in the nucleotide pool, DNA, or RNA is the base most prone to oxidation. The oxidatively-derived products of G consistently observed in high yields from hydroxyl radical, carbonate radical, or singlet oxygen oxidations under conditions modeling the cellular reducing environment are discussed. The major G base oxidation products are 8-oxo-7,8-dihydroguanine (OG), 5-carboxamido-5-formamido-2-iminohydantoin (2Ih), spiroiminodihydantoin (Sp), and 5-guanidinohydantoin (Gh). The yields of these products show dependency on the oxidant and the reaction context that includes nucleoside, single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and G-quadruplex DNA (G4-DNA) structures. Upon formation of these products in cells, they are recognized by the DNA glycosylases in the base excision repair (BER) pathway. This review focuses on initiation of BER by the mammalian Nei-like1-3 (NEIL1-3) glycosylases for removal of 2Ih, Sp, and Gh. The unique ability of the human NEILs to initiate removal of the hydantoins in ssDNA, bulge-DNA, bubble-DNA, dsDNA, and G4-DNA is outlined. Additionally, when Gh exists in a G4 DNA found in a gene promoter, NEIL-mediated repair is modulated by the plasticity of the G4-DNA structure provided by additional G-runs flanking the sequence. On the basis of these observations and cellular studies from the literature, the interplay between DNA oxidation and BER to alter gene expression is discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

DOEpatents

Gardner, Shea N [San Leandro, CA; Mariella, Jr., Raymond P.; Christian, Allen T [Tracy, CA; Young, Jennifer A [Berkeley, CA; Clague, David S [Livermore, CA

2011-01-18

A method of fabricating a DNA molecule of user-defined sequence. The method comprises the steps of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an even or odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths. In one embodiment starting sequence fragments are of different lengths, n, n+1, n+2, etc.

Insights into soil carbon dynamics across climatic gradients from carbon-pool specific radiocarbon analyses

NASA Astrophysics Data System (ADS)

van der Voort, Tessa Sophia; Hagedorn, Frank; McIntyre, Cameron; Zell, Claudia; Eglinton, Timothy Ian

2017-04-01

Soil carbon constitutes the largest terrestrial reservoir of organic carbon, and therefore understanding the mechanisms and drivers of carbon stabilization is crucial, especially in the framework of climate change. The understanding of the dependence of soil organic turnover in specific carbon pools as related to e.g. climate, soil texture and mineralogy is limited. In this framework, radiocarbon constitutes a uniquely powerful tool that help to unravel carbon dynamics from decadal to millennial timescales. This project combines bulk and pool-specific radiocarbon analyses in the top and deep soil on a wide range of forested soils that span a large climatic gradient (MAT 1.3-9.2°C, MAP 600 to 2100 mm m-2y-1). These well-studies sites are part of the Long-Term Forest Ecosystem Research (LWF) program of the Swiss Federal Institute for Forest, Snow and Landscape research (WSL). This study aims to combine the insights gained from bulk and pool-specific turnover to environmental conditions and molecular composition of soil carbon. The pools investigated span the mineral-associated (occluded and heavy fractions from density fractionation) and potentially water-soluble (free light fractions from density fractionation and water extractable organic carbon) organic carbon fractions. Pool-specific radiocarbon work is augmented by the measurement of abundance of compounds such as alkanes, fatty acids and lignin phenols on a subset of samples. Initial results show disparate patterns depending on soil type and in particular soil texture, which could be indicative of various stabilization mechanisms in different soils. Overall, this study provides new insights into the controls of soil organic matter dynamics as related to environmental conditions, in particular in specific sub-pools of carbon.

Synthetic Spike-in Standards Improve Run-Specific Systematic Error Analysis for DNA and RNA Sequencing

PubMed Central

Zook, Justin M.; Samarov, Daniel; McDaniel, Jennifer; Sen, Shurjo K.; Salit, Marc

2012-01-01

While the importance of random sequencing errors decreases at higher DNA or RNA sequencing depths, systematic sequencing errors (SSEs) dominate at high sequencing depths and can be difficult to distinguish from biological variants. These SSEs can cause base quality scores to underestimate the probability of error at certain genomic positions, resulting in false positive variant calls, particularly in mixtures such as samples with RNA editing, tumors, circulating tumor cells, bacteria, mitochondrial heteroplasmy, or pooled DNA. Most algorithms proposed for correction of SSEs require a data set used to calculate association of SSEs with various features in the reads and sequence context. This data set is typically either from a part of the data set being “recalibrated” (Genome Analysis ToolKit, or GATK) or from a separate data set with special characteristics (SysCall). Here, we combine the advantages of these approaches by adding synthetic RNA spike-in standards to human RNA, and use GATK to recalibrate base quality scores with reads mapped to the spike-in standards. Compared to conventional GATK recalibration that uses reads mapped to the genome, spike-ins improve the accuracy of Illumina base quality scores by a mean of 5 Phred-scaled quality score units, and by as much as 13 units at CpG sites. In addition, since the spike-in data used for recalibration are independent of the genome being sequenced, our method allows run-specific recalibration even for the many species without a comprehensive and accurate SNP database. We also use GATK with the spike-in standards to demonstrate that the Illumina RNA sequencing runs overestimate quality scores for AC, CC, GC, GG, and TC dinucleotides, while SOLiD has less dinucleotide SSEs but more SSEs for certain cycles. We conclude that using these DNA and RNA spike-in standards with GATK improves base quality score recalibration. PMID:22859977

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

PubMed Central

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

2016-01-01

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

Technique for quantitative RT-PCR analysis directly from single muscle fibers.

PubMed

Wacker, Michael J; Tehel, Michelle M; Gallagher, Philip M

2008-07-01

The use of single-cell quantitative RT-PCR has greatly aided the study of gene expression in fields such as muscle physiology. For this study, we hypothesized that single muscle fibers from a biopsy can be placed directly into the reverse transcription buffer and that gene expression data can be obtained without having to first extract the RNA. To test this hypothesis, biopsies were taken from the vastus lateralis of five male subjects. Single muscle fibers were isolated and underwent RNA isolation (technique 1) or placed directly into reverse transcription buffer (technique 2). After cDNA conversion, individual fiber cDNA was pooled and quantitative PCR was performed using primer-probes for beta(2)-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, insulin-like growth factor I receptor, and glucose transporter subtype 4. The no RNA extraction method provided similar quantitative PCR data as that of the RNA extraction method. A third technique was also tested in which we used one-quarter of an individual fiber's cDNA for PCR (not pooled) and the average coefficient of variation between fibers was <8% (cycle threshold value) for all genes studied. The no RNA extraction technique was tested on isolated muscle fibers using a gene known to increase after exercise (pyruvate dehydrogenase kinase 4). We observed a 13.9-fold change in expression after resistance exercise, which is consistent with what has been previously observed. These results demonstrate a successful method for gene expression analysis directly from single muscle fibers.

Construction and Evaluation of Normalized cDNA Libraries Enriched with Full-Length Sequences for Rapid Discovery of New Genes from Sisal (Agave sisalana Perr.) Different Developmental Stages

PubMed Central

Zhou, Wen-Zhao; Zhang, Yan-Mei; Lu, Jun-Ying; Li, Jun-Feng

2012-01-01

To provide a resource of sisal-specific expressed sequence data and facilitate this powerful approach in new gene research, the preparation of normalized cDNA libraries enriched with full-length sequences is necessary. Four libraries were produced with RNA pooled from Agave sisalana multiple tissues to increase efficiency of normalization and maximize the number of independent genes by SMART™ method and the duplex-specific nuclease (DSN). This procedure kept the proportion of full-length cDNAs in the subtracted/normalized libraries and dramatically enhanced the discovery of new genes. Sequencing of 3875 cDNA clones of libraries revealed 3320 unigenes with an average insert length about 1.2 kb, indicating that the non-redundancy of libraries was about 85.7%. These unigene functions were predicted by comparing their sequences to functional domain databases and extensively annotated with Gene Ontology (GO) terms. Comparative analysis of sisal unigenes and other plant genomes revealed that four putative MADS-box genes and knotted-like homeobox (knox) gene were obtained from a total of 1162 full-length transcripts. Furthermore, real-time PCR showed that the characteristics of their transcripts mainly depended on the tight expression regulation of a number of genes during the leaf and flower development. Analysis of individual library sequence data indicated that the pooled-tissue approach was highly effective in discovering new genes and preparing libraries for efficient deep sequencing. PMID:23202944

Genetic variation in Mediterranean Helichrysum italicum (Asteraceae; Gnaphalieae): do disjunct populations of subsp. microphyllum have a common origin?

PubMed

Galbany-Casals, M; Blanco-Moreno, J M; Garcia-Jacas, N; Breitwieser, I; Smissen, R D

2011-07-01

The yellow-flowered everlasting daisy Helichrysum italicum (Asteraceae, Gnaphalieae) is widely distributed in the Mediterranean basin, where it grows in continuous and widespread populations in diverse open habitats. Helichrysum italicum subsp. microphyllum has a disjunct distribution in the Balearic Islands (Majorca and Dragonera), Corsica, Sardinia, Crete and Cyprus. Numerous morphological intermediates between subsp. italicum and subsp. microphyllum are known from Corsica, where the two subspecies co-occur. The aims of the study were to investigate if subsp. microphyllum has a common origin, constituting an independent gene pool from subsp. italicum, or if the morphological differences between subsp. microphyllum and subsp. italicum have arisen independently in different locations from a common wider gene pool. Our analyses of AFLP, cpDNA sequences and morphological characters show that there is geographic structure to the genetic variation within H. italicum, with eastern and western Mediterranean groups, which do not correspond with the division into subsp. microphyllum and subsp. italicum as currently circumscribed. Local selection on quantitative trait loci provides sufficient explanation for the morphological divergence observed and is consistent with genetic data. Within the western Mediterranean group of the species we found considerable polymorphism in chloroplast DNA sequences among and within some populations. Comparison with chloroplast DNA sequences from other Helichrysum species showed that some chloroplast haplotypes are shared across species. © 2010 German Botanical Society and The Royal Botanical Society of the Netherlands.

Singlet Oxygen-Mediated Oxidation during UVA Radiation Alters the Dynamic of Genomic DNA Replication

PubMed Central

Graindorge, Dany; Martineau, Sylvain; Machon, Christelle; Arnoux, Philippe; Guitton, Jérôme; Francesconi, Stefania; Frochot, Céline; Sage, Evelyne; Girard, Pierre-Marie

2015-01-01

UVA radiation (320–400 nm) is a major environmental agent that can exert its deleterious action on living organisms through absorption of the UVA photons by endogenous or exogenous photosensitizers. This leads to the production of reactive oxygen species (ROS), such as singlet oxygen (1O2) and hydrogen peroxide (H2O2), which in turn can modify reversibly or irreversibly biomolecules, such as lipids, proteins and nucleic acids. We have previously reported that UVA-induced ROS strongly inhibit DNA replication in a dose-dependent manner, but independently of the cell cycle checkpoints activation. Here, we report that the production of 1O2 by UVA radiation leads to a transient inhibition of replication fork velocity, a transient decrease in the dNTP pool, a quickly reversible GSH-dependent oxidation of the RRM1 subunit of ribonucleotide reductase and sustained inhibition of origin firing. The time of recovery post irradiation for each of these events can last from few minutes (reduction of oxidized RRM1) to several hours (replication fork velocity and origin firing). The quenching of 1O2 by sodium azide prevents the delay of DNA replication, the decrease in the dNTP pool and the oxidation of RRM1, while inhibition of Chk1 does not prevent the inhibition of origin firing. Although the molecular mechanism remains elusive, our data demonstrate that the dynamic of replication is altered by UVA photosensitization of vitamins via the production of singlet oxygen. PMID:26485711

Selfish Little Circles: Transmission Bias and Evolution of Large Deletion-Bearing Mitochondrial DNA in Caenorhabditis briggsae Nematodes

PubMed Central

Clark, Katie A.; Howe, Dana K.; Gafner, Kristin; Kusuma, Danika; Ping, Sita; Estes, Suzanne; Denver, Dee R.

2012-01-01

Selfish DNA poses a significant challenge to genome stability and organismal fitness in diverse eukaryotic lineages. Although selfish mitochondrial DNA (mtDNA) has known associations with cytoplasmic male sterility in numerous gynodioecious plant species and is manifested as petite mutants in experimental yeast lab populations, examples of selfish mtDNA in animals are less common. We analyzed the inheritance and evolution of mitochondrial DNA bearing large heteroplasmic deletions including nad5 gene sequences (nad5Δ mtDNA), in the nematode Caenorhabditis briggsae. The deletion is widespread in C. briggsae natural populations and is associated with deleterious organismal effects. We studied the inheritance patterns of nad5Δ mtDNA using eight sets of C. briggsae mutation-accumulation (MA) lines, each initiated from a different natural strain progenitor and bottlenecked as single hermaphrodites across generations. We observed a consistent and strong drive toward higher levels of deletion-bearing molecules in the heteroplasmic pool of mtDNA after ten generations of bottlenecking. Our results demonstrate a uniform transmission bias whereby nad5Δ mtDNA accumulates to higher levels relative to intact mtDNA in multiple genetically diverse natural strains of C. briggsae. We calculated an average 1% per-generation transmission bias for deletion-bearing mtDNA relative to intact genomes. Our study, coupled with known deleterious phenotypes associated with high deletion levels, shows that nad5Δ mtDNA are selfish genetic elements that have evolved in natural populations of C. briggsae, offering a powerful new system to study selfish mtDNA dynamics in metazoans. PMID:22859984

Age-related decline in mitochondrial DNA copy number in isolated human pancreatic islets.

PubMed

Cree, L M; Patel, S K; Pyle, A; Lynn, S; Turnbull, D M; Chinnery, P F; Walker, M

2008-08-01

Pancreatic beta cell function has been shown to decline with age in man. Depletion of mitochondrial DNA (mtDNA) copy number is associated with impaired insulin secretion in pancreatic beta cell lines, and decreased mtDNA copy number has been observed with age in skeletal muscle in man. We investigated whether mtDNA copy number decreases with age in human pancreatic beta cells, which might in turn contribute to the age-related decline in insulin secretory capacity. We quantified mtDNA copy number in isolated human islet preparations from 15 pancreas donors aged between 17 and 75 years. Islets (n = 20) were individually hand-picked and pooled from each donor isolate for the quantification of mtDNA copy number and deleted mtDNA (%), which were determined using real-time PCR methods. There was a significant negative correlation between mtDNA copy number and islet donor age (r = -0.53, p = 0.044). mtDNA copy number was significantly decreased in islet preparations from donors aged > or =50 years (n = 8) compared with those aged <50 years (n = 7) (median [interquartile range]: 418 [236-503] vs 596 [554-729] mtDNA copy number/diploid genome; p = 0.032). None of the islet preparations harboured high levels of deleted mtDNA affecting the major arc. Given the correlation between mtDNA content and respiratory chain activity, the age-related decrease in mtDNA copy number that we observed in human pancreatic islet preparations may contribute to the age-dependent decline in pancreatic beta cell insulin secretory capacity.

Investigation on the correlation between energy deposition and clustered DNA damage induced by low-energy electrons.

PubMed

Liu, Wei; Tan, Zhenyu; Zhang, Liming; Champion, Christophe

2018-05-01

This study presents the correlation between energy deposition and clustered DNA damage, based on a Monte Carlo simulation of the spectrum of direct DNA damage induced by low-energy electrons including the dissociative electron attachment. Clustered DNA damage is classified as simple and complex in terms of the combination of single-strand breaks (SSBs) or double-strand breaks (DSBs) and adjacent base damage (BD). The results show that the energy depositions associated with about 90% of total clustered DNA damage are below 150 eV. The simple clustered DNA damage, which is constituted of the combination of SSBs and adjacent BD, is dominant, accounting for 90% of all clustered DNA damage, and the spectra of the energy depositions correlating with them are similar for different primary energies. One type of simple clustered DNA damage is the combination of a SSB and 1-5 BD, which is denoted as SSB + BD. The average contribution of SSB + BD to total simple clustered DNA damage reaches up to about 84% for the considered primary energies. In all forms of SSB + BD, the SSB + BD including only one base damage is dominant (above 80%). In addition, for the considered primary energies, there is no obvious difference between the average energy depositions for a fixed complexity of SSB + BD determined by the number of base damage, but average energy depositions increase with the complexity of SSB + BD. In the complex clustered DNA damage constituted by the combination of DSBs and BD around them, a relatively simple type is a DSB combining adjacent BD, marked as DSB + BD, and it is of substantial contribution (on average up to about 82%). The spectrum of DSB + BD is given mainly by the DSB in combination with different numbers of base damage, from 1 to 5. For the considered primary energies, the DSB combined with only one base damage contributes about 83% of total DSB + BD, and the average energy deposition is about 106 eV. However, the energy deposition increases with the complexity of clustered DNA damage, and therefore, the clustered DNA damage with high complexity still needs to be considered in the study of radiation biological effects, in spite of their small contributions to all clustered DNA damage.

Melting Efficiency During Plasma Arc Welding

NASA Technical Reports Server (NTRS)

McClure, J.C.; Evans, D. M.; Tang, W.; Nunes, A. C.

1999-01-01

A series of partial penetration Variable Polarity Plasma Arc welds were made at equal power but various combinations of current and voltage on 2219 aluminum. Arc Efficiency was measured calorimetrically and ranged between 48% and 66%. Melting efficiency depends on the weld pool shape. Increased current increases the melting efficiency as it increases the depth to width ratio of the weld pool. Higher currents are thought to raise arc pressure and depress the liquid at the bottom of the weld pool causing a more nearly two dimensional heat flow condition.

Microbial Composition of Near-Boiling Silica-Depositing Thermal Springs throughout Yellowstone National Park

PubMed Central

Blank, Carrine E.; Cady, Sherry L.; Pace, Norman R.

2002-01-01

The extent of hyperthermophilic microbial diversity associated with siliceous sinter (geyserite) was characterized in seven near-boiling silica-depositing springs throughout Yellowstone National Park using environmental PCR amplification of small-subunit rRNA genes (SSU rDNA), large-subunit rDNA, and the internal transcribed spacer (ITS). We found that Thermocrinis ruber, a member of the order Aquificales, is ubiquitous, an indication that primary production in these springs is driven by hydrogen oxidation. Several other lineages with no known close relatives were identified that branch among the hyperthermophilic bacteria. Although they all branch deep in the bacterial tree, the precise phylogenetic placement of many of these lineages is unresolved at this time. While some springs contained a fair amount of phylogenetic diversity, others did not. Within the same spring, communities in the subaqueous environment were not appreciably different than those in the splash zone at the edge of the pool, although a greater number of phylotypes was found along the pool's edge. Also, microbial community composition appeared to have little correlation with the type of sinter morphology. The number of cell morphotypes identified by fluorescence in situ hybridization and scanning electron microscopy was greater than the number of phylotypes in SSU clone libraries. Despite little variation in Thermocrinis ruber SSU sequences, abundant variation was found in the hypervariable ITS region. The distribution of ITS sequence types appeared to be correlated with distinct morphotypes of Thermocrinis ruber in different pools. Therefore, species- or subspecies-level divergences are present but not detectable in highly conserved SSU sequences. PMID:12324363

A lysigenic programmed cell death-dependent process shapes schizogenously formed aerenchyma in the stems of the waterweed Egeria densa

PubMed Central

Bartoli, G.; Forino, L. M. C.; Durante, M.; Tagliasacchi, A. M.

2015-01-01

Background and Aims Plant adaptation to submergence can include the formation of prominent aerenchyma to facilitate gas exchange. The aim of this study was to characterize the differentiation of the constitutive aerenchyma in the stem of the aquatic macrophyte Egeria densa (Hydrocharitaceae) and to verify if any form of cell death might be involved. Methods Plants were collected from a pool in a botanical garden. Aerenchyma differentiation and apoptotic hallmarks were investigated by light microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay coupled with genomic DNA extraction and gel electrophoresis (DNA laddering assay). Cell viability and the occurrence of peroxides and nitric oxide (NO) were determined histochemically using specific fluorogenic probes. Key Results Aerenchyma differentiation started from a hexagonally packed pre-aerenchymatic tissue and, following a basipetal and centripetal developmental pattern, produced a honeycomb arrangement. After an early schizogenous differentiation process, a late lysigenous programmed cell death- (PCD) dependent mechanism occurred. This was characterized by a number of typical apoptotic hallmarks, including DNA fragmentation, chromatin condensation, apoptotic-like bodies, partial cell wall lysis and plasmolysis. In addition, local increases in H2O2 and NO were observed and quantified. Conclusions The differentiation of cortical aerenchyma in the stem of E. densa is a complex process, consisting of a combination of an early schizogenous differentiation mechanism and a late lysigenous PCD-dependent process. The PCD remodels the architecture of the gas spaces previously formed schizogenously, and also results in a reduction of O2-consuming cells and in recycling of material derived from the lysigenic dismantling of the cells. PMID:26002256

A lysigenic programmed cell death-dependent process shapes schizogenously formed aerenchyma in the stems of the waterweed Egeria densa.

PubMed

Bartoli, G; Forino, L M C; Durante, M; Tagliasacchi, A M

2015-07-01

Plant adaptation to submergence can include the formation of prominent aerenchyma to facilitate gas exchange. The aim of this study was to characterize the differentiation of the constitutive aerenchyma in the stem of the aquatic macrophyte Egeria densa (Hydrocharitaceae) and to verify if any form of cell death might be involved. Plants were collected from a pool in a botanical garden. Aerenchyma differentiation and apoptotic hallmarks were investigated by light microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay coupled with genomic DNA extraction and gel electrophoresis (DNA laddering assay). Cell viability and the occurrence of peroxides and nitric oxide (NO) were determined histochemically using specific fluorogenic probes. Aerenchyma differentiation started from a hexagonally packed pre-aerenchymatic tissue and, following a basipetal and centripetal developmental pattern, produced a honeycomb arrangement. After an early schizogenous differentiation process, a late lysigenous programmed cell death- (PCD) dependent mechanism occurred. This was characterized by a number of typical apoptotic hallmarks, including DNA fragmentation, chromatin condensation, apoptotic-like bodies, partial cell wall lysis and plasmolysis. In addition, local increases in H2O2 and NO were observed and quantified. The differentiation of cortical aerenchyma in the stem of E. densa is a complex process, consisting of a combination of an early schizogenous differentiation mechanism and a late lysigenous PCD-dependent process. The PCD remodels the architecture of the gas spaces previously formed schizogenously, and also results in a reduction of O2-consuming cells and in recycling of material derived from the lysigenic dismantling of the cells. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A Polymerase Chain Reaction-Based Method for Isolating Clones from a Complimentary DNA Library in Sheep

PubMed Central

Friis, Thor Einar; Stephenson, Sally; Xiao, Yin; Whitehead, Jon

2014-01-01

The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes. PMID:24447069

75 FR 70102 - Airworthiness Directives; The Boeing Company Model 777-200, -200LR, -300, and -300ER Series...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-17

... ground, uncontained fuel leakage could result in pooling, and pooling combined with an ignition source... Airplanes, Attention: Data & Services Management, P.O. Box 3707, MC 2H-65, Seattle, Washington 98124-2207... Management Facility, U.S. Department of Transportation, Docket Operations, M-30, West Building Ground Floor...

In Search of Genes Associated with Risk for Psychopathic Tendencies in Children: A Two-Stage Genome-Wide Association Study of Pooled DNA

ERIC Educational Resources Information Center

Viding, Essi; Hanscombe, Ken B.; Curtis, Charles J. C.; Davis, Oliver S. P.; Meaburn, Emma L.; Plomin, Robert

2010-01-01

Background: Quantitative genetic data from our group indicates that antisocial behaviour (AB) is strongly heritable when coupled with psychopathic, callous-unemotional (CU) personality traits. We have also demonstrated that the genetic influences for AB and CU overlap considerably. We conducted a genome-wide association scan that capitalises on…

Defining the transcriptome assembly and its use for genome dynamics and transcriptome profiling studies in pigeonpea (Cajanus cajan L.)

USDA-ARS?s Scientific Manuscript database

This study reports generation of large-scale genomic resources for pigeonpea, a so-called ‘orphan crop species’ of the semi-arid tropic regions. Roche FLX/454 sequencing was carried out on a normalized cDNA pool prepared from 31 tissues produced 494,353 short transcript reads (STRs). Cluster analysi...

Evidence for a genetic discontinuity between Neandertals and 24,000-year-old anatomically modern Europeans.

PubMed

Caramelli, David; Lalueza-Fox, Carles; Vernesi, Cristiano; Lari, Martina; Casoli, Antonella; Mallegni, Francesco; Chiarelli, Brunetto; Dupanloup, Isabelle; Bertranpetit, Jaume; Barbujani, Guido; Bertorelle, Giorgio

2003-05-27

During the late Pleistocene, early anatomically modern humans coexisted in Europe with the anatomically archaic Neandertals for some thousand years. Under the recent variants of the multiregional model of human evolution, modern and archaic forms were different but related populations within a single evolving species, and both have contributed to the gene pool of current humans. Conversely, the Out-of-Africa model considers the transition between Neandertals and anatomically modern humans as the result of a demographic replacement, and hence it predicts a genetic discontinuity between them. Following the most stringent current standards for validation of ancient DNA sequences, we typed the mtDNA hypervariable region I of two anatomically modern Homo sapiens sapiens individuals of the Cro-Magnon type dated at about 23 and 25 thousand years ago. Here we show that the mtDNAs of these individuals fall well within the range of variation of today's humans, but differ sharply from the available sequences of the chronologically closer Neandertals. This discontinuity is difficult to reconcile with the hypothesis that both Neandertals and early anatomically modern humans contributed to the current European gene pool.

Aiding pest control management of long-tailed macaques (Macaca fascicularis fascicularis) in Malaysia by using molecular markers of mitochondrial DNA

NASA Astrophysics Data System (ADS)

Abdul-Latiff, M. A. B.; Abdul-Patah, P.; Yaakop, S.; Md-Zain, B. M.

2017-10-01

The long-tailed macaques (Macaca fascicularis fascicularis) has been the center of human wildlife conflict in Malaysia since 1970s. This well-adapted and opportunistic primates have been dominating wide range of habitat in Malaysia such as primary and secondary forest, mangrove, as well as human settlements. The conventional practices of translocation by the authorities are threatening the uniqueness of gene pool for this species and ironically contradicting with the ultimate purpose of genetic conservation of this species. The objectives of this study is to determine the level of genetic separation between populations of long-tailed macaques, primarily focusing on populations distributed in northern Peninsular Malaysia. A total of 954 base pairs of control regions mtDNA was sequenced and analyzed from 27 samples of M. fascicularis. The results exhibited a highly homogenous state of populations for long-tailed macaques genetically and this ultimately indicate unsuitable management and planning in terms of pest control management of the species. Authorities are suggested to translocate the species at least within the state boundaries to avoid homogeneity of gene pools for the particular species.

DNA copy number, including telomeres and mitochondria, assayed using next-generation sequencing.

PubMed

Castle, John C; Biery, Matthew; Bouzek, Heather; Xie, Tao; Chen, Ronghua; Misura, Kira; Jackson, Stuart; Armour, Christopher D; Johnson, Jason M; Rohl, Carol A; Raymond, Christopher K

2010-04-16

DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number. We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual. The described assay outputs absolute copy number, outputs an error estimate (p-value), and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads.

Murine cytomegalovirus: detection of latent infection by nucleic acid hybridization technique.

PubMed Central

Cheung, K S; Huang, E S; Lang, D J

1980-01-01

The technique of nucleic acid hybridization was used to detect the presence of murine cytomegalovirus (MCMV)-specific deoxyribonucleic acid (DNA) in cell cultures and salivary gland tissues. The presence of approximately 4.5 and 0.2 genome equivalents per cell of MCMV-specific DNA was identified in cultures of salivary (ISG2) and prostate gland (IP) cells, respectively. These cells, derived from animals with experimentally induced latent infections, were negative for virus-specific antigens by immunofluorescence and on electron microscopy revealed no visible evidence of the presence of herpesviruses. A cell line derived from the salivary gland of an uninoculated animal (NSG2) was also found to possess MCMV-specific DNA (0.2 genome equivalents per cell). For this reason, salivary gland tissues from uninoculated animals supplied as "specific pathogen-free" mice by three commercial sources were tested upon arrival for the presence of MCMC-specific DNA. MCMV-specific DNA was detectable in pooled salivary gland extracts from uninoculated animals derived from two commercial sources. All of these animals were seronegative and virus negative by conventional infectivity assays. PMID:6247281

Nanoparticle-labeled DNA capture elements for detection and identification of biological agents

NASA Astrophysics Data System (ADS)

Kiel, Johnathan L.; Holwitt, Eric A.; Parker, Jill E.; Vivekananda, Jeevalatha; Franz, Veronica

2004-12-01

Aptamers, synthetic DNA capture elements (DCEs), can be made chemically or in genetically engineered bacteria. DNA capture elements are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare or terrorism agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. The probes can then be conjugated to Quantum Dots and super paramagnetic nanoparticles. The former provide intense, bleach-resistant fluorescent detection of bioagent and the latter provide a means to collect the bioagents with a magnet. The fluorescence can be detected in a flow cytometer, in a fluorescence plate reader, or with a fluorescence microscope. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. DCEs can easily distinguish Bacillus anthracis from its nearest relatives, Bacillus cereus and Bacillus thuringiensis. Development of a high through-put process is currently being investigated.

News from the west: ancient DNA from a French megalithic burial chamber.

PubMed

Deguilloux, Marie-France; Soler, Ludovic; Pemonge, Marie-Hélène; Scarre, Chris; Joussaume, Roger; Laporte, Luc

2011-01-01

Recent paleogenetic studies have confirmed that the spread of the Neolithic across Europe was neither genetically nor geographically uniform. To extend existing knowledge of the mitochondrial European Neolithic gene pool, we examined six samples of human skeletal material from a French megalithic long mound (c.4200 cal BC). We retrieved HVR-I sequences from three individuals and demonstrated that in the Neolithic period the mtDNA haplogroup N1a, previously only known in central Europe, was as widely distributed as western France. Alternative scenarios are discussed in seeking to explain this result, including Mesolithic ancestry, Neolithic demic diffusion, and long-distance matrimonial exchanges. In light of the limited Neolithic ancient DNA (aDNA) data currently available, we observe that all three scenarios appear equally consistent with paleogenetic and archaeological data. In consequence, we advocate caution in interpreting aDNA in the context of the Neolithic transition in Europe. Nevertheless, our results strengthen conclusions demonstrating genetic discontinuity between modern and ancient Europeans whether through migration, demographic or selection processes, or social practices. Copyright © 2010 Wiley-Liss, Inc.

DNA copy number, including telomeres and mitochondria, assayed using next-generation sequencing

PubMed Central

2010-01-01

Background DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number. Results We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual. Conclusion The described assay outputs absolute copy number, outputs an error estimate (p-value), and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads. PMID:20398377

Immunogenicity of combination DNA vaccines for Rift Valley fever virus, tick-borne encephalitis virus, Hantaan virus, and Crimean Congo hemorrhagic fever virus.

PubMed

Spik, Kristin; Shurtleff, Amy; McElroy, Anita K; Guttieri, Mary C; Hooper, Jay W; SchmalJohn, Connie

2006-05-22

DNA vaccines for Rift Valley fever virus (RVFV), Crimean Congo hemorrhagic fever virus (CCHFV), tick-borne encephalitis virus (TBEV), and Hantaan virus (HTNV), were tested in mice alone or in various combinations. The bunyavirus vaccines (RVFV, CCHFV, and HTNV) expressed Gn and Gc genes, and the flavivirus vaccine (TBEV) expressed the preM and E genes. All vaccines were delivered by gene gun. The TBEV DNA vaccine and the RVFV DNA vaccine elicited similar levels of antibodies and protected mice from challenge when delivered alone or in combination with other DNAs. Although in general, the HTNV and CCHFV DNA vaccines were not very immunogenic in mice, there were no major differences in performance when given alone or in combination with the other vaccines.

Stroke Treatment Academic Industry Roundtable Recommendations for Individual Data Pooling Analyses in Stroke.

PubMed

Lees, Kennedy R; Khatri, Pooja

2016-08-01

Pooled analysis of individual patient data from stroke trials can deliver more precise estimates of treatment effect, enhance power to examine prespecified subgroups, and facilitate exploration of treatment-modifying influences. Analysis plans should be declared, and preferably published, before trial results are known. For pooling trials that used diverse analytic approaches, an ordinal analysis is favored, with justification for considering deaths and severe disability jointly. Because trial pooling is an incremental process, analyses should follow a sequential approach, with statistical adjustment for iterations. Updated analyses should be published when revised conclusions have a clinical implication. However, caution is recommended in declaring pooled findings that may prejudice ongoing trials, unless clinical implications are compelling. All contributing trial teams should contribute to leadership, data verification, and authorship of pooled analyses. Development work is needed to enable reliable inferences to be drawn about individual drug or device effects that contribute to a pooled analysis, versus a class effect, if the treatment strategy combines ≥2 such drugs or devices. Despite the practical challenges, pooled analyses are powerful and essential tools in interpreting clinical trial findings and advancing clinical care. © 2016 American Heart Association, Inc.

The UNITAID Patent Pool Initiative: Bringing Patents Together for the Common Good.

PubMed

Bermudez, Jorge; 't Hoen, Ellen

2010-01-19

Developing and delivering appropriate, affordable, well-adapted medicines for HIV/AIDS remains an urgent challenge: as first-line therapies fail, increasing numbers of people require costly second-line therapy; one-third of ARVs are not available in pediatric formulations; and certain key first- and second-line triple fixed-dose combinations do not exist or sufficient suppliers are lacking. UNITAID aims to help solve these problems through an innovative initiative for the collective management of intellectual property (IP) rights - a patent pool for HIV medicines. The idea behind a patent pool is that patent holders - companies, governments, researchers or universities - voluntarily offer, under certain conditions, the IP related to their inventions to the patent pool. Any company that wants to use the IP to produce or develop medicines can seek a license from the pool against the payment of royalties, and may then produce the medicines for use in developing countries (conditional upon meeting agreed quality standards). The patent pool will be a voluntary mechanism, meaning its success will largely depend on the willingness of pharmaceutical companies to participate and commit their IP to the pool. Generic producers must also be willing to cooperate. The pool has the potential to provide benefits to all.

Further evidence of an Amerindian contribution to the Polynesian gene pool on Easter Island.

PubMed

Thorsby, E; Flåm, S T; Woldseth, B; Dupuy, B M; Sanchez-Mazas, A; Fernandez-Vina, M A

2009-06-01

Available evidence suggests a Polynesian origin of the Easter Island population. We recently found that some native Easter Islanders also carried some common American Indian (Amerindian) human leukocyte antigen (HLA) alleles, which probably were introduced before Europeans discovered the island in 1722. In this study, we report molecular genetic investigations of 21 other selected native Easter Islanders. Analysis of mitochondrial DNA and Y chromosome markers showed no traces of an Amerindian contribution. However, high-resolution genomic HLA typing showed that two individuals carried some other common Amerindian HLA alleles, different from those found in our previous investigations. The new data support our previous evidence of an Amerindian contribution to the gene pool on Easter Island.

Nucleotide exchange and excision technology DNA shuffling and directed evolution.

PubMed

Speck, Janina; Stebel, Sabine C; Arndt, Katja M; Müller, Kristian M

2011-01-01

Remarkable success in optimizing complex properties within DNA and proteins has been achieved by directed evolution. In contrast to various random mutagenesis methods and high-throughput selection methods, the number of available DNA shuffling procedures is limited, and protocols are often difficult to adjust. The strength of the nucleotide exchange and excision technology (NExT) DNA shuffling described here is the robust, efficient, and easily controllable DNA fragmentation step based on random incorporation of the so-called 'exchange nucleotides' by PCR. The exchange nucleotides are removed enzymatically, followed by chemical cleavage of the DNA backbone. The oligonucleotide pool is reassembled into full-length genes by internal primer extension, and the recombined gene library is amplified by standard PCR. The technique has been demonstrated by shuffling a defined gene library of chloramphenicol acetyltransferase variants using uridine as fragmentation defining exchange nucleotide. Substituting 33% of the dTTP with dUTP in the incorporation PCR resulted in shuffled clones with an average parental fragment size of 86 bases and revealed a mutation rate of only 0.1%. Additionally, a computer program (NExTProg) has been developed that predicts the fragment size distribution depending on the relative amount of the exchange nucleotide.

Evaluating the feasibility of using candidate DNA barcodes in discriminating species of the large Asteraceae family.

PubMed

Gao, Ting; Yao, Hui; Song, Jingyuan; Zhu, Yingjie; Liu, Chang; Chen, Shilin

2010-10-26

Five DNA regions, namely, rbcL, matK, ITS, ITS2, and psbA-trnH, have been recommended as primary DNA barcodes for plants. Studies evaluating these regions for species identification in the large plant taxon, which includes a large number of closely related species, have rarely been reported. The feasibility of using the five proposed DNA regions was tested for discriminating plant species within Asteraceae, the largest family of flowering plants. Among these markers, ITS2 was the most useful in terms of universality, sequence variation, and identification capability in the Asteraceae family. The species discriminating power of ITS2 was also explored in a large pool of 3,490 Asteraceae sequences that represent 2,315 species belonging to 494 different genera. The result shows that ITS2 correctly identified 76.4% and 97.4% of plant samples at the species and genus levels, respectively. In addition, ITS2 displayed a variable ability to discriminate related species within different genera. ITS2 is the best DNA barcode for the Asteraceae family. This approach significantly broadens the application of DNA barcoding to resolve classification problems in the family Asteraceae at the genera and species levels.

High-throughput analysis of T-DNA location and structure using sequence capture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inagaki, Soichi; Henry, Isabelle M.; Lieberman, Meric C.

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously,more » using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. As a result, our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.« less

High-throughput analysis of T-DNA location and structure using sequence capture

DOE PAGES

Inagaki, Soichi; Henry, Isabelle M.; Lieberman, Meric C.; ...

2015-10-07

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously,more » using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. As a result, our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.« less

High sample throughput genotyping for estimating C-lineage introgression in the dark honeybee: an accurate and cost-effective SNP-based tool.

PubMed

Henriques, Dora; Browne, Keith A; Barnett, Mark W; Parejo, Melanie; Kryger, Per; Freeman, Tom C; Muñoz, Irene; Garnery, Lionel; Highet, Fiona; Jonhston, J Spencer; McCormack, Grace P; Pinto, M Alice

2018-06-04

The natural distribution of the honeybee (Apis mellifera L.) has been changed by humans in recent decades to such an extent that the formerly widest-spread European subspecies, Apis mellifera mellifera, is threatened by extinction through introgression from highly divergent commercial strains in large tracts of its range. Conservation efforts for A. m. mellifera are underway in multiple European countries requiring reliable and cost-efficient molecular tools to identify purebred colonies. Here, we developed four ancestry-informative SNP assays for high sample throughput genotyping using the iPLEX Mass Array system. Our customized assays were tested on DNA from individual and pooled, haploid and diploid honeybee samples extracted from different tissues using a diverse range of protocols. The assays had a high genotyping success rate and yielded accurate genotypes. Performance assessed against whole-genome data showed that individual assays behaved well, although the most accurate introgression estimates were obtained for the four assays combined (117 SNPs). The best compromise between accuracy and genotyping costs was achieved when combining two assays (62 SNPs). We provide a ready-to-use cost-effective tool for accurate molecular identification and estimation of introgression levels to more effectively monitor and manage A. m. mellifera conservatories.

Multiple incentives in encouraging car pool formation on a university campus

PubMed Central

Jacobs, Harvey E.; Fairbanks, David; Poche, Cheryl E.; Bailey, Jon S.

1982-01-01

The effects of a combined token economy and reserved parking program were evaluated as a means of increasing car pooling among students on a university campus. Following a baseline period, students in two experimental parking lots were notified of the availability of reserved parking and coupons redeemable for 25 cents in merchandise for each occupant of automobiles containing two or more people. Two control lots also were monitored for a comparison of treatment effects. Results indicated variable increases in car pooling in the two experimental lots and no increase in the control lots. Removal of the 25-cent coupons from one treatment lot (reserved parking only) did not adversely affect rates of car pooling. An analysis of individual automobile occupancy rates among car poolers indicated distinct patterns of car pooling and underscored the importance of individual subject data analysis in the present situation. A cost-benefit analysis indicated that even moderately effective car pool programs can be cost-effective on both a public and personal level. PMID:16795653

Methods to maximise recovery of environmental DNA from water samples.

PubMed

Hinlo, Rheyda; Gleeson, Dianne; Lintermans, Mark; Furlan, Elise

2017-01-01

The environmental DNA (eDNA) method is a detection technique that is rapidly gaining credibility as a sensitive tool useful in the surveillance and monitoring of invasive and threatened species. Because eDNA analysis often deals with small quantities of short and degraded DNA fragments, methods that maximize eDNA recovery are required to increase detectability. In this study, we performed experiments at different stages of the eDNA analysis to show which combinations of methods give the best recovery rate for eDNA. Using Oriental weatherloach (Misgurnus anguillicaudatus) as a study species, we show that various combinations of DNA capture, preservation and extraction methods can significantly affect DNA yield. Filtration using cellulose nitrate filter paper preserved in ethanol or stored in a -20°C freezer and extracted with the Qiagen DNeasy kit outperformed other combinations in terms of cost and efficiency of DNA recovery. Our results support the recommendation to filter water samples within 24hours but if this is not possible, our results suggest that refrigeration may be a better option than freezing for short-term storage (i.e., 3-5 days). This information is useful in designing eDNA detection of low-density invasive or threatened species, where small variations in DNA recovery can signify the difference between detection success or failure.

Composition and metabolism of fecal microbiota from normal and overweight children are differentially affected by melibiose, raffinose and raffinose-derived fructans.

PubMed

Adamberg, Kaarel; Adamberg, Signe; Ernits, Karin; Larionova, Anneli; Voor, Tiia; Jaagura, Madis; Visnapuu, Triinu; Alamäe, Tiina

2018-06-20

The aim of the study was to investigate the metabolism of non-digestible oligo- and polysaccharides by fecal microbiota, using isothermal microcalorimetry. The five tested substrates were raffinose, melibiose, a mixture of oligo- and polysaccharides produced from raffinose by levansucrase, levan synthesized from raffinose, and levan from timothy grass. Two inocula were comprised of pooled fecal samples from overweight or normal-weight children, from healthy adult volunteers and a pure culture of Bacteroides thetaiotaomicron as a reference bacterium for colon microbiota. The growth was analyzed based on the heat evolution curves, and the production of organic acids and gases. Taxonomic profiles of the microbiota were assessed by 16S rDNA sequencing. Raffinose and melibiose promoted the growth of bifidobacteria in all fecal pools. Several pool-specific substrate-related responses to raffinose and melibiose were revealed. Lactate-producing bacteria (Streptococcus and Enterococcus) became enriched in the pool of overweight children resulting in lactic acid as the major fermentation product on short saccharides. Acetic and butyric acids were prevalent at fermentation in the normal-weight pool coinciding with the enrichment of Catenibacterium. In the adult pool, the specific promotion of Bacteroides and Lachnospiraceae by levans was disclosed. In the fecal pool of normal-weight children, levans stimulated the growth of Senegalimassilia and Lachnoclostridium and this particular pool also showed the highest maximum heat production rate at levan fermentation. Levans and raffinose-derived oligosaccharides, but not raffinose and melibiose were completely fermented by a pure culture of Bacteroides thetaiotaomicron. The main conclusion from the study is that fecal microbiota of normal and overweight children have different compositions and they respond in specific manners to non-digestible oligo- and polysaccharides: raffinose, melibiose, raffinose-derived oligosaccharides and levans. The potential of the tested saccharides to support a healthy balance of colon microbiota requires further studies. Copyright © 2018. Published by Elsevier Ltd.

[Improving venous tone and capillary sealing. Effect of a combination of Ruscus extract and hesperidine methyl chalcone in healthy probands in heat stress].

PubMed

Rudofsky, G

1989-06-30

The drug combination of Ruscus-extract and hesperidine methyl chalcone (HMC) involves two basic mechanisms in the treatment of venous diseases: increase in venous tonicity and edema protection. This was shown in a double-blind study on 20 healthy volunteers by comparing the effectiveness of the individual substances, the combination and a placebo on the venous hemodynamics and the volume of the foot. Ruscus-extract augments the tonicity of the venous wall. This is expressed by a decrease in venous capacity (p less than 0.01), a reduction in the blood pool in the lower leg under orthostatic conditions, and a decrease in tissue volume of the foot and ankle (p less than 0.01). HMC lowers the capillary filtration rate (p less than 0.01) but augmented the blood pool. The increase in blood volume can be explained by dehydration of the tissue of the lower leg lowering the pressure of tissue on the venous system and increasing the blood pool in the limb. After administration of the combination, the blood volume was between the Ruscus and HMC volumes, while the effects on filtration rate, venous capacity and tissue volume corresponded to the changes seen after administration of HMC and Ruscus extract alone.

[The estimation of possibilities for the application of the laser capture microdissection technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA].

PubMed

Ivanov, P L; Leonov, S N; Zemskova, E Iu

2012-01-01

The present study was designed to estimate the possibilities of application of the laser capture microdissection (LCM) technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA. The experimental method employed for the purpose was the multiplex multilocus analysis of autosomal DNA polymorphism in the preparations of buccal epitheliocytes obtained by LCM. The key principles of the study were the application of physical methods for contrast enhancement of the micropreparations (such as phase-contrast microscopy and dark-field microscopy) and PCR-compatible cell lysis. Genotyping was carried out with the use of AmpFISTR Minifiler TM PCR Amplification Kits ("Applied Biosynthesis", USA). It was shown that the technique employed in the present study ensures reliable genotyping of human chromosomal DNA in the pooled preparations containing 10-20 dissected diploid cells each. This result fairly well agrees with the calculated sensitivity of the method. A few practical recommendations are offered.

Inherited occipital hypoplasia/syringomyelia in the cavalier King Charles spaniel: experiences in setting up a worldwide DNA collection.

PubMed

Rusbridge, Clare; Knowler, Penny; Rouleau, Guy A; Minassian, Berge A; Rothuizen, Jan

2005-01-01

Inherited diseases commonly emerge within pedigree dog populations, often due to use of repeatedly bred carrier sire(s) within a small gene pool. Accurate family records are usually available making linkage analysis possible. However, there are many factors that are intrinsically difficult about collecting DNA and collating pedigree information from a large canine population. The keys to a successful DNA collection program include (1) the need to establish and maintain support from the pedigree breed clubs and pet owners; (2) committed individual(s) who can devote the considerable amount of time and energy to coordinating sample collection and communicating with breeders and clubs; and (3) providing means by which genotypic and phenotypic information can be easily collected and stored. In this article we described the clinical characteristics of inherited occipital hypoplasia/syringomyelia (Chiari type I malformation) in the cavalier King Charles spaniel and our experiences in establishing a pedigree and DNA database to study the disease.

Nuclear DNA sequences from the Middle Pleistocene Sima de los Huesos hominins.

PubMed

Meyer, Matthias; Arsuaga, Juan-Luis; de Filippo, Cesare; Nagel, Sarah; Aximu-Petri, Ayinuer; Nickel, Birgit; Martínez, Ignacio; Gracia, Ana; Bermúdez de Castro, José María; Carbonell, Eudald; Viola, Bence; Kelso, Janet; Prüfer, Kay; Pääbo, Svante

2016-03-24

A unique assemblage of 28 hominin individuals, found in Sima de los Huesos in the Sierra de Atapuerca in Spain, has recently been dated to approximately 430,000 years ago. An interesting question is how these Middle Pleistocene hominins were related to those who lived in the Late Pleistocene epoch, in particular to Neanderthals in western Eurasia and to Denisovans, a sister group of Neanderthals so far known only from southern Siberia. While the Sima de los Huesos hominins share some derived morphological features with Neanderthals, the mitochondrial genome retrieved from one individual from Sima de los Huesos is more closely related to the mitochondrial DNA of Denisovans than to that of Neanderthals. However, since the mitochondrial DNA does not reveal the full picture of relationships among populations, we have investigated DNA preservation in several individuals found at Sima de los Huesos. Here we recover nuclear DNA sequences from two specimens, which show that the Sima de los Huesos hominins were related to Neanderthals rather than to Denisovans, indicating that the population divergence between Neanderthals and Denisovans predates 430,000 years ago. A mitochondrial DNA recovered from one of the specimens shares the previously described relationship to Denisovan mitochondrial DNAs, suggesting, among other possibilities, that the mitochondrial DNA gene pool of Neanderthals turned over later in their history.

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

PubMed Central

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

1998-01-01

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

Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere.

PubMed

Schouten, Henk J; Vande Geest, Henri; Papadimitriou, Sofia; Bemer, Marian; Schaart, Jan G; Smulders, Marinus J M; Perez, Gabino Sanchez; Schijlen, Elio

2017-03-01

Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.

Structure of Escherichia coli dGTP Triphosphohydrolase: A Hexameric Enzyme with DNA Effector Molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Deepa; Gawel, Damian; Itsko, Mark

The Escherichia coli dgt gene encodes a dGTP triphosphohydrolase whose detailed role still remains to be determined. Deletion of dgt creates a mutator phenotype, indicating that the dGTPase has a fidelity role, possibly by affecting the cellular dNTP pool. In the present paper, we have investigated the structure of the Dgt protein at 3.1-Å resolution. One of the obtained structures revealed a protein hexamer that contained two molecules of single-stranded DNA. The presence of DNA caused significant conformational changes in the enzyme, including in the catalytic site of the enzyme. Dgt preparations lacking DNA were able to bind single-stranded DNAmore » with high affinity (K d ~ 50 nM). DNA binding positively affected the activity of the enzyme: dGTPase activity displayed sigmoidal (cooperative) behavior without DNA but hyperbolic (Michaelis-Menten) kinetics in its presence, consistent with a specific lowering of the apparent K m for dGTP. A mutant Dgt enzyme was also created containing residue changes in the DNA binding cleft. This mutant enzyme, whereas still active, was incapable of DNA binding and could no longer be stimulated by addition of DNA. We also created an E. coli strain containing the mutant dgt gene on the chromosome replacing the wild-type gene. The mutant also displayed a mutator phenotype. Finally, our results provide insight into the allosteric regulation of the enzyme and support a physiologically important role of DNA binding.« less

Structure of Escherichia coli dGTP Triphosphohydrolase: A Hexameric Enzyme with DNA Effector Molecules

DOE PAGES

Singh, Deepa; Gawel, Damian; Itsko, Mark; ...

2015-02-18

The Escherichia coli dgt gene encodes a dGTP triphosphohydrolase whose detailed role still remains to be determined. Deletion of dgt creates a mutator phenotype, indicating that the dGTPase has a fidelity role, possibly by affecting the cellular dNTP pool. In the present paper, we have investigated the structure of the Dgt protein at 3.1-Å resolution. One of the obtained structures revealed a protein hexamer that contained two molecules of single-stranded DNA. The presence of DNA caused significant conformational changes in the enzyme, including in the catalytic site of the enzyme. Dgt preparations lacking DNA were able to bind single-stranded DNAmore » with high affinity (K d ~ 50 nM). DNA binding positively affected the activity of the enzyme: dGTPase activity displayed sigmoidal (cooperative) behavior without DNA but hyperbolic (Michaelis-Menten) kinetics in its presence, consistent with a specific lowering of the apparent K m for dGTP. A mutant Dgt enzyme was also created containing residue changes in the DNA binding cleft. This mutant enzyme, whereas still active, was incapable of DNA binding and could no longer be stimulated by addition of DNA. We also created an E. coli strain containing the mutant dgt gene on the chromosome replacing the wild-type gene. The mutant also displayed a mutator phenotype. Finally, our results provide insight into the allosteric regulation of the enzyme and support a physiologically important role of DNA binding.« less

The steric gate of DNA polymerase ι regulates ribonucleotide incorporation and deoxyribonucleotide fidelity.

PubMed

Donigan, Katherine A; McLenigan, Mary P; Yang, Wei; Goodman, Myron F; Woodgate, Roger

2014-03-28

Accurate DNA synthesis in vivo depends on the ability of DNA polymerases to select dNTPs from a nucleotide pool dominated by NTPs. High fidelity replicative polymerases have evolved to efficiently exclude NTPs while copying long stretches of undamaged DNA. However, to bypass DNA damage, cells utilize specialized low fidelity polymerases to perform translesion DNA synthesis (TLS). Of interest is human DNA polymerase ι (pol ι), which has been implicated in TLS of oxidative and UV-induced lesions. Here, we evaluate the ability of pol ι to incorporate NTPs during DNA synthesis. pol ι incorporates and extends NTPs opposite damaged and undamaged template bases in a template-specific manner. The Y39A "steric gate" pol ι mutant is considerably more active in the presence of Mn(2+) compared with Mg(2+) and exhibits a marked increase in NTP incorporation and extension, and surprisingly, it also exhibits increased dNTP base selectivity. Our results indicate that a single residue in pol ι is able to discriminate between NTPs and dNTPs during DNA synthesis. Because wild-type pol ι incorporates NTPs in a template-specific manner, certain DNA sequences may be "at risk" for elevated mutagenesis during pol ι-dependent TLS. Molecular modeling indicates that the constricted active site of wild-type pol ι becomes more spacious in the Y39A variant. Therefore, the Y39A substitution not only permits incorporation of ribonucleotides but also causes the enzyme to favor faithful Watson-Crick base pairing over mutagenic configurations.

Trigger Factor can antagonize both SecB and DnaK/DnaJ chaperone functions in Escherichia coli

PubMed Central

Ullers, Ronald S.; Ang, Debbie; Schwager, Françoise; Georgopoulos, Costa; Genevaux, Pierre

2007-01-01

Polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytoplasmic, integral membrane, or exported proteins. In Escherichia coli, the chaperones SecB, Trigger Factor (TF), and DnaK are key players in this process. Here, we report that, as with dnaK or dnaJ mutants, a secB null strain exhibits a strong cold-sensitive (Cs) phenotype. Through suppressor analyses, we found that inactivating mutations in the tig gene encoding TF fully relieve both the Cs phenotype and protein aggregation observed in the absence of SecB. This antagonistic effect of TF depends on its ribosome-binding and chaperone activities but unrelated to its peptidyl-prolyl cis/trans isomerase (PPIase) activity. Furthermore, in contrast to the previously known synergistic action of TF and DnaK/DnaJ above 30°C, a tig null mutation partially suppresses the Cs phenotype exhibited by a compromised DnaK/DnaJ chaperone machine. The antagonistic role of TF is further exemplified by the fact that the secB dnaJ double mutant is viable only in the absence of TF. Finally, we show that, in the absence of TF, more SecA and ribosomes are associated with the inner membrane, suggesting that the presence of TF directly or indirectly interferes with the process of cotranslational protein targeting to the Sec translocon. PMID:17360615

Multicenter evaluation of a commercial multiplex polymerase chain reaction test for screening plasma donations for parvovirus B19 DNA and hepatitis A virus RNA.

PubMed

Koppelman, Marco H G M; Cuijpers, H Theo M; Wessberg, Susanna; Valkeajärvi, Anne; Pichl, Lutz; Schottstedt, Volkmar; Saldanha, John

2012-07-01

Three European laboratories evaluated the TaqScreen DPX test (DPX test), a multiplex nucleic acid test assay for the simultaneous detection and quantitation of parvovirus B19 (B19V) DNA and the detection of hepatitis A virus (HAV) RNA. The 95% limit of detection of the test for B19V and HAV was determined using the respective WHO International Standards. The reproducibility of the test was evaluated by testing replicate samples of B19V at log 4.0 and 40 IU/mL and HAV at 5 IU/mL. The accuracy of the DPX test for B19V was evaluated by replicate testing of B19V samples containing log 3.0, log 4.0, and log 5.0 IU/mL. Panels of B19V Genotypes 1, 2, and 3 and HAV genotypes were evaluated. Cross-contamination was evaluated. For comparison of the DPX test and the established tests, the sites tested plasma samples in pools of either 96 or 480 donations. The mean 95% lower limits of detection of the three laboratories for B19V and HAV were 20.30 and 1.85 IU/mL. The test showed good reproducibility with the major part of the variance of the test being attributed to intermediate assay variation. The test showed great accuracy for B19V, especially at log 4.0 IU/mL. Spiking of test pools of 480 donations and manufacturing pools with log 4.0 IU/mL B19 DNA and 4 IU/mL HAV RNA showed that the DPX assay was robust. The test was able to detect the three genotypes of B19V and HAV genotypes. No cross-contamination was seen. Test results of routine samples correlated well with those of the established tests. The DPX test is a robust and sensitive test for the detection of B19V and HAV in plasma samples. The quantitative B19V results obtained with the test are accurate, and the test is able to detect all the known genotypes of B19V and HAV and fulfills all the European Pharmacopoeia and Food and Drug Administration requirements for a B19V and HAV test for screening of plasma donations and samples from plasma pools for manufacture. © 2012 American Association of Blood Banks.

The combination of dimethoxycurcumin with DNA methylation inhibitor enhances gene re-expression of promoter-methylated genes and antagonizes their cytotoxic effect

PubMed Central

Hassan, Hazem E.; Keita, Jean-Arnaud; Narayan, Lawrence; Brady, Sean M.; Frederick, Richard; Carlson, Samuel; C. Glass, Karen; Natesan, Senthil; Buttolph, Thomm; Fandy, Tamer E.

2016-01-01

ABSTRACT Curcumin and its analogs exhibited antileukemic activity either as single agent or in combination therapy. Dimethoxycurcumin (DMC) is a more metabolically stable curcumin analog that was shown to induce the expression of promoter-methylated genes without reversing DNA methylation. Accordingly, co-treatment with DMC and DNA methyltransferase (DNMT) inhibitors could hypothetically enhance the re-expression of promoter-methylated tumor suppressor genes. In this study, we investigated the cytotoxic effects and epigenetic changes associated with the combination of DMC and the DNMT inhibitor decitabine (DAC) in primary leukemia samples and cell lines. The combination demonstrated antagonistic cytotoxic effects and was minimally cytotoxic to primary leukemia cells. The combination did not affect the metabolic stability of DMC. Although the combination enhanced the downregulation of nuclear DNMT proteins, the hypomethylating activity of the combination was not increased significantly compared to DAC alone. On the other hand, the combination significantly increased H3K27 acetylation (H3K27Ac) compared to the single agents near the promoter region of promoter-methylated genes. Furthermore, sequential chromatin immunoprecipitation (ChIP) and DNA pyrosequencing of the chromatin-enriched H3K27Ac did not show any significant decrease in DNA methylation compared to other regions. Consequently, the enhanced induction of promoter-methylated genes by the combination compared to DAC alone is mediated by a mechanism that involves increased histone acetylation and not through potentiation of the DNA hypomethylating activity of DAC. Collectively, our results provide the mechanistic basis for further characterization of this combination in leukemia animal models and early phase clinical trials. PMID:27588609

Singular and combined effects of blowdown, salvage logging, and wildfire on forest floor and soil mercury pools

Treesearch

Carl P.J. Mitchell; Randall K. Kolka; Shawn Fraver

2012-01-01

A number of factors influence the amount of mercury (Hg) in forest floors and soils, including deposition, volatile emission, leaching, and disturbances such as fire. Currently the impact on soil Hg pools from other widespread forest disturbances such as blowdown and management practices like salvage logging are unknown. Moreover, ecological and biogeochemical...

Discussions on the Design of the Pool Landscape in the Rain Garden Construction

NASA Astrophysics Data System (ADS)

Zou, Shuzhen; Zhu, Yirong; Wei, Chaojun; Tao, Biaohong

2018-03-01

With rapid urbanization, the environmental problems are becoming increasingly prominent and diversified ecological landscape designs consequently appear with the rain garden landscape design as a typical. Based on the introduction to rain garden ecological functions and in combination with domestic and international rain garden landscape design cases, this paper discussed the rain garden pool landscape design.

DIVA V2.0

DOE Office of Scientific and Technical Information (OSTI.GOV)

CHEN, JOANNA; SIMIRENKO, LISA; TAPASWI, MANJIRI

The DIVA software interfaces a process in which researchers design their DNA with a web-based graphical user interface, submit their designs to a central queue, and a few weeks later receive their sequence-verified clonal constructs. Each researcher independently designs the DNA to be constructed with a web-based BioCAD tool, and presses a button to submit their designs to a central queue. Researchers have web-based access to their DNA design queues, and can track the progress of their submitted designs as they progress from "evaluation", to "waiting for reagents", to "in progress", to "complete". Researchers access their completed constructs through themore » central DNA repository. Along the way, all DNA construction success/failure rates are captured in a central database. Once a design has been submitted to the queue, a small number of dedicated staff evaluate the design for feasibility and provide feedback to the responsible researcher if the design is either unreasonable (e.g., encompasses a combinatorial library of a billion constructs) or small design changes could significantly facilitate the downstream implementation process. The dedicated staff then use DNA assembly design automation software to optimize the DNA construction process for the design, leveraging existing parts from the DNA repository where possible and ordering synthetic DNA where necessary. SynTrack software manages the physical locations and availability of the various requisite reagents and process inputs (e.g., DNA templates). Once all requisite process inputs are available, the design progresses from "waiting for reagents" to "in progress" in the design queue. Human-readable and machine-parseable DNA construction protocols output by the DNA assembly design automation software are then executed by the dedicated staff exploiting lab automation devices wherever possible. Since the all employed DNA construction methods are sequence-agnostic, standardized (utilize the same enzymatic master mixes and reaction conditions), completely independent DNA construction tasks can be aggregated into the same multi-well plates and pursued in parallel. The resulting sets of cloned constructs can then be screened by high-throughput next-gen sequencing platforms for sequence correctness. A combination of long read-length (e.g., PacBio) and paired-end read platforms (e.g., Illumina) would be exploited depending the particular task at hand (e.g., PacBio might be sufficient to screen a set of pooled constructs with significant gene divergence). Post sequence verification, designs for which at least one correct clone was identified will progress to a "complete" status, while designs for which no correct clones wereidentified will progress to a "failure" status. Depending on the failure mode (e.g., no transformants), and how many prior attempts/variations of assembly protocol have been already made for a given design, subsequent attempts may be made or the design can progress to a "permanent failure" state. All success and failure rate information will be captured during the process, including at which stage a given clonal construction procedure failed (e.g., no PCR product) and what the exact failure was (e.g. assembly piece 2 missing). This success/failure rate data can be leveraged to refine the DNA assembly design process.« less

Rates of Spontaneous Mutation in Bacteriophage T4 Are Independent of Host Fidelity Determinants

PubMed Central

Santos, M. E.; Drake, J. W.

1994-01-01

Bacteriophage T4 encodes most of the genes whose products are required for its DNA metabolism, and host (Escherichia coli) genes can only infrequently complement mutationally inactivated T4 genes. We screened the following host mutator mutations for effects on spontaneous mutation rates in T4: mutT (destruction of aberrant dGTPs), polA, polB and polC (DNA polymerases), dnaQ (exonucleolytic proofreading), mutH, mutS, mutL and uvrD (methyl-directed DNA mismatch repair), mutM and mutY (excision repair of oxygen-damaged DNA), mutA (function unknown), and topB and osmZ (affecting DNA topology). None increased T4 spontaneous mutation rates within a resolving power of about twofold (nor did optA, which is not a mutator but overexpresses a host dGTPase). Previous screens in T4 have revealed strong mutator mutations only in the gene encoding the viral DNA polymerase and proofreading 3'-exonuclease, plus weak mutators in several polymerase accessory proteins or determinants of dNTP pool sizes. T4 maintains a spontaneous mutation rate per base pair about 30-fold greater than that of its host. Thus, the joint high fidelity of insertion by T4 DNA polymerase and proofreading by its associated 3'-exonuclease appear to determine the T4 spontaneous mutation rate, whereas the host requires numerous additional systems to achieve high replication fidelity. PMID:7851754

DNA damage in an animal model of maple syrup urine disease.

PubMed

Scaini, Giselli; Jeremias, Isabela C; Morais, Meline O S; Borges, Gabriela D; Munhoz, Bruna P; Leffa, Daniela D; Andrade, Vanessa M; Schuck, Patrícia F; Ferreira, Gustavo C; Streck, Emilio L

2012-06-01

Maple syrup urine disease is an inborn error of metabolism caused by a severe deficiency of the branched chain alpha-ketoacid dehydrogenase complex. Neurological dysfunction is a common finding in patients with maple syrup urine disease. However, the mechanisms underlying the neuropathology of brain damage in this disorder are poorly understood. In this study, we investigated whether acute or chronic administration of a branched chain amino acid pool (leucine, isoleucine and valine) causes transient DNA damage, as determined by the alkaline comet assay, in the brain and blood of rats during development and whether antioxidant treatment prevented the alterations induced by branched chain amino acids. Our results showed that the acute administration of branched chain amino acids increased the DNA damage frequency and damage index in the hippocampus. However, the chronic administration of branched chain amino acids increased the DNA damage frequency and damage index in both the hippocampus and the striatum, and the antioxidant treatment was able to prevent DNA damage in the hippocampus and striatum. The present study demonstrated that metabolite accumulation in MSUD induces DNA damage in the hippocampus and striatum and that it may be implicated in the neuropathology observed in the affected patients. We demonstrated that the effect of antioxidant treatment (N-acetylcysteine plus deferoxamine) prevented DNA damage, suggesting the involvement of oxidative stress in DNA damage. Copyright © 2012 Elsevier Inc. All rights reserved.

Synergistic effect of ultrasound and PEI on DNA transfection in vitro

PubMed Central

Deshpande, Mangesh C.; Prausnitz, Mark R.

2007-01-01

Ultrasound and poly(ethylenimine) (PEI) have each separately been shown to increase DNA transfection efficiency. This study tested the hypothesis that the combination of ultrasound and PEI can have a synergistic effect to increase DNA transfection. This in vitro study assessed transfection efficiency of two different DNA plasmids encoding green fluorescent protein and firefly luciferase in two different cells types, a primary culture of human aortic smooth muscle cells and an immortal line of human prostrate cancer cells. We found that ultrasound sonication increased transfection up to 18-fold, DNA complexation with PEI increased transfection up to 90-fold, and the combination of ultrasound and PEI synergistically increased transfection up to 200-fold, which resulted in reporter gene expression by 34% of cells. Kinetic measurements found that the effects of ultrasound alone acted quickly, whereas increased transfection by PEI either alone or in combination with ultrasound strongly benefited from a 4-h incubation with the DNA plasmid after sonication. Although serum reduced absolute expression levels, it did not affect the relative increase in transfection when ultrasound was added to PEI enhancement. Flow cytometry measurements showed that sonication increased intracellular uptake of labelled DNA complexed to PEI by 55% relative to PEI complexation alone. Electrophoresis assay showed no damage to DNA or PEI-DNA complexes after sonication. Overall, these results suggest that the combination of ultrasound and PEI can have a synergistic effect to increase DNA transfection. PMID:17258835

Checkpoint kinase 1 inhibition sensitises transformed cells to dihydroorotate dehydrogenase inhibition

PubMed Central

Arnould, Stéphanie; Rodier, Geneviève; Matar, Gisèle; Vincent, Charles; Pirot, Nelly; Delorme, Yoann; Berthet, Charlène; Buscail, Yoan; Noël, Jean Yohan; Lachambre, Simon; Jarlier, Marta; Bernex, Florence; Delpech, Hélène; Vidalain, Pierre Olivier; Janin, Yves L.; Theillet, Charles; Sardet, Claude

2017-01-01

Reduction in nucleotide pools through the inhibition of mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) has been demonstrated to effectively reduce cancer cell proliferation and tumour growth. The current study sought to investigate whether this antiproliferative effect could be enhanced by combining Chk1 kinase inhibition. The pharmacological activity of DHODH inhibitor teriflunomide was more selective towards transformed mouse embryonic fibroblasts than their primary or immortalised counterparts, and this effect was amplified when cells were subsequently exposed to PF477736 Chk1 inhibitor. Flow cytometry analyses revealed substantial accumulations of cells in S and G2/M phases, followed by increased cytotoxicity which was characterised by caspase 3-dependent induction of cell death. Associating PF477736 with teriflunomide also significantly sensitised SUM159 and HCC1937 human triple negative breast cancer cell lines to dihydroorotate dehydrogenase inhibition. The main characteristic of this effect was the sustained accumulation of teriflunomide-induced DNA damage as cells displayed increased phospho serine 139 H2AX (γH2AX) levels and concentration-dependent phosphorylation of Chk1 on serine 345 upon exposure to the combination as compared with either inhibitor alone. Importantly a similar significant increase in cell death was observed upon dual siRNA mediated depletion of Chk1 and DHODH in both murine and human cancer cell models. Altogether these results suggest that combining DHODH and Chk1 inhibitions may be a strategy worth considering as a potential alternative to conventional chemotherapies. PMID:29221122

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia

PubMed Central

Martin, Jaclyn; Carrillo, Yisel; Talley, Justin L.; Ochoa-Corona, Francisco M.

2018-01-01

Background The importance of tick and flea-borne rickettsia infections is increasingly recognized worldwide. While increased focus has shifted in recent years to the development of point-of-care diagnostics for various vector-borne diseases in humans and animals, little research effort has been devoted to their integration into vector surveillance and control programs, particularly in resource-challenged countries. One technology which may be helpful for large scale vector surveillance initiatives is loop-mediated isothermal amplification (LAMP). The aim of this study was to develop a LAMP assay to detect spotted fever group (SFG) rickettsia DNA from field-collected ticks and fleas and compare with published end-point PCR results. Methodology/Principal findings A Spotted Fever Group rickettsia-specific loop-mediated isothermal amplification (SFGR-LAMP) assay was developed using primers based on a region of the R. rickettsii 17kDa protein gene. The sensitivity, specificity, and reproducibility of the assay were evaluated. The assay was then compared with the results of end-point PCR assays for pooled tick and flea samples obtained from field-based surveillance studies. The sensitivity of the SFGR-LAMP assay was 0.00001 ng/μl (25μl volume) which was 10 times more sensitive than the 17kDa protein gene end-point PCR used as the reference method. The assay only recognized gDNA from SFG and transitional group (TRG) rickettsia species tested but did not detect gDNA from typhus group (TG) rickettsia species or closely or distantly related bacterial species. The SFGR-LAMP assay detected the same positives from a set of pooled tick and flea samples detected by end-point PCR in addition to two pooled flea samples not detected by end-point PCR. Conclusions/significance To our knowledge, this is the first study to develop a functional LAMP assay to initially screen for SFG and TRG rickettsia pathogens in field-collected ticks and fleas. With a high sensitivity and specificity, the results indicate the potential use as a field-based surveillance tool for tick and flea-borne rickettsial pathogens in resource-challenged countries. PMID:29390021

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia.

PubMed

Noden, Bruce H; Martin, Jaclyn; Carrillo, Yisel; Talley, Justin L; Ochoa-Corona, Francisco M

2018-01-01

The importance of tick and flea-borne rickettsia infections is increasingly recognized worldwide. While increased focus has shifted in recent years to the development of point-of-care diagnostics for various vector-borne diseases in humans and animals, little research effort has been devoted to their integration into vector surveillance and control programs, particularly in resource-challenged countries. One technology which may be helpful for large scale vector surveillance initiatives is loop-mediated isothermal amplification (LAMP). The aim of this study was to develop a LAMP assay to detect spotted fever group (SFG) rickettsia DNA from field-collected ticks and fleas and compare with published end-point PCR results. A Spotted Fever Group rickettsia-specific loop-mediated isothermal amplification (SFGR-LAMP) assay was developed using primers based on a region of the R. rickettsii 17kDa protein gene. The sensitivity, specificity, and reproducibility of the assay were evaluated. The assay was then compared with the results of end-point PCR assays for pooled tick and flea samples obtained from field-based surveillance studies. The sensitivity of the SFGR-LAMP assay was 0.00001 ng/μl (25μl volume) which was 10 times more sensitive than the 17kDa protein gene end-point PCR used as the reference method. The assay only recognized gDNA from SFG and transitional group (TRG) rickettsia species tested but did not detect gDNA from typhus group (TG) rickettsia species or closely or distantly related bacterial species. The SFGR-LAMP assay detected the same positives from a set of pooled tick and flea samples detected by end-point PCR in addition to two pooled flea samples not detected by end-point PCR. To our knowledge, this is the first study to develop a functional LAMP assay to initially screen for SFG and TRG rickettsia pathogens in field-collected ticks and fleas. With a high sensitivity and specificity, the results indicate the potential use as a field-based surveillance tool for tick and flea-borne rickettsial pathogens in resource-challenged countries.

Accuracy of non-invasive prenatal testing using cell-free DNA for detection of Down, Edwards and Patau syndromes: a systematic review and meta-analysis.

PubMed

Taylor-Phillips, Sian; Freeman, Karoline; Geppert, Julia; Agbebiyi, Adeola; Uthman, Olalekan A; Madan, Jason; Clarke, Angus; Quenby, Siobhan; Clarke, Aileen

2016-01-18

To measure test accuracy of non-invasive prenatal testing (NIPT) for Down, Edwards and Patau syndromes using cell-free fetal DNA and identify factors affecting accuracy. Systematic review and meta-analysis of published studies. PubMed, Ovid Medline, Ovid Embase and the Cochrane Library published from 1997 to 9 February 2015, followed by weekly autoalerts until 1 April 2015. English language journal articles describing case-control studies with ≥ 15 trisomy cases or cohort studies with ≥ 50 pregnant women who had been given NIPT and a reference standard. 41, 37 and 30 studies of 2012 publications retrieved were included in the review for Down, Edwards and Patau syndromes. Quality appraisal identified high risk of bias in included studies, funnel plots showed evidence of publication bias. Pooled sensitivity was 99.3% (95% CI 98.9% to 99.6%) for Down, 97.4% (95.8% to 98.4%) for Edwards, and 97.4% (86.1% to 99.6%) for Patau syndrome. The pooled specificity was 99.9% (99.9% to 100%) for all three trisomies. In 100,000 pregnancies in the general obstetric population we would expect 417, 89 and 40 cases of Downs, Edwards and Patau syndromes to be detected by NIPT, with 94, 154 and 42 false positive results. Sensitivity was lower in twin than singleton pregnancies, reduced by 9% for Down, 28% for Edwards and 22% for Patau syndrome. Pooled sensitivity was also lower in the first trimester of pregnancy, in studies in the general obstetric population, and in cohort studies with consecutive enrolment. NIPT using cell-free fetal DNA has very high sensitivity and specificity for Down syndrome, with slightly lower sensitivity for Edwards and Patau syndrome. However, it is not 100% accurate and should not be used as a final diagnosis for positive cases. CRD42014014947. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Quantitative species-level ecology of reef fish larvae via metabarcoding.

PubMed

Kimmerling, Naama; Zuqert, Omer; Amitai, Gil; Gurevich, Tamara; Armoza-Zvuloni, Rachel; Kolesnikov, Irina; Berenshtein, Igal; Melamed, Sarah; Gilad, Shlomit; Benjamin, Sima; Rivlin, Asaph; Ohavia, Moti; Paris, Claire B; Holzman, Roi; Kiflawi, Moshe; Sorek, Rotem

2018-02-01

The larval pool of coral reef fish has a crucial role in the dynamics of adult fish populations. However, large-scale species-level monitoring of species-rich larval pools has been technically impractical. Here, we use high-throughput metabarcoding to study larval ecology in the Gulf of Aqaba, a region that is inhabited by >500 reef fish species. We analysed 9,933 larvae from 383 samples that were stratified over sites, depth and time. Metagenomic DNA extracted from pooled larvae was matched to a mitochondrial cytochrome c oxidase subunit I barcode database compiled for 77% of known fish species within this region. This yielded species-level reconstruction of the larval community, allowing robust estimation of larval spatio-temporal distributions. We found significant correlations between species abundance in the larval pool and in local adult assemblages, suggesting a major role for larval supply in determining local adult densities. We documented larval flux of species whose adults were never documented in the region, suggesting environmental filtering as the reason for the absence of these species. Larvae of several deep-sea fishes were found in shallow waters, supporting their dispersal over shallow bathymetries, potentially allowing Lessepsian migration into the Mediterranean Sea. Our method is applicable to any larval community and could assist coral reef conservation and fishery management efforts.

Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch.

PubMed

Poulain, Adeline; Perret, Sylvie; Malenfant, Félix; Mullick, Alaka; Massie, Bernard; Durocher, Yves

2017-08-10

To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHO BRI/rcTA ) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHO BRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHO BRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Can soy intake affect serum uric acid level? Pooled analysis from two 6-month randomized controlled trials among Chinese postmenopausal women with prediabetes or prehypertension.

PubMed

Liu, Z M; Ho, C S; Chen, Y M; Woo, J

2015-02-01

Hyperuricemia is a recognized risk factor for cardiovascular diseases. Soy foods contain a moderate amount of purine and may predispose to raised serum uric acid (UA). However, no study has examined the long-term effect of soy intake on UA levels. We examined whether consumption of soy foods and isoflavone extracts for 6 months altered serum UA. The analysis included two randomized controlled trials (soy protein trial and whole soy trial) among total 450 postmenopausal women with either prehypertension or prediabetes. We conducted a pooled analysis by combining participants from both the soy flour and soy protein groups (combined soy foods group), participants from both the isoflavone and daidzein groups (combined isoflavone group) and participants from both milk placebo groups. Fasting venous samples were obtained at baseline and the end of the trial for serum UA analysis. In the pooled data, 417 subjects completed the study according to protocol. The baseline serum UA levels were comparable among the three combined groups. There was a lower decrease in UA levels among women in the combined soy foods group compared with women in the other two groups (p = 0.028 and 0.026). The net decrease and % decrease in UA were 14.5 μmol/L (95 % CI 1.93-25.6, p = 0.023) or 4.9 % (95 % CI 1.3-8.5 %, p = 0.023) between the combined soy foods group and placebo group. Among Chinese postmenopausal women with either prehypertension or prediabetes, soy intake did not increase urate levels.

Cell-free DNA testing in the maternal blood in high-risk pregnancies after first-trimester combined screening.

PubMed

Persico, Nicola; Boito, Simona; Ischia, Benedetta; Cordisco, Adalgisa; De Robertis, Valentina; Fabietti, Isabella; Periti, Enrico; Volpe, Paolo; Fedele, Luigi; Rembouskos, Georgios

2016-03-01

The objective of this study was to investigate a strategy for clinical implementation of cell-free DNA (cfDNA) testing in high-risk pregnancies after first-trimester combined screening. In 259 singleton pregnancies undergoing invasive testing after first-trimester combined screening, a maternal blood sample was sent to the laboratory Natera for cfDNA testing using a single-nucleotide polymorphism-based methodology. The cfDNA test provided a result in 249 (96.1%) pregnancies and, among these, identified as being at high risk 35 of 36 cases of trisomy 21, 13 of 13 with trisomy 18, five of five with trisomy 13 and three of four with sex chromosome aneuploidies. A policy of performing an invasive test in women with a combined risk of ≥1 in 10 or NT ≥4 mm and offering cfDNA testing to the remaining cases would detect all cases of trisomy 21, 18 or 13, 80% of sex aneuploidies and 62.5% of other defects and would avoid an invasive procedure in 82.4% of euploid fetuses. In high-risk pregnancies after combined screening, a policy of selecting a subgroup for invasive testing and another for cfDNA testing would substantially reduce the number of invasive procedures and retain the ability to diagnose most of the observed aneuploidies. © 2016 John Wiley & Sons, Ltd.

Improved EGFR mutation detection using combined exosomal RNA and circulating tumor DNA in NSCLC patient plasma

PubMed Central

Krug, A K; Enderle, D; Karlovich, C; Priewasser, T; Bentink, S; Spiel, A; Brinkmann, K; Emenegger, J; Grimm, D G; Castellanos-Rizaldos, E; Goldman, J W; Sequist, L V; Soria, J -C; Camidge, D R; Gadgeel, S M; Wakelee, H A; Raponi, M; Noerholm, M; Skog, J

2018-01-01

Abstract Background A major limitation of circulating tumor DNA (ctDNA) for somatic mutation detection has been the low level of ctDNA found in a subset of cancer patients. We investigated whether using a combined isolation of exosomal RNA (exoRNA) and cell-free DNA (cfDNA) could improve blood-based liquid biopsy for EGFR mutation detection in non-small-cell lung cancer (NSCLC) patients. Patients and methods Matched pretreatment tumor and plasma were collected from 84 patients enrolled in TIGER-X (NCT01526928), a phase 1/2 study of rociletinib in mutant EGFR NSCLC patients. The combined isolated exoRNA and cfDNA (exoNA) was analyzed blinded for mutations using a targeted next-generation sequencing panel (EXO1000) and compared with existing data from the same samples using analysis of ctDNA by BEAMing. Results For exoNA, the sensitivity was 98% for detection of activating EGFR mutations and 90% for EGFR T790M. The corresponding sensitivities for ctDNA by BEAMing were 82% for activating mutations and 84% for T790M. In a subgroup of patients with intrathoracic metastatic disease (M0/M1a; n = 21), the sensitivity increased from 26% to 74% for activating mutations (P = 0.003) and from 19% to 31% for T790M (P = 0.5) when using exoNA for detection. Conclusions Combining exoRNA and ctDNA increased the sensitivity for EGFR mutation detection in plasma, with the largest improvement seen in the subgroup of M0/M1a disease patients known to have low levels of ctDNA and poses challenges for mutation detection on ctDNA alone. Clinical Trials NCT01526928 PMID:29216356

Plant cell nucleolus as a hot spot for iron.

PubMed

Roschzttardtz, Hannetz; Grillet, Louis; Isaure, Marie-Pierre; Conéjéro, Geneviève; Ortega, Richard; Curie, Catherine; Mari, Stéphane

2011-08-12

Many central metabolic processes require iron as a cofactor and take place in specific subcellular compartments such as the mitochondrion or the chloroplast. Proper iron allocation in the different organelles is thus critical to maintain cell function and integrity. To study the dynamics of iron distribution in plant cells, we have sought to identify the different intracellular iron pools by combining three complementary imaging approaches, histochemistry, micro particle-induced x-ray emission, and synchrotron radiation micro X-ray fluorescence. Pea (Pisum sativum) embryo was used as a model in this study because of its large cell size and high iron content. Histochemical staining with ferrocyanide and diaminobenzidine (Perls/diaminobenzidine) strongly labeled a unique structure in each cell, which co-labeled with the DNA fluorescent stain DAPI, thus corresponding to the nucleus. The unexpected presence of iron in the nucleus was confirmed by elemental imaging using micro particle-induced x-ray emission. X-ray fluorescence on cryo-sectioned embryos further established that, quantitatively, the iron concentration found in the nucleus was higher than in the expected iron-rich organelles such as plastids or vacuoles. Moreover, within the nucleus, iron was particularly accumulated in a subcompartment that was identified as the nucleolus as it was shown to transiently disassemble during cell division. Taken together, our data uncover an as yet unidentified although abundant iron pool in the cell, which is located in the nuclei of healthy, actively dividing plant tissues. This result paves the way for the discovery of a novel cellular function for iron related to nucleus/nucleolus-associated processes.

DNA migration mechanism analyses for applications in capillary and microchip electrophoresis

PubMed Central

Forster, Ryan E.; Hert, Daniel G.; Chiesl, Thomas N.; Fredlake, Christopher P.; Barron, Annelise E.

2009-01-01

In 2009, electrophoretically driven DNA separations in slab gels and capillaries have the sepia tones of an old-fashioned technology in the eyes of many, even while they remain ubiquitously used, fill a unique niche, and arguably have yet to reach their full potential. For comic relief, what is old becomes new again: agarose slab gel separations are used to prepare DNA samples for “next-gen” sequencing platforms (e.g., the Illumina and 454 machines)—dsDNA molecules within a certain size range are “cut out” of a gel and recovered for subsequent “massively parallel” pyrosequencing. In this review, we give a Barron lab perspective on how our comprehension of DNA migration mechanisms in electrophoresis has evolved, since the first reports of DNA separations by CE (∼1989) until now, 20 years later. Fused silica capillaries, and borosilicate glass and plastic microchips, quietly offer increasing capacities for fast (and even “ultra-fast”), efficient DNA separations. While the channel-by-channel scaling of both old and new electrophoresis platforms provides key flexibility, it requires each unique DNA sample to be prepared in its own micro- or nanovolume. This Achille's heel of electrophoresis technologies left an opening through which pooled-sample, next-gen DNA sequencing technologies rushed. We shall see, over time, whether sharpening understanding of transitions in DNA migration modes in crosslinked gels, nanogel solutions, and uncrosslinked polymer solutions will allow electrophoretic DNA analysis technologies to flower again. Microchannel electrophoresis, after a quiet period of metamorphosis, may emerge sleeker and more powerful, to claim its own important niche applications. PMID:19582705

Incorporating incorporating economic models into seasonal pool conservation planning

USGS Publications Warehouse

Freeman, Robert C.; Bell, Kathleen P.; Calhoun, Aram J.K.; Loftin, Cyndy

2012-01-01

Massachusetts, New Jersey, Connecticut, and Maine have adopted regulatory zones around seasonal (vernal) pools to conserve terrestrial habitat for pool-breeding amphibians. Most amphibians require access to distinct seasonal habitats in both terrestrial and aquatic ecosystems because of their complex life histories. These habitat requirements make them particularly vulnerable to land uses that destroy habitat or limit connectivity (or permeability) among habitats. Regulatory efforts focusing on breeding pools without consideration of terrestrial habitat needs will not ensure the persistence of pool-breeding amphibians. We used GIS to combine a discrete-choice, parcel-scale economic model of land conversion with a landscape permeability model based on known habitat requirements of wood frogs (Lithobates sylvaticus) in Maine (USA) to examine permeability among habitat elements for alternative future scenarios. The economic model predicts future landscapes under different subdivision open space and vernal pool regulatory requirements. Our model showed that even “no build” permit zones extending 76 m (250 ft) outward from the pool edge were insufficient to assure permeability among required habitat elements. Furthermore, effectiveness of permit zones may be inconsistent due to interactions with other growth management policies, highlighting the need for local and state planning for the long-term persistence of pool-breeding amphibians in developing landscapes.

The UNITAID Patent Pool Initiative: Bringing Patents Together for the Common Good

PubMed Central

Bermudez, Jorge; 't Hoen, Ellen

2010-01-01

Developing and delivering appropriate, affordable, well-adapted medicines for HIV/AIDS remains an urgent challenge: as first-line therapies fail, increasing numbers of people require costly second-line therapy; one-third of ARVs are not available in pediatric formulations; and certain key first- and second-line triple fixed-dose combinations do not exist or sufficient suppliers are lacking. UNITAID aims to help solve these problems through an innovative initiative for the collective management of intellectual property (IP) rights – a patent pool for HIV medicines. The idea behind a patent pool is that patent holders - companies, governments, researchers or universities - voluntarily offer, under certain conditions, the IP related to their inventions to the patent pool. Any company that wants to use the IP to produce or develop medicines can seek a license from the pool against the payment of royalties, and may then produce the medicines for use in developing countries (conditional upon meeting agreed quality standards). The patent pool will be a voluntary mechanism, meaning its success will largely depend on the willingness of pharmaceutical companies to participate and commit their IP to the pool. Generic producers must also be willing to cooperate. The pool has the potential to provide benefits to all. PMID:20309404

Random integration of SV40 in SV40-transformed, immortalized human fibroblasts.

PubMed

Hara, H; Kaji, H

1987-02-01

We have studied the relationship between immortalization of SV40-transformed human embryonic fibroblasts and their SV40 integration sites. From several independently transformed cell pools, we have isolated clones which do not harbor unintegrated SV40 DNA. We have analysed whole-cell DNA from these clones, using the Southern blot method. Our results suggest that no specific integration sites in the cellular genome exist which are a prerequisite for the immortalization process. Although some integration sites were found to be predominant in pre-crisis clones, they could not be detected in the post-crisis clones. This suggests that none of these predominating sites is selected for during the crisis period.

Integration of DNA barcoding for the initial recordings of Lessepsian fishes: a case study of the Indo-Pacific slender ponyfish Equulites elongatus.

PubMed

Sakinan, S; Karahan, A; Ok, M

2017-03-01

In this study, the DNA barcode of a regional Lessepsian sighting of the slender ponyfish Equulites elongatus is integrated with morphometric and meristic descriptors as a case study to address further identification problems in the Mediterranean Sea. The study also aims to contribute to the regional mitochondrial cytochrome oxidase I information pool, to support other potential uses. The initial sighting of E. elongatus from the north-eastern Mediterranean coast of Turkey is provided from a trawl survey on 3 June 2015, where 76 specimens were captured during a 15 min tow. © 2016 The Fisheries Society of the British Isles.

Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens

PubMed Central

2018-01-01

Many implementations of pooled screens in mammalian cells rely on linking an element of interest to a barcode, with the latter subsequently quantitated by next generation sequencing. However, substantial uncoupling between these paired elements during lentiviral production has been reported, especially as the distance between elements increases. We detail that PCR amplification is another major source of uncoupling, and becomes more pronounced with increased amounts of DNA template molecules and PCR cycles. To lessen uncoupling in systems that use paired elements for detection, we recommend minimizing the distance between elements, using low and equal template DNA inputs for plasmid and genomic DNA during PCR, and minimizing the number of PCR cycles. We also present a vector design for conducting combinatorial CRISPR screens that enables accurate barcode-based detection with a single short sequencing read and minimal uncoupling. PMID:29799876

Nanofluidic Device with Embedded Nanopore

NASA Astrophysics Data System (ADS)

Zhang, Yuning; Reisner, Walter

2014-03-01

Nanofluidic based devices are robust methods for biomolecular sensing and single DNA manipulation. Nanopore-based DNA sensing has attractive features that make it a leading candidate as a single-molecule DNA sequencing technology. Nanochannel based extension of DNA, combined with enzymatic or denaturation-based barcoding schemes, is already a powerful approach for genome analysis. We believe that there is revolutionary potential in devices that combine nanochannels with nanpore detectors. In particular, due to the fast translocation of a DNA molecule through a standard nanopore configuration, there is an unfavorable trade-off between signal and sequence resolution. With a combined nanochannel-nanopore device, based on embedding a nanopore inside a nanochannel, we can in principle gain independent control over both DNA translocation speed and sensing signal, solving the key draw-back of the standard nanopore configuration. We demonstrate that we can detect - using fluorescent microscopy - successful translocation of DNA from the nanochannel out through the nanopore, a possible method to 'select' a given barcode for further analysis. We also show that in equilibrium DNA will not escape through an embedded sub-persistence length nanopore until a certain voltage bias is added.

Meta-analysis of the predictive value of DNA aneuploidy in malignant transformation of oral potentially malignant disorders.

PubMed

Alaizari, Nader A; Sperandio, Marcelo; Odell, Edward W; Peruzzo, Daiane; Al-Maweri, Sadeq A

2018-02-01

DNA aneuploidy is an imbalance of chromosomal DNA content that has been highlighted as a predictor of biological behavior and risk of malignant transformation. To date, DNA aneuploidy in oral potentially malignant diseases (OPMD) has been shown to correlate strongly with severe dysplasia and high-risk lesions that appeared non-dysplastic can be identified by ploidy analysis. Nevertheless, the prognostic value of DNA aneuploidy in predicting malignant transformation of OPMD remains to be validated. The aim of this meta-analysis was to assess the role of DNA aneuploidy in predicting malignant transformation in OPMD. The questions addressed were (i) Is DNA aneuploidy a useful marker to predict malignant transformation in OPMD? (ii) Is DNA diploidy a useful negative marker of malignant transformation in OPMD? These questions were addressed using the PECO method. Five studies assessing aneuploidy as a risk marker of malignant change were pooled into the meta-analysis. Aneuploidy was found to be associated with a 3.12-fold increased risk to progress into cancer (RR=3.12, 95% CI 1.86-5.24). Based on the five studies meta-analyzed, "no malignant progression" was more likely to occur in DNA diploid OPMD by 82% when compared to aneuploidy (RR=0.18, 95% CI 0.08-0.41). In conclusion, aneuploidy is a useful marker of malignant transformation in OPMD, although a diploid result should be interpreted with caution. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The response of ecosystem carbon pools to management approaches in loblolly pine (Pinus taeda L.) plantations

NASA Astrophysics Data System (ADS)

Vogel, J. G.; Bacon, A. R.; Bracho, R. G.; Gonzalez-Benecke, C. A.; Fox, T. D.; Laviner, M. A.; Kane, M.; Burkhart, H.; Martin, T.; Will, R.; Ross, C. W.; Grunwald, S.; Jokela, E. J.; Meek, C.

2016-12-01

Extending from Virginia to east Texas in the southeastern United States, managed pine plantations are an important component of the region's carbon cycle. An objective of the Pine Integrated Network: Education, Mitigation, and Adaptation project (PINEMAP) is to improve estimates of how ecosystem carbon pools respond to the management strategies used to increase the growth of loblolly pine plantations. Experimental studies (108 total) that have been used to understand plantation productivity and stand dynamics by university-forest industry cooperatives were measured for the carbon stored in the trees, roots, coarse-wood, detritus in soil, forest floor, understory and soils to 1-meter. The age of the studied plantations ranged from 4-26 years at the time of sampling, with 26 years very near the period when these plantations are commonly harvested. Across all study sites, 455 experimental plots were measured. The average C storage across all pools, sites, and treatments was 192 Mg C ha-1, with the average percentage of the total coming from soil (44%), tree biomass (40%), forest floor (8%), root (5%), soil detritus (2%), understory biomass (1%), and coarse-wood (<1%) pools. Plots had as a treatment either fertilization, competition control, and stand density control (thinning), and every possible combination of treatments including `no treatment'. A paired plot analysis was used where two plots at a site were examined for relative differences caused by a single treatment and these differences averaged across the region. Thinning as a stand-alone treatment significantly reduced forest floor mass by 60%, and the forest floor in the thinned plus either competition control or fertilization was 18.9% and 19.2% less, respectively, than unthinned stands combined with the same treatments. Competition control increased C storage in tree biomass by 12% and thinning decreased tree biomass by 32%. Thinning combined with fertilization had lower soil carbon (0-1 m) than unthinned-fertilized plots (22%), although the replication for this combination was relatively low (n=6). Overall these results suggest that maintaining higher tree densities increases ecosystem carbon storage across multiple pools of C in loblolly pine plantations.

Upregulated Genes In Sporadic, Idiopathic Pulmonary Arterial Hypertension

PubMed Central

Edgar, Alasdair J; Chacón, Matilde R; Bishop, Anne E; Yacoub, Magdi H; Polak, Julia M

2006-01-01

Background To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by suppression subtractive hybridisation (SSH). Methods Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test. Results We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase. Conclusion Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively. PMID:16390543

The congruence between matrilineal genetic (mtDNA) and geographic diversity of Iranians and the territorial populations

PubMed Central

Bahmanimehr, Ardeshir; Eskandari, Ghafar; Nikmanesh, Fatemeh

2015-01-01

Objective(s): From the ancient era, emergence of Agriculture in the connecting region of Mesopotamia and the Iranian plateau at the foothills of the Zagros Mountains, made Iranian gene pool as an important source of populating the region. It has differentiated the population spread and different language groups. In order to trace the maternal genetic affinity between Iranians and other populations of the area and to establish the place of Iranians in a broad framework of ethnically and linguistically diverse groups of Middle Eastern and South Asian populations, a comparative study of territorial groups was designed and used in the population statistical analysis. Materials and Methods: Mix of 616 samples was sequenced for complete mtDNA or hyper variable regions in this study. A published dataset of neighboring populations was used as a comparison in the Iranian matrilineal lineage study based on mtDNA haplogroups. Results: Statistical analyses data, demonstrate a close genetic structure of all Iranian populations, thus suggesting their origin from a common maternal ancestral gene pool and show that the diverse maternal genetic structure does not reflect population differentiation in the region in their language. Conclusion: In the aggregate of the eastward spreads of proto-Elamo-Dravidian language from the Southwest region of Iran, the Elam province, a reasonable degree of homogeneity has been observed among Iranians in this study. The approach will facilitate our perception of the more detailed relationship of the ethnic groups living in Iran with the other ancient peoples of the area, testing linguistic hypothesis and population movements. PMID:25810873

Coxiella burnetii and Rickettsia conorii: Two zoonotic pathogens in peridomestic rodents and their ectoparasites in Nigeria.

PubMed

Kamani, Joshua; Baneth, Gad; Gutiérrez, Ricardo; Nachum-Biala, Yaarit; Mumcuoglu, Kosta Y; Harrus, Shimon

2018-01-01

Rodents are hosts of numerous pathogenic agents of public health importance globally. Their ability to harbor these pathogens without showing overt clinical signs of disease has epidemiologic consequences. In some rural settings in Nigeria, humans and rodents do not only share feeds and abode, but the latter may end up on the table of the former as a source of protein, thereby increasing the risks of disease transmission. Molecular assays were used to detect and characterize two agents of zoonotic importance, Coxiella burnetii and Rickettsia spp. in 194 peridomestic rodents captured in a peri-urban setting in Nigeria, and 32 pools of ectoparasites removed from them, to determine their possible role in the epidemiology of these diseases in this country. Targeting and characterizing the insertion sequence IS1111, C. burnetii DNA was detected in 4 out of 194 (2.1%) rodents comprising 3 out of 121 (2.5%) Rattus norvegicus and 1 out of 48 (2.1%) Rattus rattus screened in this study. Rickettsia spp. DNA was detected in two Rhipicephalus sanginueus sensu lato pools (i.e. RT1 and RT4) using the citrate synthase (gltA) gene and further characterized by amplification and sequence analysis of six genes to determine their identity. The RT1 sample consistently gave 98-100% identity to Rickettsia conorii str. Malish 7 for the various genes and loci studied. However, the identity of RT4 could not be definitively determined due to variable identities to different Rickettsia spp. according to the gene or loci under consideration. Further isolation study to determine if the RT4 characterized is a new variant or a mixture of sequences of different rickettsiae within the pool will be worthwhile. Copyright © 2017 Elsevier GmbH. All rights reserved.

Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay

PubMed Central

Jin, Zhao; Di Rienzi, Sara C.; Janzon, Anders; Werner, Jeff J.; Angenent, Largus T.; Dangl, Jeffrey L.; Fowler, Douglas M.

2015-01-01

Metagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen to Escherichia coli in vitro. The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate. PMID:26637602

Cations Form Sequence Selective Motifs within DNA Grooves via a Combination of Cation-Pi and Ion-Dipole/Hydrogen Bond Interactions

PubMed Central

Stewart, Mikaela; Dunlap, Tori; Dourlain, Elizabeth; Grant, Bryce; McFail-Isom, Lori

2013-01-01

The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl+) and the polarized first hydration shell waters of divalent cations (Mg2+, Ca2+) form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves. PMID:23940752

Cations form sequence selective motifs within DNA grooves via a combination of cation-pi and ion-dipole/hydrogen bond interactions.

PubMed

Stewart, Mikaela; Dunlap, Tori; Dourlain, Elizabeth; Grant, Bryce; McFail-Isom, Lori

2013-01-01

The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl⁺) and the polarized first hydration shell waters of divalent cations (Mg²⁺, Ca²⁺) form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves.