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.

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

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.

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.

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

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/.

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

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.

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

PubMed

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

2015-01-01

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

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.

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 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

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.

Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria

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

Liu, Hualan; Price, Morgan N.; Waters, Robert Jordan

Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term “magic pools.” Within a magic pool, each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. Tomore » identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use this efficient vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylumBacteroidetes. IMPORTANCEMolecular genetics is indispensable for interrogating the physiology of bacteria. However, the development of a functional genetic system for any given bacterium can be time-consuming. Here, we present a streamlined approach for identifying an effective transposon mutagenesis system for a new bacterium. Our strategy first involves the construction of hundreds of different transposon vector variants, which we term a “magic pool.” The efficacy of each vector in a magic pool is monitored in parallel using a unique DNA barcode that is introduced into each vector design. Using archived DNA “parts,” we next reassemble an effective vector for making a whole-genome transposon mutant library that is suitable for large-scale interrogation of gene function using competitive growth assays. Here, we demonstrate the utility of the magic pool system to make mutant libraries in five genera of bacteria.« less

Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria

DOE PAGES

Liu, Hualan; Price, Morgan N.; Waters, Robert Jordan; ...

2018-01-16

Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term “magic pools.” Within a magic pool, each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. Tomore » identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use this efficient vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylumBacteroidetes. IMPORTANCEMolecular genetics is indispensable for interrogating the physiology of bacteria. However, the development of a functional genetic system for any given bacterium can be time-consuming. Here, we present a streamlined approach for identifying an effective transposon mutagenesis system for a new bacterium. Our strategy first involves the construction of hundreds of different transposon vector variants, which we term a “magic pool.” The efficacy of each vector in a magic pool is monitored in parallel using a unique DNA barcode that is introduced into each vector design. Using archived DNA “parts,” we next reassemble an effective vector for making a whole-genome transposon mutant library that is suitable for large-scale interrogation of gene function using competitive growth assays. Here, we demonstrate the utility of the magic pool system to make mutant libraries in five genera of bacteria.« less

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)…

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.

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

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.

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

Mitochondrial deoxyribonucleoside triphosphate pools in thymidine kinase 2 deficiency.

PubMed

Saada, Ann; Ben-Shalom, Efrat; Zyslin, Rivka; Miller, Chaya; Mandel, Hanna; Elpeleg, Orly

2003-10-24

Deficiency of mitochondrial thymidine kinase (TK2) is associated with mitochondrial DNA (mtDNA) depletion and manifests by severe skeletal myopathy in infancy. In order to elucidate the pathophysiology of this condition, mitochondrial deoxyribonucleoside triphosphate (dNTP) pools were determined in patients' fibroblasts. Despite normal mtDNA content and cytochrome c oxidase (COX) activity, mitochondrial dNTP pools were imbalanced. Specifically, deoxythymidine triphosphate (dTTP) content was markedly decreased, resulting in reduced dTTP:deoxycytidine triphosphate ratio. These findings underline the importance of balanced mitochondrial dNTP pools for mtDNA synthesis and may serve as the basis for future therapeutic interventions.

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.

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…

Microbial life in Champagne Pool, a geothermal spring in Waiotapu, New Zealand.

PubMed

Hetzer, Adrian; Morgan, Hugh W; McDonald, Ian R; Daughney, Christopher J

2007-07-01

Surveys of Champagne Pool, one of New Zealand's largest terrestrial hot springs and rich in arsenic ions and compounds, have been restricted to geological and geochemical descriptions, and a few microbiological studies applying culture-independent methods. In the current investigation, a combination of culture and culture-independent approaches were chosen to determine microbial density and diversity in Champagne Pool. Recovered total DNA and adenosine 5'-triphosphate (ATP) content of spring water revealed relatively low values compared to other geothermal springs within New Zealand and are in good agreement with low cell numbers of 5.6 +/- 0.5 x 10(6) cells/ml obtained for Champagne Pool water samples by 4',6-diamidino-2-phenylindole (DAPI) staining. Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA (small-subunit ribosomal nucleic acid) gene clone library analyses of environmental DNA indicated the abundance of Sulfurihydrogenibium, Sulfolobus, and Thermofilum-like populations in Champagne Pool. From these results, media were selected to target the enrichment of hydrogen-oxidizing and sulfur-dependent microorganisms. Three isolates were successfully obtained having 16S rRNA gene sequences with similarities of approximately 98% to Thermoanaerobacter tengcongensis, 94% to Sulfurihydrogenibium azorense, and 99% to Thermococcus waiotapuensis, respectively.

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.

Priming with a simplified intradermal HIV-1 DNA vaccine regimen followed by boosting with recombinant HIV-1 MVA vaccine is safe and immunogenic: a phase IIa randomized clinical trial.

PubMed

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

2015-01-01

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

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

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.

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.

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.

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.

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...

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

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.

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.

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

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

Kanzler, Daniel, E-mail: daniel.kanzler@bam.de, E-mail: christina.mueller@bam.de; Müller, Christina, E-mail: daniel.kanzler@bam.de, E-mail: christina.mueller@bam.de; Pitkänen, Jorma, E-mail: jorma.pitkanen@posiva.fi

2015-03-31

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

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

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.

Where is DNA preserved in soil organic matter?

NASA Astrophysics Data System (ADS)

Zaccone, Claudio; Beneduce, Luciano; Plaza, César

2015-04-01

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

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

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

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.

Dual Targeting Biomimetic Liposomes for Paclitaxel/DNA Combination Cancer Treatment

PubMed Central

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

2014-01-01

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

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

Combined therapy with cyclophosphamide and DNA preparation inhibits the tumor growth in mice

PubMed Central

Alyamkina, Ekaterina A; Dolgova, Evgenia V; Likhacheva, Anastasia S; Rogachev, Vladimir A; Sebeleva, Tamara E; Nikolin, Valeriy P; Popova, Nelly A; Orishchenko, Konstantin E; Strunkin, Dmitriy N; Chernykh, Elena R; Zagrebelniy, Stanislav N; Bogachev, Sergei S; Shurdov, Mikhail A

2009-01-01

Background When cyclophosphamide and preparations of fragmented exogenous genomic double stranded DNA were administered in sequence, the regressive effect on the tumor was synergic: this combined treatment had a more pronounced effect than cyclophosphamide alone. Our further studies demonstrated that exogenous DNA stimulated the maturation and specific activities of dendritic cells. This suggests that cyclophosphamide, combined with DNA, leads to an immune response to the tumors that were grafted into the subjects post treatment. Methods Three-month old CBA/Lac mice were used in the experiments. The mice were injected with cyclosphamide (200 mkg per 1 kg body weight) and genomic DNA (of human, mouse or salmon sperm origin). The DNA was administered intraperitoneally or subcutaneously. After 23 to 60 days, one million tumor cells were intramuscularly grafted into the mice. In the final experiment, the mice were pre-immunized by subcutaneous injections of 20 million repeatedly thawed and frozen tumor cells. Changes in tumor growth were determined by multiplying the three perpendicular diameters (measured by caliper). Students' t-tests were used to determine the difference between tumor growth and average survival rate between the mouse groups and the controls. Results An analysis of varying treatments with cyclophosphamide and exogenous DNA, followed by tumor grafting, provided evidence that this combined treatment had an immunizing effect. This inhibitory effect in mice was analyzed in an experiment with the classical immunization of a tumor homogenate. The strongest inhibitory action on a transplanted graft was created through the following steps: cyclophosphamide at 200 mg/kg of body weight administered as a pretreatment; 6 mg fragmented exogenous DNA administered over the course of 3 days; tumor homogenate grafted 10 days following the final DNA injection. Conclusion Fragmented exogenous DNA injected with cyclophosphamide inhibits the growth of tumors that are

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

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.

Cell-free DNA testing after combined test: factors affecting the uptake.

PubMed

Maiz, Nerea; Alzola, Irune; Murua, Emerson J; Rodríguez Santos, Javier

2016-11-01

First, to assess what was the uptake of cell free DNA (cfDNA) testing after a combined test and the maternal and fetal factors that influenced this decision, and second, to assess the uptake and factors that influence the choice of invasive testing. This observational retrospective study included 1083 singleton pregnancies who had a combined test for screening for Down syndrome between 11 (+) (0) and 13 (+) (6) weeks. Multivariate logistic regression analysis was used to determine which factors affected the uptake of cfDNA test and invasive testing among risk for trisomies 21, 18, and 13, maternal characteristics and fetal nuchal translucency (NT) thickness. Two-hundred fifty-seven (23.7%) women had a cfDNA test, 89 (8.2%) had an invasive test, and 737 (68.1%) had no further test. The uptake of cfDNA increased with the risk for trisomies (p < 0.001), maternal age (p = 0.013), and was higher in nulliparous women (p = 0.004). The uptake of invasive test increased with the risk for trisomies (p < 0.001) and NT thickness (p < 0.001). This study shows that the uptake of cfDNA testing increases with the risk for trisomies, maternal age, and is higher in nulliparous, whereas the uptake of invasive testing increases with the risk for trisomies and NT thickness.

Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase

PubMed Central

Roberts, Victoria A.; Pique, Michael E.; Hsu, Simon; Li, Sheng; Slupphaug, Geir; Rambo, Robert P.; Jamison, Jonathan W.; Liu, Tong; Lee, Jun H.; Tainer, John A.; Ten Eyck, Lynn F.; Woods, Virgil L.

2012-01-01

X-ray crystallography provides excellent structural data on protein–DNA interfaces, but crystallographic complexes typically contain only small fragments of large DNA molecules. We present a new approach that can use longer DNA substrates and reveal new protein–DNA interactions even in extensively studied systems. Our approach combines rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). DXMS identifies solvent-exposed protein surfaces; docking is used to create a 3-dimensional model of the protein–DNA interaction. We investigated the enzyme uracil-DNA glycosylase (UNG), which detects and cleaves uracil from DNA. UNG was incubated with a 30 bp DNA fragment containing a single uracil, giving the complex with the abasic DNA product. Compared with free UNG, the UNG–DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210–220 and 251–264. Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG–DNA crystallographic structures. Our results can be explained by separation of the two DNA strands on one side of the active site. These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway. PMID:22492624

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

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

Flow cytometric detection of micronuclei by combined staining of DNA and membranes

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

Wessels, J.M.; Nuesse, M.

1995-03-01

A new staining method is presented for flow cytometric measurement of micronuclei (MN) in cell cultures and human lymphocytes using membrane-specific fluorescent dyes in addition to DNA staining. Several combinations of fluorescent membrane and DNA dyes were studied for a better discrimination of MN from debris in a suspension of nuclei and micronuclei. For staining of membranes, the lipophilic dyes 2-hydroxyethyl-7,12,17-tris(methoxyethyl)porphycene (HEPn) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were used in combination with ethidium bromide (EB), proflavine (PF), and Hoechst 33258 (HO). Due to their spectral properties, HO or EB combined with HEPn were not as suitable for the discrimination of MN frommore » debris as was HEPn in combination with PF. With HEPn in combination with PF, however, additional noise was found at low fluorescence intensities, probably due to free fluorescent dye molecules in the solution. The optimal simultaneous staining of membranes and DNA was obtained using a combination of DPH and EB. The induction of MN in Chinese hamster and mouse NIH-3T3 cells by UV-B illumination was studied with this new staining technique. UV-B illumination (280-360 nm) induced MN in both cell lines. Chinese hamster cells were found to be more sensitive to these wavelengths. Illumination with wavelengths above 360 nm did not induce MN in either cell line. The results obtained from human lymphocytes using the combination of EB or DPH were comparable to the results obtained with the combination of EB and HO. 23 refs., 7 figs.« less

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

PubMed

Hsu, Ching-Shan; Huang, Da-Ji

2015-02-01

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

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.

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.

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.

Imaging of DNA and Protein by SFM and Combined SFM-TIRF Microscopy.

PubMed

Grosbart, Małgorzata; Ristić, Dejan; Sánchez, Humberto; Wyman, Claire

2018-01-01

Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nm resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. In addition, even from static images, the different forms observed and their relative distributions can be used to deduce the variety and stability of different complexes that are necessarily involved in dynamic processes. Recently available instruments that combine fluorescence with topographic imaging allow the identification of specific molecular components in complex assemblies, which broadens the applications and increases the information obtained from direct imaging of molecular complexes. We describe here basic methods for preparing samples of proteins, DNA and complexes of the two for topographic imaging and quantitative analysis. We also describe special considerations for combined fluorescence and topographic imaging of molecular complexes.

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.

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.

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

Power analysis of QTL detection in half-sib families using selective DNA pooling

PubMed Central

Baro, Jesús Á; Carleos, Carlos; Corral, Norberto; López, Teresa; Cañón, Javier

2001-01-01

Individual loci of economic importance (QTL) can be detected by comparing the inheritance of a trait and the inheritance of loci with alleles readily identifiable by laboratory methods (genetic markers). Data on allele segregation at the individual level are costly and alternatives have been proposed that make use of allele frequencies among progeny, rather than individual genotypes. Among the factors that may affect the power of the set up, the most important are those intrinsic to the QTL: the additive effect of the QTL, and its dominance, and distance between markers and QTL. Other factors are relative to the choice of animals and markers, such as the frequency of the QTL and marker alleles among dams and sires. Data collection may affect the detection power through the size of half-sib families, selection rate within families, and the technical error incurred when estimating genetic frequencies. We present results for a sensitivity analysis for QTL detection using pools of DNA from selected half-sibs. Simulations showed that conclusive detection may be achieved with families of at least 500 half-sibs if sires are chosen on the criteria that most of their marker alleles are either both missing, or one is fixed, among dams. PMID:11403746

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.

Design of association studies with pooled or un-pooled next-generation sequencing data.

PubMed

Kim, Su Yeon; Li, Yingrui; Guo, Yiran; Li, Ruiqiang; Holmkvist, Johan; Hansen, Torben; Pedersen, Oluf; Wang, Jun; Nielsen, Rasmus

2010-07-01

Most common hereditary diseases in humans are complex and multifactorial. Large-scale genome-wide association studies based on SNP genotyping have only identified a small fraction of the heritable variation of these diseases. One explanation may be that many rare variants (a minor allele frequency, MAF <5%), which are not included in the common genotyping platforms, may contribute substantially to the genetic variation of these diseases. Next-generation sequencing, which would allow the analysis of rare variants, is now becoming so cheap that it provides a viable alternative to SNP genotyping. In this paper, we present cost-effective protocols for using next-generation sequencing in association mapping studies based on pooled and un-pooled samples, and identify optimal designs with respect to total number of individuals, number of individuals per pool, and the sequencing coverage. We perform a small empirical study to evaluate the pooling variance in a realistic setting where pooling is combined with exon-capturing. To test for associations, we develop a likelihood ratio statistic that accounts for the high error rate of next-generation sequencing data. We also perform extensive simulations to determine the power and accuracy of this method. Overall, our findings suggest that with a fixed cost, sequencing many individuals at a more shallow depth with larger pool size achieves higher power than sequencing a small number of individuals in higher depth with smaller pool size, even in the presence of high error rates. Our results provide guidelines for researchers who are developing association mapping studies based on next-generation sequencing. (c) 2010 Wiley-Liss, Inc.

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.

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

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/.

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.

DNA electrotransfer into the skin using a combination of one high- and one low-voltage pulse.

PubMed

Pavselj, N; Préat, V

2005-09-02

Electroporation is an effective alternative to viral methods to significantly improve DNA transfection after intradermal and topical delivery. The aim of the study was to check whether a combination of a short high-voltage pulse (HV) to permeabilize the skin cells and a long low-voltage pulse (LV) to transfer DNA by electrophoresis was more efficient to enhance DNA expression than conventional repeated HV or LV pulses alone after intradermal injection of DNA plasmid. GFP and luciferase expressions in the skin were enhanced by HV+LV protocol as compared to HV or LV pulses alone. The expression lasted for up to 10 days. Consistently, HV+LV protocol induced a higher Th2 immune response against ovalbumin than HV or LV pulses. Standard methods were used to assess the effect of electric pulses on skin: the application of a combination of HV and LV pulses on rat skin fold delivered by plate electrodes was well tolerated. These data demonstrate that a combination of one HV (700 to 1000 V/cm; 100 micros) followed by one LV (140 to 200 V/cm; 400 ms) is an efficient electroporation protocol to enhance DNA expression in the skin.

Characterizing convective cold pools: Characterizing Convective Cold Pools

DOE PAGES

Drager, Aryeh J.; van den Heever, Susan C.

2017-05-09

Cold pools produced by convective storms play an important role in Earth's climate system. However, a common framework does not exist for objectively identifying convective cold pools in observations and models. The present study investigates convective cold pools within a simulation of tropical continental convection that uses a cloud-resolving model with a coupled land-surface model. Multiple variables are assessed for their potential in identifying convective cold pool boundaries, and a novel technique is developed and tested for identifying and tracking cold pools in numerical model simulations. This algorithm is based on surface rainfall rates and radial gradients in the densitymore » potential temperature field. The algorithm successfully identifies near-surface cold pool boundaries and is able to distinguish between connected cold pools. Once cold pools have been identified and tracked, composites of cold pool evolution are then constructed, and average cold pool properties are investigated. Wet patches are found to develop within the centers of cold pools where the ground has been soaked with rainwater. These wet patches help to maintain cool surface temperatures and reduce cold pool dissipation, which has implications for the development of subsequent convection.« less

Characterizing convective cold pools: Characterizing Convective Cold Pools

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

Drager, Aryeh J.; van den Heever, Susan C.

Cold pools produced by convective storms play an important role in Earth's climate system. However, a common framework does not exist for objectively identifying convective cold pools in observations and models. The present study investigates convective cold pools within a simulation of tropical continental convection that uses a cloud-resolving model with a coupled land-surface model. Multiple variables are assessed for their potential in identifying convective cold pool boundaries, and a novel technique is developed and tested for identifying and tracking cold pools in numerical model simulations. This algorithm is based on surface rainfall rates and radial gradients in the densitymore » potential temperature field. The algorithm successfully identifies near-surface cold pool boundaries and is able to distinguish between connected cold pools. Once cold pools have been identified and tracked, composites of cold pool evolution are then constructed, and average cold pool properties are investigated. Wet patches are found to develop within the centers of cold pools where the ground has been soaked with rainwater. These wet patches help to maintain cool surface temperatures and reduce cold pool dissipation, which has implications for the development of subsequent convection.« less

Combining atomic force and fluorescence microscopy for analysis of quantum-dot labeled protein–DNA complexes

PubMed Central

Ebenstein, Yuval; Gassman, Natalie; Kim, Soohong; Weiss, Shimon

2011-01-01

Atomic force microscopy (AFM) and fluorescence microscopy are widely used for the study of protein-DNA interactions. While AFM excels in its ability to elucidate structural detail and spatial arrangement, it lacks the ability to distinguish between similarly sized objects in a complex system. This information is readily accessible to optical imaging techniques via site-specific fluorescent labels, which enable the direct detection and identification of multiple components simultaneously. Here, we show how the utilization of semiconductor quantum dots (QDs), serving as contrast agents for both AFM topography and fluorescence imaging, facilitates the combination of both imaging techniques, and with the addition of a flow based DNA extension method for sample deposition, results in a powerful tool for the study of protein-DNA complexes. We demonstrate the inherent advantages of this novel combination of techniques by imaging individual RNA polymerases (RNAP) on T7 genomic DNA. PMID:19452448

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

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.

13 CFR 120.611 - Pools backing Pool Certificates.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 13 Business Credit and Assistance 1 2011-01-01 2011-01-01 false Pools backing Pool Certificates. 120.611 Section 120.611 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION BUSINESS LOANS Secondary Market Certificates § 120.611 Pools backing Pool Certificates. (a) Pool characteristics. As set...

13 CFR 120.611 - Pools backing Pool Certificates.

Code of Federal Regulations, 2010 CFR

2010-01-01

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

A phylogenetic study of Laeliinae (Orchidaceae) based on combined nuclear and plastid DNA sequences

PubMed Central

van den Berg, Cássio; Higgins, Wesley E.; Dressler, Robert L.; Whitten, W. Mark; Soto-Arenas, Miguel A.; Chase, Mark W.

2009-01-01

Background and Aims Laeliinae are a neotropical orchid subtribe with approx. 1500 species in 50 genera. In this study, an attempt is made to assess generic alliances based on molecular phylogenetic analysis of DNA sequence data. Methods Six DNA datasets were gathered: plastid trnL intron, trnL-F spacer, matK gene and trnK introns upstream and dowstream from matK and nuclear ITS rDNA. Data were analysed with maximum parsimony (MP) and Bayesian analysis with mixed models (BA). Key Results Although relationships between Laeliinae and outgroups are well supported, within the subtribe sequence variation is low considering the broad taxonomic range covered. Localized incongruence between the ITS and plastid trees was found. A combined tree followed the ITS trees more closely, but the levels of support obtained with MP were low. The Bayesian analysis recovered more well-supported nodes. The trees from combined MP and BA allowed eight generic alliances to be recognized within Laeliinae, all of which show trends in morphological characters but lack unambiguous synapomorphies. Conclusions By using combined plastid and nuclear DNA data in conjunction with mixed-models Bayesian inference, it is possible to delimit smaller groups within Laeliinae and discuss general patterns of pollination and hybridization compatibility. Furthermore, these small groups can now be used for further detailed studies to explain morphological evolution and diversification patterns within the subtribe. PMID:19423551

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

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

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

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

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

MUTYH mediates the toxicity of combined DNA 6-thioguanine and UVA radiation

PubMed Central

De Luca, Gabriele; Leopardi, Paola; Mancuso, Maria Teresa; Casorelli, Ida; Pichierri, Pietro; Karran, Peter; Bignami, Margherita

2015-01-01

The therapeutic thiopurines, including the immunosuppressant azathioprine (Aza) cause the accumulation of the UVA photosensitizer 6-thioguanine (6-TG) in the DNA of the patients' cells. DNA 6-TG and UVA are synergistically cytotoxic and their interaction causes oxidative damage. The MUTYH DNA glycosylase participates in the base excision repair of oxidized DNA bases. Using Mutyh-nullmouse fibroblasts (MEFs) we examined whether MUTYH provides protection against the lethal effects of combined DNA 6-TG/UVA. Surprisingly, Mutyh-null MEFs were more resistant than wild-type MEFs, despite accumulating higher levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG). Their enhanced 6-TG/UVA resistance reflected the absence of the MUTYH protein and MEFs expressing enzymatically-dead human variants were as sensitive as wild-type cells. Consistent with their enhanced resistance, Mutyh-null cells sustained fewer DNA strand breaks and lower levels of chromosomal damage after 6-TG/UVA. Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, Mutyh-null cells failed to arrest in S-phase at late time points. MUTYH-dependent toxicity was also apparent in vivo. Mutyh−/−mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG. Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh−/− mice whereas similarly treated wild-type animals remained tumor-free. PMID:25638157

MUTYH mediates the toxicity of combined DNA 6-thioguanine and UVA radiation.

PubMed

Grasso, Francesca; Ruggieri, Vitalba; De Luca, Gabriele; Leopardi, Paola; Mancuso, Maria Teresa; Casorelli, Ida; Pichierri, Pietro; Karran, Peter; Bignami, Margherita

2015-04-10

The therapeutic thiopurines, including the immunosuppressant azathioprine (Aza) cause the accumulation of the UVA photosensitizer 6-thioguanine (6-TG) in the DNA of the patients' cells. DNA 6-TG and UVA are synergistically cytotoxic and their interaction causes oxidative damage. The MUTYH DNA glycosylase participates in the base excision repair of oxidized DNA bases. Using Mutyh-nullmouse fibroblasts (MEFs) we examined whether MUTYH provides protection against the lethal effects of combined DNA 6-TG/UVA. Surprisingly, Mutyh-null MEFs were more resistant than wild-type MEFs, despite accumulating higher levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG).Their enhanced 6-TG/UVA resistance reflected the absence of the MUTYH protein and MEFs expressing enzymatically-dead human variants were as sensitive as wild-type cells. Consistent with their enhanced resistance, Mutyh-null cells sustained fewer DNA strand breaks and lower levels of chromosomal damage after 6-TG/UVA. Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, Mutyh-null cells failed to arrest in S-phase at late time points. MUTYH-dependent toxicity was also apparent in vivo. Mutyh-/- mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG. Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh-/- mice whereas similarly treated wild-type animals remained tumor-free.

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.

A combined method for DNA analysis and radiocarbon dating from a single sample.

PubMed

Korlević, Petra; Talamo, Sahra; Meyer, Matthias

2018-03-07

Current protocols for ancient DNA and radiocarbon analysis of ancient bones and teeth call for multiple destructive samplings of a given specimen, thereby increasing the extent of undesirable damage to precious archaeological material. Here we present a method that makes it possible to obtain both ancient DNA sequences and radiocarbon dates from the same sample material. This is achieved by releasing DNA from the bone matrix through incubation with either EDTA or phosphate buffer prior to complete demineralization and collagen extraction utilizing the acid-base-acid-gelatinization and ultrafiltration procedure established in most radiocarbon dating laboratories. Using a set of 12 bones of different ages and preservation conditions we demonstrate that on average 89% of the DNA can be released from sample powder with minimal, or 38% without any, detectable collagen loss. We also detect no skews in radiocarbon dates compared to untreated samples. Given the different material demands for radiocarbon dating (500 mg of bone/dentine) and DNA analysis (10-100 mg), combined DNA and collagen extraction not only streamlines the sampling process but also drastically increases the amount of DNA that can be recovered from limited sample material.

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.

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

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

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

[Immune response induced by HIV DNA vaccine combined with recombinant adeno-associated virus].

PubMed

Liu, Yan-zheng; Zhou, Ling; Wang, Qi; Ye, Shu-qing; Li, Hong-xia; Zeng, Yi

2004-09-01

HIV-1 DNA vaccine and recombinant adeno-associated virus (rAAV) expressing gagV3 gene of HIV-1 subtype B were constructed and BALB/c mice were immunized by vaccination regimen consisting of consecutive priming with DNA vaccine and boosting with rAAV vaccine; the CTL and antibody response were detected and compared with those induced by DNA vaccine or rAAV vaccine separately. HIV-1 subtype B gagV3 gene was inserted into the polyclonal site of plasmid pCI-neo, DNA vaccine pCI-gagV3 was thereby constructed; pCI-gagV3 was transfected into p815 cells, G-418-resistant cells were obtained through screening transfected cells with G418, the expression of HIV-1 antigen in G-418-resistant cells was detected by EIA; BALB/c mice were immunized with pCI-gagV3 and the immune response was tested; BALB/c mouse immunized with pCI-gagV3 and combined with rAAV expressing the same gagV3 genes were tested for antibody level in sera by EIA method and cytotoxicity response by LDH method. pCI-gagV3 could express HIV-1 gene in p815 cells; pCI-gagV3 could induce HIV-1 specific humoral and cell-mediated immune response in BALB/c mice. The HIV-1 specific antibody level was 1/20; when the ratio of effector cells: target cells was 50:1, the average specific cytotoxicity was 41.7%; there was no evident increase in the antibody level induced by pCI-gagV3 combined with rAAV, but there was increase in CTL response, the average specific cytotoxicity was 61.3% when effector cells: target cells ratio was 50:1. HIV-1 specific cytotoxicity in BALB/c mice can be increased by immunization of BALB/c mice with DNA vaccine combined with rAAV vaccine.

Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe.

PubMed

Ingianni, A; Floris, M; Palomba, P; Madeddu, M A; Quartuccio, M; Pompei, R

2001-10-01

Listeria monocytogenes is a frequent contaminant of water and foods. Its rapid detection is needed before some foods can be prepared for marketing. In this work L. monocytogenes has been searched for in foods, by a combination of polymerase chain reaction (PCR) and a DNA probe. Both PCR and the probe were prepared for recognizing a specific region of the internalin gene, which is responsible for the production of one of the most important pathogenic factors of Listeria. The combined use of PCR and the DNA probe was used for the detection of L. monocytogenes in over 180 environmental and food samples. Several detection methods were compared in this study, namely conventional culture methods; direct PCR; PCR after an enrichment step; a DNA probe alone; a DNA probe after enrichment and another commercially available gene-probe. Finally PCR and the DNA probe were used in series on all the samples collected. When the DNA probe was associated with the PCR, specific and accurate detection of listeria in the samples could be obtained in about a working-day. The present molecular method showed some advantages in terms of rapidity and specificity in comparison to the other aforementioned tests. In addition, it resulted as being easy to handle, even for non-specialized personnel in small diagnostic microbiology laboratories. Copyright 2001 Academic Press.

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

Efficacy and Safety of Nivolumab Alone or in Combination With Ipilimumab in Patients With Mucosal Melanoma: A Pooled Analysis.

PubMed

D'Angelo, Sandra P; Larkin, James; Sosman, Jeffrey A; Lebbé, Celeste; Brady, Benjamin; Neyns, Bart; Schmidt, Henrik; Hassel, Jessica C; Hodi, F Stephen; Lorigan, Paul; Savage, Kerry J; Miller, Wilson H; Mohr, Peter; Marquez-Rodas, Ivan; Charles, Julie; Kaatz, Martin; Sznol, Mario; Weber, Jeffrey S; Shoushtari, Alexander N; Ruisi, Mary; Jiang, Joel; Wolchok, Jedd D

2017-01-10

Purpose Mucosal melanoma is an aggressive malignancy with a poor response to conventional therapies. The efficacy and safety of nivolumab (a programmed death-1 checkpoint inhibitor), alone or combined with ipilimumab (a cytotoxic T-lymphocyte antigen-4 checkpoint inhibitor), have not been reported in this rare melanoma subtype. Patients and Methods Data were pooled from 889 patients who received nivolumab monotherapy in clinical studies, including phase III trials; 86 (10%) had mucosal melanoma and 665 (75%) had cutaneous melanoma. Data were also pooled for patients who received nivolumab combined with ipilimumab (n = 35, mucosal melanoma; n = 326, cutaneous melanoma). Results Among patients who received nivolumab monotherapy, median progression-free survival was 3.0 months (95% CI, 2.2 to 5.4 months) and 6.2 months (95% CI, 5.1 to 7.5 months) for mucosal and cutaneous melanoma, with objective response rates of 23.3% (95% CI, 14.8% to 33.6%) and 40.9% (95% CI, 37.1% to 44.7%), respectively. Median progression-free survival in patients treated with nivolumab combined with ipilimumab was 5.9 months (95% CI, 2.8 months to not reached) and 11.7 months (95% CI, 8.9 to 16.7 months) for mucosal and cutaneous melanoma, with objective response rates of 37.1% (95% CI, 21.5% to 55.1%) and 60.4% (95% CI, 54.9% to 65.8%), respectively. For mucosal and cutaneous melanoma, respectively, the incidence of grade 3 or 4 treatment-related adverse events was 8.1% and 12.5% for nivolumab monotherapy and 40.0% and 54.9% for combination therapy. Conclusion To our knowledge, this is the largest analysis of data for anti-programmed death-1 therapy in mucosal melanoma to date. Nivolumab combined with ipilimumab seemed to have greater efficacy than either agent alone, and although the activity was lower in mucosal melanoma, the safety profile was similar between subtypes.

Efficacy and Safety of Nivolumab Alone or in Combination With Ipilimumab in Patients With Mucosal Melanoma: A Pooled Analysis

PubMed Central

Larkin, James; Sosman, Jeffrey A.; Lebbé, Celeste; Brady, Benjamin; Neyns, Bart; Schmidt, Henrik; Hassel, Jessica C.; Hodi, F. Stephen; Lorigan, Paul; Savage, Kerry J.; Miller, Wilson H.; Mohr, Peter; Marquez-Rodas, Ivan; Charles, Julie; Kaatz, Martin; Sznol, Mario; Weber, Jeffrey S.; Shoushtari, Alexander N.; Ruisi, Mary; Jiang, Joel; Wolchok, Jedd D.

2017-01-01

Purpose Mucosal melanoma is an aggressive malignancy with a poor response to conventional therapies. The efficacy and safety of nivolumab (a programmed death-1 checkpoint inhibitor), alone or combined with ipilimumab (a cytotoxic T-lymphocyte antigen-4 checkpoint inhibitor), have not been reported in this rare melanoma subtype. Patients and Methods Data were pooled from 889 patients who received nivolumab monotherapy in clinical studies, including phase III trials; 86 (10%) had mucosal melanoma and 665 (75%) had cutaneous melanoma. Data were also pooled for patients who received nivolumab combined with ipilimumab (n = 35, mucosal melanoma; n = 326, cutaneous melanoma). Results Among patients who received nivolumab monotherapy, median progression-free survival was 3.0 months (95% CI, 2.2 to 5.4 months) and 6.2 months (95% CI, 5.1 to 7.5 months) for mucosal and cutaneous melanoma, with objective response rates of 23.3% (95% CI, 14.8% to 33.6%) and 40.9% (95% CI, 37.1% to 44.7%), respectively. Median progression-free survival in patients treated with nivolumab combined with ipilimumab was 5.9 months (95% CI, 2.8 months to not reached) and 11.7 months (95% CI, 8.9 to 16.7 months) for mucosal and cutaneous melanoma, with objective response rates of 37.1% (95% CI, 21.5% to 55.1%) and 60.4% (95% CI, 54.9% to 65.8%), respectively. For mucosal and cutaneous melanoma, respectively, the incidence of grade 3 or 4 treatment-related adverse events was 8.1% and 12.5% for nivolumab monotherapy and 40.0% and 54.9% for combination therapy. Conclusion To our knowledge, this is the largest analysis of data for anti–programmed death-1 therapy in mucosal melanoma to date. Nivolumab combined with ipilimumab seemed to have greater efficacy than either agent alone, and although the activity was lower in mucosal melanoma, the safety profile was similar between subtypes. PMID:28056206

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.

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

PubMed

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

2015-08-01

DNA is a type of potential biomaterials for drug delivery due to its nanoscale geometry, loading capacity of therapeutics, biocompatibility, and biodegradability. Unfortunately, DNA is easily degraded by DNases in the body circulation and has low intracellular uptake. In the present study, we selected three cationic polymers polyethylenimine (PEI), hexadecyl trimethyl ammonium bromide (CTAB), and low-density lipoprotein (LDL) receptor targeted peptide (RLT), to modify DNA and improve the issues. A potent anti-tumor anthracycline-doxorubicin (DOX) was intercalated into DNA non-covalently and the DOX/DNA was then combined with PEI, CTAB, and RLT, respectively. Compact nanocomplexes were formed by electrostatic interaction and could potentially protect DNA from DNases. More importantly, RLT had the potential to enhance intracellular uptake by LDL receptor mediated endocytosis. In a series of in vitro experiments, RLT complexed DNA enhanced intracellular delivery of DOX, increased tumor cell death and intracellular ROS production, and reduced intracellular elimination of DOX. All results suggested that the easily prepared and targeted RLT/DNA nanocomplexes had great potential to be developed into a formulation for doxorubicin with enhanced anti-tumor activity. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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.

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.

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

Identification of DNA-binding proteins by combining auto-cross covariance transformation and ensemble learning.

PubMed

Liu, Bin; Wang, Shanyi; Dong, Qiwen; Li, Shumin; Liu, Xuan

2016-04-20

DNA-binding proteins play a pivotal role in various intra- and extra-cellular activities ranging from DNA replication to gene expression control. With the rapid development of next generation of sequencing technique, the number of protein sequences is unprecedentedly increasing. Thus it is necessary to develop computational methods to identify the DNA-binding proteins only based on the protein sequence information. In this study, a novel method called iDNA-KACC is presented, which combines the Support Vector Machine (SVM) and the auto-cross covariance transformation. The protein sequences are first converted into profile-based protein representation, and then converted into a series of fixed-length vectors by the auto-cross covariance transformation with Kmer composition. The sequence order effect can be effectively captured by this scheme. These vectors are then fed into Support Vector Machine (SVM) to discriminate the DNA-binding proteins from the non DNA-binding ones. iDNA-KACC achieves an overall accuracy of 75.16% and Matthew correlation coefficient of 0.5 by a rigorous jackknife test. Its performance is further improved by employing an ensemble learning approach, and the improved predictor is called iDNA-KACC-EL. Experimental results on an independent dataset shows that iDNA-KACC-EL outperforms all the other state-of-the-art predictors, indicating that it would be a useful computational tool for DNA binding protein identification. .

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

USGS Publications Warehouse

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

2012-01-01

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

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.

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.

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

PubMed Central

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

2015-01-01

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

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.

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

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

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

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

NASA Astrophysics Data System (ADS)

Samanta, Anirban; Medintz, Igor L.

2016-04-01

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

Combined effects of metal complexation and size expansion in the electronic structure of DNA base pairs

NASA Astrophysics Data System (ADS)

Brancolini, Giorgia; Di Felice, Rosa

2011-05-01

Novel DNA derivatives have been recently investigated in the pursuit of modified DNA duplexes to tune the electronic structure of DNA-based assemblies for nanotechnology applications. Size-expanded DNAs (e.g., xDNA) and metalated DNAs (M-DNA) may enhance stacking interactions and induce metallic conductivity, respectively. Here we explore possible ways of tailoring the DNA electronic structure by combining the aromatic size expansion with the metal-doping. We select the salient structures from our recent study on natural DNA pairs complexed with transition metal ions and consider the equivalent model configurations for xDNA pairs. We present the results of density functional theory electronic structure calculations of the metalated expanded base-pairs with various localized basis sets and exchange-correlation functionals. Implicit solvent and coordination water molecules are also included. Our results indicate that the effect of base expansion is largest in Ag-xGC complexes, while Cu-xGC complexes are the most promising candidates for nanowires with enhanced electron transfer and also for on-purpose modification of the DNA double-helix for signal detection.

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

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

Barends, Thomas R. M., E-mail: thomas.barends@mpimf-heidelberg.mpg.de; Brosi, Richard W. W.; Steinmetz, Andrea

2013-08-01

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

Combined loss of three DNA damage response pathways renders C. elegans intolerant to light.

PubMed

van Bostelen, Ivo; Tijsterman, Marcel

2017-06-01

Infliction of DNA damage initiates a complex cellular reaction - the DNA damage response - that involves both signaling and DNA repair networks with many redundancies and parallel pathways. Here, we reveal the three strategies that the simple multicellular eukaryote, C. elegans, uses to deal with DNA damage induced by light. Separately inactivating repair or replicative bypass of photo-lesions results in cellular hypersensitivity towards UV-light, but impeding repair of replication associated DNA breaks does not. Yet, we observe an unprecedented synergistic relationship when these pathways are inactivated in combination. C. elegans mutants that lack nucleotide excision repair (NER), translesion synthesis (TLS) and alternative end joining (altEJ) grow undisturbed in the dark, but become sterile when grown in light. Even exposure to very low levels of normal daylight impedes animal growth. We show that NER and TLS operate to suppress the formation of lethal DNA breaks that require polymerase theta-mediated end joining (TMEJ) for their repair. Our data testifies to the enormous genotoxicity of light and to the demand of multiple layers of protection against an environmental threat that is so common. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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

PubMed Central

2012-01-01

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

Combined branched-DNA and conventional HBV PCR assays for detection of serum HBV-DNA in hepatitis B e antigen-positive chronic hepatitis B patients.

PubMed

Ozdarendeli, Aykut; Toroman, Zulal Asci; Bulut, Yasemin; Demirbag, Kutbeddin; Kalkan, Ahmet; Ozden, Mehmet; Kilic, Suleyman Sirri

2006-01-01

Monitoring of HBV replication level is very useful for the management of patients with chronic HBV. However, the use of the correct tools to quantify HBV-DNA levels in serum and monitor the replication of HBV is of paramount importance in terms of diagnosis, and antiviral treatment of patients with chronic HBV infection. The aim of this study was to combine the bDNA assay and HBV PCR to improve detection of viremia the patients with HBeAg-positive chronic hepatitis B infection. In this study, 67 HBeAg-positive chronic hepatitis B patients were analyzed to determine viremia level using bDNA and HBV PCR assays. Sixty-four patients with HBeAg-positive chronic hepatitis B showed positivity by conventional HBV PCR, whereas 56 subjects with HBeAg-positive chronic hepatitis B showed HBV-DNA levels by bDNA. The results indicated that it is reasonable to use the bDNA assay to determine HBV replicative activity first, and use conventional HBV-PCR for HBeAg-positive chronic hepatitis B patient samples that are negative in bDNA assay.

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.

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.

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

13 CFR 120.1704 - Pool Loans eligible for Pooling.

Code of Federal Regulations, 2010 CFR

2010-01-01

..., construction or renovation of an aquarium, zoo, golf course, or swimming pool; or (iv) To a business covered by... zoos—712130 (“Zoos and Botanical Gardens”). (b) SBA review of a Pool Loan prior to pool formation. SBA...

13 CFR 120.1704 - Pool Loans eligible for Pooling.

Code of Federal Regulations, 2013 CFR

2013-01-01

..., construction or renovation of an aquarium, zoo, golf course, or swimming pool; or (iv) To a business covered by... zoos—712130 (“Zoos and Botanical Gardens”). (b) SBA review of a Pool Loan prior to pool formation. SBA...

13 CFR 120.1704 - Pool Loans eligible for Pooling.

Code of Federal Regulations, 2011 CFR

2011-01-01

..., construction or renovation of an aquarium, zoo, golf course, or swimming pool; or (iv) To a business covered by... zoos—712130 (“Zoos and Botanical Gardens”). (b) SBA review of a Pool Loan prior to pool formation. SBA...

13 CFR 120.1704 - Pool Loans eligible for Pooling.

Code of Federal Regulations, 2014 CFR

2014-01-01

..., construction or renovation of an aquarium, zoo, golf course, or swimming pool; or (iv) To a business covered by... zoos—712130 (“Zoos and Botanical Gardens”). (b) SBA review of a Pool Loan prior to pool formation. SBA...

13 CFR 120.1704 - Pool Loans eligible for Pooling.

Code of Federal Regulations, 2012 CFR

2012-01-01

..., construction or renovation of an aquarium, zoo, golf course, or swimming pool; or (iv) To a business covered by... zoos—712130 (“Zoos and Botanical Gardens”). (b) SBA review of a Pool Loan prior to pool formation. SBA...

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

ECS DAAC Data Pools

NASA Astrophysics Data System (ADS)

Kiebuzinski, A. B.; Bories, C. M.; Kalluri, S.

2002-12-01

As part of its Earth Observing System (EOS), NASA supports operations for several satellites including Landsat 7, Terra, and Aqua. ECS (EOSDIS Core System) is a vast archival and distribution system and includes several Distributed Active Archive Centers (DAACs) located around the United States. EOSDIS reached a milestone in February when its data holdings exceeded one petabyte (1,000 terabytes) in size. It has been operational since 1999 and originally was intended to serve a large community of Earth Science researchers studying global climate change. The Synergy Program was initiated in 2000 with the purpose of exploring and expanding the use of remote sensing data beyond the traditional research community to the applications community including natural resource managers, disaster/emergency managers, urban planners and others. This included facilitating data access at the DAACs to enable non-researchers to exploit the data for their specific applications. The combined volume of data archived daily across the DAACs is of the order of three terabytes. These archived data are made available to the research community and to general users of ECS data. Currently, the average data volume distributed daily is two terabytes, which combined with an ever-increasing need for timely access to these data, taxes the ECS processing and archival resources for more real-time use than was previously intended for research purposes. As a result, the delivery of data sets to users was being delayed in many cases, to unacceptable limits. Raytheon, under the auspices of the Synergy Program, investigated methods at making data more accessible at a lower cost of resources (processing and archival) at the DAACs. Large on-line caches (as big as 70 Terabytes) of data were determined to be a solution that would allow users who require contemporary data to access them without having to pull it from the archive. These on-line caches are referred to as "Data Pools." In the Data Pool concept

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.

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.

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.

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.

An integrated strategy combining DNA walking and NGS to detect GMOs.

PubMed

Fraiture, Marie-Alice; Herman, Philippe; Papazova, Nina; De Loose, Marc; Deforce, Dieter; Ruttink, Tom; Roosens, Nancy H

2017-10-01

Recently, we developed a DNA walking system for the detection and characterization of a broad spectrum of GMOs in routine analysis of food/feed matrices. Here, we present a new version with improved throughput and sensitivity by coupling the DNA walking system to Pacific Bioscience® Next-generation sequencing technology. The performance of the new strategy was thoroughly assessed through several assays. First, we tested its detection and identification capability on grains with high or low GMO content. Second, the potential impacts of food processing were investigated using rice noodle samples. Finally, GMO mixtures and a real-life sample were analyzed to illustrate the applicability of the proposed strategy in routine GMO analysis. In all tested samples, the presence of multiple GMOs was unambiguously proven by the characterization of transgene flanking regions and the combinations of elements that are typical for transgene constructs. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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.

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.

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

PubMed Central

Asanov, Alexander; Zepeda, Angélica; Vaca, Luis

2012-01-01

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

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.

What Combined Measurements From Structures and Imaging Tell Us About DNA Damage Responses

PubMed Central

Brosey, Chris A.; Ahmed, Zamal; Lees-Miller, Susan P.; Tainer, John A.

2017-01-01

DNA damage outcomes depend upon the efficiency and fidelity of DNA damage responses (DDRs) for different cells and damage. As such, DDRs represent tightly regulated prototypical systems for linking nanoscale biomolecular structure and assembly to the biology of genomic regulation and cell signaling. However, the dynamic and multifunctional nature of DDR assemblies can render elusive the correlation between the structures of DDR factors and specific biological disruptions to the DDR when these structures are altered. In this chapter, we discuss concepts and strategies for combining structural, biophysical, and imaging techniques to investigate DDR recognition and regulation, and thus bridge sequence-level structural biochemistry to quantitative biological outcomes visualized in cells. We focus on representative DDR responses from PARP/PARG/AIF damage signaling in DNA single-strand break repair and nonhomologous end joining complexes in double-strand break repair. Methods with exemplary experimental results are considered with a focus on strategies for probing flexibility, conformational changes, and assembly processes that shape a predictive understanding of DDR mechanisms in a cellular context. Integration of structural and imaging measurements promises to provide foundational knowledge to rationally control and optimize DNA damage outcomes for synthetic lethality and for immune activation with resulting insights for biology and cancer interventions. PMID:28668129

Pool Purification

NASA Technical Reports Server (NTRS)

1988-01-01

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

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.

Formation of the southern Bay of Bengal cold pool

NASA Astrophysics Data System (ADS)

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

2016-09-01

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

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

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.

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.

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.

Exciting Pools

ERIC Educational Resources Information Center

Wright, Bradford L.

1975-01-01

Advocates the creation of swimming pool oscillations as part of a general investigation of mechanical oscillations. Presents the equations, procedure for deriving the slosh modes, and methods of period estimation for exciting swimming pool oscillations. (GS)

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…

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.

Apparatus for draining lower drywell pool water into suppresion pool in boiling water reactor

DOEpatents

Gluntz, Douglas M.

1996-01-01

An apparatus which mitigates temperature stratification in the suppression pool water caused by hot water drained into the suppression pool from the lower drywell pool. The outlet of a spillover hole formed in the inner bounding wall of the suppression pool is connected to and in flow communication with one end of piping. The inlet end of the piping is above the water level in the suppression pool. The piping is routed down the vertical downcomer duct and through a hole formed in the thin wall separating the downcomer duct from the suppression pool water. The piping discharge end preferably has an elevation at or near the bottom of the suppression pool and has a location in the horizontal plane which is removed from the point where the piping first emerges on the suppression pool side of the inner bounding wall of the suppression pool. This enables water at the surface of the lower drywell pool to flow into and be discharged at the bottom of the suppression pool.

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

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

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.

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.

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

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

[Applylication of new type combined fragments: nrDNA ITS+ nad 1-intron 2 for identification of Dendrobium species of Fengdous].

PubMed

Geng, Li-xia; Zheng, Rui; Ren, Jie; Niu, Zhi-tao; Sun, Yu-long; Xue, Qing-yun; Liu, Wei; Ding, Xiao-yu

2015-08-01

In this study, 17 kinds of Dendrobium species of Fengdous including 39 individuals were collected from 4 provinces. Mitochondrial gene sequences co I, nad 5, nad 1-intron 2 and chloroplast gene sequences rbcL, matK amd psbA-trnH were amplified from these materials, as well as nrDNA ITS. Furthermore, suitable sequences for identification of Dendrobium species of Fengdous were screened by K-2-P and P-distance. The results showed that during the mentioned 7 sequences, nrDNA ITS, nad 1-intron 2 and psbA-trnH which had a high degree of variability could be used to identify Dendrobium species of Fengdous. However, single fragment could not be used to distinguish D. moniliforme and D. huoshanense. Moreover, compared to other combined fragments, new type combined fragments nrDNA ITS+nad 1-intron 2 was more effective in identifying the original plants of Dendrobium species and could be used to identify D. huoshanense and D. moniliforme. Besides, according to the UPGMA tree constructed with nrDNA ITS+nad 1-intron 2, 3 inspected Dendrobium plants were identified as D. huoshanense, D. moniliforme and D. officinale, respectively. This study identified Dendrobium species of Fengdous by combined fragments nrDNA ITS+nad 1-intron 2 for the first time, which provided a more effective basis for identification of Dendrobium species. And this study will be helpful for regulating the market of Fengdous.

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

Combination of DNA-based and conventional methods to detect human leukocyte antigen polymorphism and its use for paternity testing.

PubMed

Kereszturya, László; Rajczya, Katalin; Lászikb, András; Gyódia, Eva; Pénzes, Mária; Falus, András; Petrányia, Gyõzõ G

2002-03-01

In cases of disputed paternity, the scientific goal is to promote either the exclusion of a falsely accused man or the affiliation of the alleged father. Until now, in addition to anthropologic characteristics, the determination of genetic markers included human leukocyte antigen gene variants; erythrocyte antigens and serum proteins were used for that reason. Recombinant DNA techniques provided a new set of highly variable genetic markers based on DNA nucleotide sequence polymorphism. From the practical standpoint, the application of these techniques to paternity testing provides greater versatility than do conventional genetic marker systems. The use of methods to detect the polymorphism of human leukocyte antigen loci significantly increases the chance of validation of ambiguous results in paternity testing. The outcome of 2384 paternity cases investigated by serologic and/or DNA-based human leukocyte antigen typing was statistically analyzed. Different cases solved by DNA typing are presented involving cases with one or two accused men, exclusions and nonexclusions, and tests of the paternity of a deceased man. The results provide evidence for the advantage of the combined application of various techniques in forensic diagnostics and emphasizes the outstanding possibilities of DNA-based assays. Representative examples demonstrate the strength of combined techniques in paternity testing.

Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection

PubMed Central

Qiu, Jieqiong; Wilson, Adam; El-Sagheer, Afaf H.; Brown, Tom

2016-01-01

A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics. PMID:27369379

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

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.

Combination Platinum-based and DNA Damage Response-targeting Cancer Therapy: Evolution and Future Directions

PubMed Central

Basourakos, Spyridon P.; Li, Likun; Aparicio, Ana M.; Corn, Paul G.; Kim, Jeri; Thompson, Timothy C.

2017-01-01

Maintenance of genomic stability is a critical determinant of cell survival and is necessary for growth and progression of malignant cells. Interstrand crosslinking (ICL) agents, including platinum-based agents, are first-line chemotherapy treatment for many solid human cancers. In malignant cells, ICL triggers the DNA damage response (DDR). When the damage burden is high and lesions cannot be repaired, malignant cells are unable to divide and ultimately undergo cell death either through mitotic catastrophe or apoptosis. The activities of ICL agents, in particular platinum-based therapies, establish a “molecular landscape,” i.e., a pattern of DNA damage that can potentially be further exploited therapeutically with DDR-targeting agents. If the molecular landscape created by platinum-based agents could be better defined at the molecular level, a systematic, mechanistic rationale(s) could be developed for the use of DDR-targeting therapies in combination/maintenance protocols for specific, clinically advanced malignancies. New therapeutic drugs such as poly(ADP-ribose) polymerase (PARP) inhibitors are examples of DDR-targeting therapies that could potentially increase the DNA damage and replication stress imposed by platinum-based agents in tumor cells and provide therapeutic benefit for patients with advanced malignancies. Recent studies have shown that the use of PARP inhibitors together with platinum-based agents is a promising therapy strategy for ovarian cancer patients with ”BRCAness”, i.e., a phenotypic characteristic of tumors that not only can involve loss-of-function mutations in either BRCA1 or BRCA2, but also encompasses the molecular features of BRCA-mutant tumors. On the basis of these promising results, additional mechanism-based studies focused on the use of various DDR-targeting therapies in combination with platinum-based agents should be considered. This review discusses, in general, (1) ICL agents, primarily platinum-based agents, that

ALOS2-Indonesia REDD+ Experiment (AIREX): Soil Pool Carbon Application

NASA Astrophysics Data System (ADS)

Raimadoya, M.; Kristijono, A.; Sudiana, N.; Sumawinata, B.; Suwardi; Santoso, E.; Mahargo, D.; Sudarman, S.; Mattikainen, M.

2015-04-01

The bilateral REDD+ agreement between Indonesia and Norway [1] has scheduled that performance based result phase will be started in 2014. Therefore, a transparent and reliable Monitoring, Reporting and V erification (MRV) system for the following carbon pools: (1) biomass, (2) dead organic matter (DOM), and (3) soil, is required to be ready prior to the performance based phase. While the biomass pool could be acquired by space-borne radar (SAR) application i.e. SAR Interferometry (In-SAR) and Polarimetric SAR Interferometry (Pol-InSAR), the method for soil pool is still needed to be developed.A study was implemented in a test site located in the pulp plantation concession of Teluk Meranti Estate, Riau Andalan Pulp and Paper (RAPP), Pelalawan District, Riau Province, Indonesia. The study was intended to evaluate the possibility to estimate soil pool carbon with radar technology. For this purpose, a combination of spaceborne SAR (ALOS/PALSAR) and Ground Penetrating Radar (200 MHz IDS 200 MHz IDS GPR) were used in this exercise.The initial result this study provides a promising outcome for improved soil pool carbon estimation in tropical peat forest condition. The volume estimation of peat soil could be measured from the combination of spaceborne SAR and GPR. Based on this volume, total carbon content can be generated. However, the application of this approach has several limitation such as: (1) GPR survey can only be implemented during the dry season, (2) Rugged Terrain Antenna (RTA) type of GPR should be used for smooth GPR survey in the surface of peat soil which covered by DOM, and (3) the map of peat soil extent by spaceborne SAR need to be improved.

Transformation of primary human embryonic kidney cells to anchorage independence by a combination of BK virus DNA and the Harvey-ras oncogene

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

Pater, A.; Pater, M.M.

Primary human embryonic kidney (HEK) cells were transformed by a focus assay with BK virus (BKV) DNA molecularly cloned at its unique EcoRI site. Both viral DNA sequences and viral tumor antigens were present and expressed in all the foci that the authors examined. However, cells isolated from foci were incapable of growth in soft agar. They then examined the transformation of HEK cells after their transfection with a combination of BKV DNA and either the normal or the activated form of the human Ha-ras oncogene (EJ c-Ha-ras-1). Only the cells transfected with a combination of BKV DNA and themore » activated form of Ha-ras DNAs were present in the transformed colonies. BKV tumor antigens and the Ha-ras p21 protein were also expressed.« less

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.

In vitro DNA binding studies of lenalidomide using spectroscopic in combination with molecular docking techniques

NASA Astrophysics Data System (ADS)

Xu, Liang; Hu, Yan-Xi; Li, Yan-Cheng; Zhang, Li; Ai, Hai-Xin; Liu, Yu-Feng; Liu, Hong-Sheng

2018-02-01

In the present work, the binding interaction between lenalidomide (LEN) and calf thymus DNA (ct-DNA) was systematically studied by using fluorescence, ultraviolet-visible (UV-vis) absorption, circular dichroism (CD) spectroscopies under imitated physiological conditions (pH = 7.4) coupled with molecular docking. It was found that LEN was bound to ct-DNA with high binding affinity (Ka = 2.308 × 105 M-1 at 283 K) through groove binding as evidenced by a slight decrease in the absorption intensity in combination with CD spectra. Thermodynamic parameters (ΔG < 0, ΔH > 0 and ΔS < 0) of the LEN-DNA system obtained at three different temperatures suggested that the binding process was spontaneous and was primarily driven by hydrogen bonds and hydrophobic interaction. Furthermore, competitive binding experiments with ethidium bromide and 4‧, 6-dia-midino-2-phenylindoleas probes showed that LEN could preferentially bind in the minor groove of double-stranded DNA. The average lifetime of LEN was calculated to be 7.645 ns. The φ of LEN was measured as 0.09 and non-radiation energy transfer between LEN and DNA had occurred. The results of the molecular docking were consistent with the experimental results. This study explored the potential applicability of the spectroscopic properties of LEN and also investigated its interactions with relevant biological targets. In addition, it will provide some theoretical references for the deep research of simultaneous administration of LEN with other drugs.

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

Diving into Cold Pools

NASA Astrophysics Data System (ADS)

van den Heever, S. C.; Grant, L. D.; Drager, A. J.

2017-12-01

Cold pools play a significant role in convective storm initiation, organization and longevity. Given their role in convective life cycles, recent efforts have been focused on improving the representation of cold pool processes within weather forecast models, as well as on developing cold pool parameterizations in order to better represent their impacts within global climate models. Understanding the physical processes governing cold pool formation, intensity and dissipation is therefore critical to these efforts. Cold pool characteristics are influenced by numerous factors, including those associated with precipitation formation and evaporation, variations in the environmental moisture and shear, and land surface interactions. The focus of this talk will be on the manner in which the surface characteristics and associated processes impact cold pool genesis and dissipation. In particular, the results from high-resolution modeling studies focusing on the role of sensible and latent heat fluxes, soil moisture and SST will be presented. The results from a recent field campaign examining cold pools over northern Colorado will also be discussed.

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.

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.

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

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)

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.

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

A Biosignature Suite from Cave Pool Precipitates, Cottonwood Cave, New Mexico

NASA Astrophysics Data System (ADS)

Melim, L. A.; Liescheidt, R.; Northup, D. E.; Spilde, M. N.; Boston, P. J.; Queen, J. M.

2009-11-01

Calcite cave pool precipitates often display a variety of potential biosignatures from the macroscopic to the submicroscopic. A fossil cave pool in Cottonwood Cave, New Mexico, exhibits older stalactites and stalagmites that are completely coated in brown, laminated calcitic crust that extends down as pool fingers and u-loops. The pool fingers and u-loops are mainly micrite to clotted micrite, some recrystallized to microspar, with some isopachous spar layers. Micrite, particularly clotted micrite, is usually interpreted by carbonate workers as microbial in origin. Scanning electron microscopy examination of etched pool fingers, u-loops, and the brown crust revealed abundant calcified microbial filaments and biofilm. Energy dispersive X-ray analysis showed that these features have excess carbon, above that found in pure calcite. Independent carbon analysis indicated that these same samples contain up to 0.2% organic carbon. Since pool fingers hang down but form underwater, we hypothesize they are biogenic with hanging microbial filaments or biofilm acting as nuclei for calcite precipitation. Because of the abundance of micrite and fossil filaments, we further hypothesize that these pendant features formed during a period of plentiful nutrients and active hydrological activity when the pool was literally dripping with microbial slime. Although each of these lines of evidence could be interpreted in other ways, their combined weight strongly suggests the cave pool precipitates in Cottonwood Cave are biogenic. These investigations can be used to help inform extraterrestrial life-detection studies.

A biosignature suite from cave pool precipitates, Cottonwood Cave, New Mexico.

PubMed

Melim, L A; Liescheidt, R; Northup, D E; Spilde, M N; Boston, P J; Queen, J M

2009-11-01

Calcite cave pool precipitates often display a variety of potential biosignatures from the macroscopic to the submicroscopic. A fossil cave pool in Cottonwood Cave, New Mexico, exhibits older stalactites and stalagmites that are completely coated in brown, laminated calcitic crust that extends down as pool fingers and u-loops. The pool fingers and u-loops are mainly micrite to clotted micrite, some recrystallized to microspar, with some isopachous spar layers. Micrite, particularly clotted micrite, is usually interpreted by carbonate workers as microbial in origin. Scanning electron microscopy examination of etched pool fingers, u-loops, and the brown crust revealed abundant calcified microbial filaments and biofilm. Energy dispersive X-ray analysis showed that these features have excess carbon, above that found in pure calcite. Independent carbon analysis indicated that these same samples contain up to 0.2% organic carbon. Since pool fingers hang down but form underwater, we hypothesize they are biogenic with hanging microbial filaments or biofilm acting as nuclei for calcite precipitation. Because of the abundance of micrite and fossil filaments, we further hypothesize that these pendant features formed during a period of plentiful nutrients and active hydrological activity when the pool was literally dripping with microbial slime. Although each of these lines of evidence could be interpreted in other ways, their combined weight strongly suggests the cave pool precipitates in Cottonwood Cave are biogenic. These investigations can be used to help inform extraterrestrial life-detection studies.

Pool spacing in forest channels

Treesearch

David R. Montgomery; John M. Buffington; Richard D. Smith; Kevin M. Schmidt; George Pess

1995-01-01

Field surveys of stream channels in forested mountain drainage basins in southeast Alaska and Washington reveal that pool spacing depends on large woody debris (LWD) loading and channel type, slope, and width. Mean pool spacing in pool-riffle, plane-bed, and forced pool-riffle channels systematically decreases from greater than 13 channel widths per pool to less than 1...

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.

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.

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.

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.

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

PubMed Central

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

2014-01-01

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

Swimming pool granuloma

MedlinePlus

... this page: //medlineplus.gov/ency/article/001357.htm Swimming pool granuloma To use the sharing features on this page, please enable JavaScript. A swimming pool granuloma is a long-term (chronic) skin ...

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

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.

A Mechanistic Model of Waterfall Plunge Pool Erosion into Bedrock

NASA Astrophysics Data System (ADS)

Scheingross, Joel S.; Lamb, Michael P.

2017-11-01

Landscapes often respond to changes in climate and tectonics through the formation and upstream propagation of knickzones composed of waterfalls. Little work has been done on the mechanics of waterfall erosion, and instead most landscape-scale models neglect waterfalls or use rules for river erosion, such as stream power, that may not be applicable to waterfalls. Here we develop a physically based model to predict waterfall plunge pool erosion into rock by abrasion from particle impacts and test the model against flume experiments. Both the model and experiments show that evolving plunge pools have initially high vertical erosion rates due to energetic particle impacts, and erosion slows and eventually ceases as pools deepen and deposition protects the pool floor from further erosion. Lateral erosion can continue after deposition on the pool floor, but it occurs at slow rates that become negligible as pools widen. Our work points to the importance of vertical drilling of successive plunge pools to drive upstream knickzone propagation in homogenous rock, rather than the classic mechanism of headwall undercutting. For a series of vertically drilling waterfalls, we find that upstream knickzone propagation is faster under higher combined water and sediment fluxes and for knickzones composed of many waterfalls that are closely spaced. Our model differs significantly from stream-power-based erosion rules in that steeper knickzones can retreat faster or more slowly depending on the number and spacing of waterfalls within a knickzone, which has implications for interpreting climatic and tectonic history through analysis of river longitudinal profiles.

Designing ARVs Patent Pool Up to Trade & Policy Evolutionary Dynamics

PubMed Central

Dionisio, Daniele; Racalbuto, Vincenzo; Messeri, Daniela

2010-01-01

Patent pools for second and third-line Fixed Dose Combination (FDC) antiretroviral drugs (ARVs) should not be delayed as they are instrumental to urgent public health needs in the under-served markets. Nonetheless, multinational originator companies still seem to perceive patent pooling for ARVs as a minefield that would offer the generic competitors lots of deeply exploitable opportunities, to the detriment of patent owner’s rights. This paper analyses the brand industry concerns, while looking for a strategy up to a really equitable and free world market, without any discrimination between end-users in wealthy and resource-limited countries. This strategy would urge partnerships between originator companies first to make newer FDC ARVs quickly available and allow patent pool agreements with generic counterparts to be negotiated straight afterwards. The patent pool strategy highlighted in this paper would assert the primacy of health over for-profit policies, while aligning with the 61st WHO’s Assembly recommendations and G7, G8 and World Trade Organisation’s warnings and pledges against trade protectionism. PMID:20200604

Combination Platinum-based and DNA Damage Response-targeting Cancer Therapy: Evolution and Future Directions.

PubMed

Basourakos, Spyridon P; Li, Likun; Aparicio, Ana M; Corn, Paul G; Kim, Jeri; Thompson, Timothy C

2017-01-01

Maintenance of genomic stability is a critical determinant of cell survival and is necessary for growth and progression of malignant cells. Interstrand crosslinking (ICL) agents, including platinum-based agents, are first-line chemotherapy treatment for many solid human cancers. In malignant cells, ICL triggers the DNA damage response (DDR). When the damage burden is high and lesions cannot be repaired, malignant cells are unable to divide and ultimately undergo cell death either through mitotic catastrophe or apoptosis. The activities of ICL agents, in particular platinum-based therapies, establish a "molecular landscape," i.e., a pattern of DNA damage that can potentially be further exploited therapeutically with DDR-targeting agents. If the molecular landscape created by platinum-based agents could be better defined at the molecular level, a systematic, mechanistic rationale(s) could be developed for the use of DDR-targeting therapies in combination/maintenance protocols for specific, clinically advanced malignancies. New therapeutic drugs such as poly(ADP-ribose) polymerase (PARP) inhibitors are examples of DDR-targeting therapies that could potentially increase the DNA damage and replication stress imposed by platinum-based agents in tumor cells and provide therapeutic benefit for patients with advanced malignancies. Recent studies have shown that the use of PARP inhibitors together with platinum-based agents is a promising therapy strategy for ovarian cancer patients with "BRCAness", i.e., a phenotypic characteristic of tumors that not only can involve loss-of-function mutations in either BRCA1 or BRCA2, but also encompasses the molecular features of BRCA-mutant tumors. On the basis of these promising results, additional mechanism-based studies focused on the use of various DDR-targeting therapies in combination with platinum-based agents should be considered. This review discusses, in general, (1) ICL agents, primarily platinum-based agents, that establish a

Identification of DNA primase inhibitors via a combined fragment-based and virtual screening

NASA Astrophysics Data System (ADS)

Ilic, Stefan; Akabayov, Sabine R.; Arthanari, Haribabu; Wagner, Gerhard; Richardson, Charles C.; Akabayov, Barak

2016-11-01

The structural differences between bacterial and human primases render the former an excellent target for drug design. Here we describe a technique for selecting small molecule inhibitors of the activity of T7 DNA primase, an ideal model for bacterial primases due to their common structural and functional features. Using NMR screening, fragment molecules that bind T7 primase were identified and then exploited in virtual filtration to select larger molecules from the ZINC database. The molecules were docked to the primase active site using the available primase crystal structure and ranked based on their predicted binding energies to identify the best candidates for functional and structural investigations. Biochemical assays revealed that some of the molecules inhibit T7 primase-dependent DNA replication. The binding mechanism was delineated via NMR spectroscopy. Our approach, which combines fragment based and virtual screening, is rapid and cost effective and can be applied to other targets.

From antidunes to step-pools

NASA Astrophysics Data System (ADS)

Recking, Alain; Leduc, Pauline

2014-05-01

Step-pools are bed morphologies that are typical in high-gradient streams , recognizable by a staircase-like longitudinal profile resulting from accumulation of cobbles and boulders that are transverse to the channel and alternating with pools containing finer sediments. Within the last two decades step-pools have been the subject of increased efforts to characterize their nature; however their origin is still in debate. Researchers have very soon suspected step-pools to be the residual form of antidunes produced during flooding, but this hypothesis was continuously contested. Other theories has been proposed, considering, that step-pool profile develops a maximum flow resistance, or that pools geometry is controlled by the energy of a falling jet, or that steps form by boulders accumulation in a channel-spanning manner. All these theories gave very satisfying results when compared with experimental data, but does it mean that the antidune theory should we abandoned? We performed new flume experiments on steep slopes to investigate the antidune origin for step-pools. Our experiments showed that step-pools can have several origins, depending on the flow conditions and sediment mixture used. In some circumstances antidunes were well observed but did not produce stable step-pools morphology. In many occasions, step-pools obtained in the flume were isolated step-pools, with no real apparent periodicity. Only a few flow and sediment conditions allowed us to reproduce trains of antidunes which stabilized at the flow recession to produce stable periodical step-pools. These conditions are presented and discussed.

Interactions between pool geometry and hydraulics

USGS Publications Warehouse

Thompson, Douglas M.; Nelson, Jonathan M.; Wohl, Ellen E.

1998-01-01

An experimental and computational research approach was used to determine interactions between pool geometry and hydraulics. A 20-m-long, 1.8-m-wide flume was used to investigate the effect of four different geometric aspects of pool shape on flow velocity. Plywood sections were used to systematically alter constriction width, pool depth, pool length, and pool exit-slope gradient, each at two separate levels. Using the resulting 16 unique geometries with measured pool velocities in four-way factorial analyses produced an empirical assessment of the role of the four geometric aspects on the pool flow patterns and hence the stability of the pool. To complement the conclusions of these analyses, a two-dimensional computational flow model was used to investigate the relationships between pool geometry and flow patterns over a wider range of conditions. Both experimental and computational results show that constriction and depth effects dominate in the jet section of the pool and that pool length exhibits an increasing effect within the recirculating-eddy system. The pool exit slope appears to force flow reattachment. Pool length controls recirculating-eddy length and vena contracta strength. In turn, the vena contracta and recirculating eddy control velocities throughout the pool.

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.

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.

13 CFR 120.1706 - Pool Originator's retained interest in Pool.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Pool Originator's retained interest in Pool. 120.1706 Section 120.1706 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION BUSINESS LOANS Establishment of SBA Secondary Market Guarantee Program for First Lien Position 504 Loan...

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

PubMed Central

2014-01-01

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

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.

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.

Observations of Cold Pool Properties during GoAmazon2014/5

NASA Astrophysics Data System (ADS)

Mayne, S. L.; Schumacher, C.; MacDonald, L.; Turner, D. D.

2017-12-01

Convectively generated cold pools are instrumental in both the development of the sub-cloud layer and the organization of deep convection. Despite this, analyses of cold pools in the tropics are constrained by a lack of observational data; insight into the phenomena therefore relies heavily on numerical models. GoAmazon2014/5, a 2-year DOE-sponsored field campaign centered on Manacapuru, Brazil in the central Amazon, provides a unique opportunity to characterize tropical cold pools and allows for the comparison of observational data with theoretical results from model cold pool simulations and parameterizations. This investigation analyzes radar, disdrometer, and profiler measurements at the DOE mobile facility site to study tropical cold pool characteristics. The Brazilian military (SIPAM) operational S-band radar in Manaus is used to provide a broad context of convective systems, while measurements from Parsivel disdrometers are used to assess drop-size distributions (DSDs) at the surface. A unique aspect of this research is the use of the Atmospheric Emitted Radiance Interferometer (AERI) instrument, which utilizes down-welling IR measurements to obtain vertical profiles of thermodynamic quantities such as temperature and water vapor in the lowest few km of the atmosphere. Combined with surface observations and sounding data, these datasets will result in a thorough investigation of the horizontal and vertical characteristics of cold pools over the tropical rain forest. Preliminary analyses of 20 events reveal a mean cold pool height of 220 m and a mean radius of approximately 8.5 km. The average cold pool experienced a temperature (specific humidity) decrease of approximately 1 K (0.4 g/kg) at the surface. The temperature decrease is consistent with modeling studies and limited observations from previous studies over the tropics. The small decrease in specific humidity is attributed to the high moisture content within the cold pools. AERI retrievals of

Second-line chemotherapy in advanced biliary cancer progressed to first-line platinum-gemcitabine combination: a multicenter survey and pooled analysis with published data.

PubMed

Fornaro, Lorenzo; Vivaldi, Caterina; Cereda, Stefano; Leone, Francesco; Aprile, Giuseppe; Lonardi, Sara; Silvestris, Nicola; Santini, Daniele; Milella, Michele; Caparello, Chiara; Musettini, Gianna; Pasquini, Giulia; Falcone, Alfredo; Brandi, Giovanni; Sperduti, Isabella; Vasile, Enrico

2015-12-23

After progression to a standard first-line platinum and gemcitabine combination (GP), there is no established second-line therapy for patients with advanced biliary tract cancers (aBTC). Indeed, literature data suggest limited activity of most second-line agents evaluated so far. We collected a large retrospective series of aBTC patients treated with second-line chemotherapy after progression to a first-line GP regimen at different Italian institutions. We then pooled the data with those reported in previous studies, which were identified with a Medline search and the on-line abstract datasets of major international oncology meetings. A total of 174 patients were included in the multicenter survey: response rate (RR) with second-line chemotherapy was low (3.4 %), with median PFS and OS of 3.0 months and 6.6 months, respectively. At multivariate analysis, preserved performance status, low CA19.9 levels and absence of distant metastases were favorable prognostic factors. Data from other five presented or published series were identified, for a total of 499 patients included in the pooled analysis. The results confirmed marginal activity of second-line chemotherapy (RR: 10.2 %), with limited efficacy in unselected patient populations (median PFS: 3.1 months; median OS: 6.3 months). The current analysis highlights the limited value of second-line chemotherapy after a first-line GP combination in aBTC. While waiting for effective biologic agents in this setting, ongoing randomized trials will identify the optimal second-line chemotherapy regimen and validate prognostic factors for individual patient management.

Energy Pooling Upconversion in Free Space and Optical Cavities

NASA Astrophysics Data System (ADS)

LaCount, Michael D.

The ability to efficiently convert the wavelength of light has value in a wide range of disciplines that include the fields of photovoltaics, plant growth, optics and medicine. The processes by which such transformations are carried out are known as upconversions and downconversions. There are several ways to up/down convert light, each with its own attributes, issues, and competing mechanisms. Most are associated with one-body or two-body processes. Three-body dynamics are also possible though, going by the names of quantum cutting (downconversion) and energy pooling (upconversion). These use virtual excited electronic states to mediate conversions as has been experimentally realized using lanthanide ions embedded in wide bandgap materials. The use of lanthanides to convert light is not ideal due to their relative scarcity, toxicity, and the limited range of light frequencies that can be absorbed and emitted. Organic molecules, on the other hand, are typically non-toxic, are made up of abundant elements, and can be designed with tailored spectral properties. At issue is whether or not they can be used to carry out efficient energy pooling, the central question to be answered in this thesis. The research presented here draws on a perturbative quantum electrodynamics framework previously established for generic energy pooling. It was used to develop a computational methodology for determining the rate of energy pooling and its competing processes. This, in turn, draws on a combination of time-dependent density functional theory, quantum electrodynamics, and perturbation theory to generate the requisite material property data. This computational model was applied to two test systems consisting of stilbene-fluorescein and hexabenzocoronene-oligothiophene. The stilbene-fluorescein system was found to have a maximum energy pooling rate efficiency (as compared to competing processes) of 17% and the hexabenzocoronene-oligothiophene system was found to have a maximum

Sparse feature learning for instrument identification: Effects of sampling and pooling methods.

PubMed

Han, Yoonchang; Lee, Subin; Nam, Juhan; Lee, Kyogu

2016-05-01

Feature learning for music applications has recently received considerable attention from many researchers. This paper reports on the sparse feature learning algorithm for musical instrument identification, and in particular, focuses on the effects of the frame sampling techniques for dictionary learning and the pooling methods for feature aggregation. To this end, two frame sampling techniques are examined that are fixed and proportional random sampling. Furthermore, the effect of using onset frame was analyzed for both of proposed sampling methods. Regarding summarization of the feature activation, a standard deviation pooling method is used and compared with the commonly used max- and average-pooling techniques. Using more than 47 000 recordings of 24 instruments from various performers, playing styles, and dynamics, a number of tuning parameters are experimented including the analysis frame size, the dictionary size, and the type of frequency scaling as well as the different sampling and pooling methods. The results show that the combination of proportional sampling and standard deviation pooling achieve the best overall performance of 95.62% while the optimal parameter set varies among the instrument classes.

Convenient yet not a convenience sample: Jury pools as experimental subject pools.

PubMed

Murray, Gregg R; Rugeley, Cynthia R; Mitchell, Dona-Gene; Mondak, Jeffery J

2013-01-01

Scholars greatly benefit from access to convenient, inexpensive data sources. Many researchers rely on student subject pools, a practice that raises concern about the "college sophomore problem," or the possibility that findings from student subjects do not generalize beyond the campus. As an accessible, low cost, and heterogeneous data source, some researchers have used subjects recruited from jury pools, which are drawn from randomly-selected citizens required by law to appear for jury duty. In this paper, we discuss the strengths and weaknesses of this approach. First, we review pragmatic considerations involving access to jury pools, substantive content, the administration of survey-experiments, and the financial costs and benefits of this approach. Next, we present evidence regarding the quality of jury pool samples in terms of response rates, diversity, and representativeness. We conclude that jury pools, given proper attention to their limitations, offer an attractive addition to the viable sources of experimental data. Copyright © 2012 Elsevier Inc. All rights reserved.

Vernal Pool Lessons and Activities.

ERIC Educational Resources Information Center

Childs, Nancy; Colburn, Betsy

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

Morphology of drying blood pools

NASA Astrophysics Data System (ADS)

Laan, Nick; Smith, Fiona; Nicloux, Celine; Brutin, David; D-Blood project Collaboration

2016-11-01

Often blood pools are found on crime scenes providing information concerning the events and sequence of events that took place on the scene. However, there is a lack of knowledge concerning the drying dynamics of blood pools. This study focuses on the drying process of blood pools to determine what relevant information can be obtained for the forensic application. We recorded the drying process of blood pools with a camera and measured the weight. We found that the drying process can be separated into five different: coagulation, gelation, rim desiccation, centre desiccation, and final desiccation. Moreover, we found that the weight of the blood pool diminishes similarly and in a reproducible way for blood pools created in various conditions. In addition, we verify that the size of the blood pools is directly related to its volume and the wettability of the surface. Our study clearly shows that blood pools dry in a reproducible fashion. This preliminary work highlights the difficult task that represents blood pool analysis in forensic investigations, and how internal and external parameters influence its dynamics. We conclude that understanding the drying process dynamics would be advancement in timeline reconstitution of events. ANR funded project: D-Blood Project.

Curcumin stably interacts with DNA hairpin through minor groove binding and demonstrates enhanced cytotoxicity in combination with FdU nucleotides.

PubMed

Ghosh, Supratim; Mallick, Sumana; Das, Upasana; Verma, Ajay; Pal, Uttam; Chatterjee, Sabyasachi; Nandy, Abhishek; Saha, Krishna D; Maiti, Nakul Chandra; Baishya, Bikash; Suresh Kumar, G; Gmeiner, William H

2018-03-01

We report, based on biophysical studies and molecular mechanical calculations that curcumin binds DNA hairpin in the minor groove adjacent to the loop region forming a stable complex. UV-Vis and fluorescence spectroscopy indicated interaction of curcumin with DNA hairpin. In this novel binding motif, two ɣ H of curcumin heptadiene chain are closely positioned to the A 16 -H8 and A 17 -H8, while G 12 -H8 is located in the close proximity of curcumin α H. Molecular dynamics (MD) simulations suggest, the complex is stabilized by noncovalent forces including; π-π stacking, H-bonding and hydrophobic interactions. Nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics simulations indicated curcumin is bound in the minor groove, while circular dichroism (CD) spectra suggested minute enhancement in base stacking and a little change in DNA helicity, without significant conformational change of DNA hairpin structure. The DNA:curcumin complex formed with FdU nucleotides rather than Thymidine, demonstrated enhanced cytotoxicity towards oral cancer cells relative to the only FdU substituted hairpin. Fluorescence co-localization demonstrated stability of the complex in biologically relevant conditions, including its cellular uptake. Acridine orange/EtBr staining further confirmed the enhanced cytotoxic effects of the complex, suggesting apoptosis as mode of cell death. Thus, curcumin can be noncovalently complexed to small DNA hairpin for cellular delivery and the complex showed increased cytotoxicity in combination with FdU nucleotides, demonstrating its potential for advanced cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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.

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.

Nanomaterials Based on DNA

PubMed Central

Seeman, Nadrian C.

2012-01-01

The combination of synthetic stable branched DNA and sticky ended cohesion has led to the development of structural DNA nanotechnology over the past 30 years. The basis of this enterprise is that it is possible to construct novel DNA-based materials by combining these features in a self-assembly protocol. Thus, simple branched molecules lead directly to the construction of polyhedra whose edges consist of double helical DNA, and whose vertices correspond to the branch points. Stiffer branched motifs can be used to produce self-assembled two-dimensional and three-dimensional periodic lattices of DNA (crystals). DNA has also been used to make a variety of nanomechanical devices, including molecules that change their shapes, and molecules that can walk along a DNA sidewalk. Devices have been incorporated into two-dimensional DNA arrangements; sequence-dependent devices are driven by increases in nucleotide pairing at each step in their machine cycles. PMID:20222824

The Madden-Julian Oscillation and the Indo-Pacific Warm Pool

NASA Astrophysics Data System (ADS)

Raymond, David J.; Fuchs, Željka

2018-04-01

A minimal model of the interaction of the Madden-Julian oscillation (MJO) with the Indo-Pacific warm pool is presented. This model is based on the linear superposition of the flow associated with a highly simplified treatment of the MJO plus the flow induced by the warm pool itself. Both of these components parameterize rainfall as proportional to the column water vapor, which in turn is governed by a linearized moisture equation in which WISHE (wind induced surface heat exchange) plays a governing role. The MJO component has maximum growth rate for planetary wavenumber 1 and is equatorially trapped with purely zonal winds. The warm pool component exhibits a complex flow pattern, differing significantly from the classical Gill model as a result of the mean easterly flow. The combination of the two produce a flow that reproduces many aspects of the observed global flow associated with the MJO.

Data Pool Description

Atmospheric Science Data Center

2016-04-29

ASDC Data Pool   Notices   • DataPool will transition from ...  • Use IE7 for FTP sessions: a) Select "View", "Open FTP site in Windows Explorer" or b) Open Windows Explorer and enter the URL for the FTP site in the address bar ...

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

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

UVA photoactivation of DNA containing halogenated thiopyrimidines induces cytotoxic DNA lesions

PubMed Central

Brem, Reto; Zhang, Xiaohui; Xu, Yao-Zhong; Karran, Peter

2015-01-01

Photochemotherapy, the combination of a photosensitiser and ultraviolet (UV) or visible light, is an effective treatment for skin conditions including cancer. The high mutagenicity and non-selectivity of photochemotherapy regimes warrants the development of alternative approaches. We demonstrate that the thiopyrimidine nucleosides 5-bromo-4-thiodeoxyuridine (SBrdU) and 5-iodo-4-thiodeoxyuridine (SIdU) are incorporated into the DNA of cultured human and mouse cells where they synergistically sensitise killing by low doses of UVA radiation. The DNA halothiopyrimidine/UVA combinations induce DNA interstrand crosslinks, DNA-protein crosslinks, DNA strand breaks, nucleobase damage and lesions that resemble UV-induced pyrimidine(6-4)pyrimidone photoproducts. These are potentially lethal DNA lesions and cells defective in their repair are hypersensitive to killing by SBrdU/UVA and SIdU/UVA. DNA SIdU and SBrdU generate lethal DNA photodamage by partially distinct mechanisms that reflect the different photolabilities of their C–I and C–Br bonds. Although singlet oxygen is involved in photolesion formation, DNA SBrdU and SIdU photoactivation does not detectably increase DNA 8-oxoguanine levels. The absence of significant collateral damage to normal guanine suggests that UVA activation of DNA SIdU or SBrdU might offer a strategy to target hyperproliferative skin conditions that avoids the extensive formation of a known mutagenic DNA lesion. PMID:25747491

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.

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

Anti-dsDNA antibodies in systemic lupus erythematosus: A combination of two quantitative methods and the ANA pattern is the most efficient strategy of detection.

PubMed

Almeida González, Delia; Roces Varela, Alfredo; Marcelino Rodríguez, Itahisa; González Vera, Alexander; Delgado Sánchez, Mónica; Aznar Esquivel, Antonio; Casañas Rodríguez, Carlos; Cabrera de León, Antonio

2015-12-01

Several methods have been used to measure anti-double-stranded DNA auto-antibody (anti-dsDNA). Our aim was to determine the most efficient strategy to test anti-dsDNA in systemic lupus erythematosus (SLE). In this study, anti-dsDNA and anti-nuclear antibody (ANA) tests were requested for 644 patients. Anti-dsDNA was tested by RIA, ELISA and CLIA in all patients. The results indicated that 78 patients had a positive anti-dsDNA test according to at least one of the methods. After a 3-year follow-up period only 26 patients were diagnosed with SLE. We evaluated each method and combination of methods. Specificity and positive predictive value (PPV) increased with the number of assay methods used (p=0.002 for trend), and PPV was 100% in patients whose results were positive by all three anti-dsDNA assay methods. The proportion of anti-dsDNA-positive patients who had SLE was highest (82%; p b 0.001) among those with a homogeneous pattern of ANA staining, followed by those with a speckled pattern. In ANA positive patients, when only RIA was considered, 59% of anti-dsDNA-positive patients had SLE, but when RIA and CLIA were both considered, all patients with positive results on both tests had SLE. The combination of RIA+CLIA in patients with homogeneous and speckled ANA staining showed a similar cost and higher sensitivity than RIA alone in ANA positive patients (p b 0.001). We conclude that the most efficient strategy was to combine simultaneously two quantitative and sensitive methods but only in patients with a homogeneous or speckled pattern of ANA staining. This approach maximized specificity and PPV, and reduced costs. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Laser microtreatment for genetic manipulations and DNA diagnostics by a combination of microbeam and photonic tweezers (laser microbeam trap)

NASA Astrophysics Data System (ADS)

Greulich, Karl-Otto; Monajembashi, Shamci; Celeda, D.; Endlich, N.; Eickhoff, Holger; Hoyer, Carsten; Leitz, G.; Weber, Gerd; Scheef, J.; Rueterjans, H.

1994-12-01

Genomes of higher organisms are larger than one typically expects. For example, the DNA of a single human cell is almost two meters long, the DNA in the human body covers the distance Earth-Sun approximately 140 times. This is often not considered in typical molecular biological approaches for DNA diagnostics, where usually only DNA of the length of a gene is investigated. Also, one basic aspect of sequencing the human genome is not really solved: the problem how to prepare the huge amounts of DNA required. Approaches from biomedical optics combined with new developments in single molecule biotechnology may at least contribute some parts of the puzzle. A large genome can be partitioned into portions comprising approximately 1% of the whole DNA using a laser microbeam. The single DNA fragment can be amplified by the polymerase chain reaction in order to obtain a sufficient amount of molecules for conventional DNA diagnostics or for analysis by octanucleotide hybridization. When not amplified by biotechnological processes, the individual DNA molecule can be visualized in the light microscope and can be manipulated and dissected with the laser microbeam trap. The DNA probes obtained by single molecule biotechnology can be employed for fluorescence in situ introduced into plant cells and subcellular structures even when other techniques fail. Since the laser microbeam trap allows to work in the interior of a cell without opening it, subcellular structures can be manipulated. For example, in algae, such structures can be moved out of their original position and used to study intracellular viscosities.

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.

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.

Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool.

PubMed

Nagy, Gergely N; Leveles, Ibolya; Vértessy, Beáta G

2014-09-01

The occurrence of modified bases in DNA is attributed to some major factors: incorporation of altered nucleotide building blocks and chemical reactions or radiation effects on bases within the DNA structure. Several enzyme families are involved in preventing the incorporation of noncanonical bases playing a 'sanitizing' role. The catalytic mechanism of action of these enzymes has been revealed for a number of representatives in clear structural and kinetic detail. In this review, we focus in detail on those examples where clear evidence has been produced using high-resolution structural studies. Comparing the protein fold and architecture of the enzyme active sites, two main classes of sanitizing deoxyribonucleoside triphosphate pyrophosphatases can be assigned that are distinguished by the site of nucleophilic attack. In enzymes associated with attack at the α-phosphorus, it is shown that coordination of the γ-phosphate group is also ensured by multiple interactions. By contrast, enzymes catalyzing attack at the β-phosphorus atom mainly coordinate the α- and the β-phosphate only. Characteristic differences are also observed with respect to the role of the metal ion cofactor (Mg(2+) ) and the coordination of nucleophilic water. Using different catalytic mechanisms embedded in different protein folds, these enzymes present a clear example of convergent evolution. © 2014 FEBS.

Combined quantum-mechanics/molecular-mechanics dynamics simulation of A-DNA double strands irradiated by ultra-low-energy carbon ions

NASA Astrophysics Data System (ADS)

Ngaojampa, C.; Nimmanpipug, P.; Yu, L. D.; Anuntalabhochai, S.; Lee, V. S.

2011-02-01

In order to promote understanding of the fundamentals of ultra-low-energy ion interaction with DNA, molecular dynamics simulations using combined quantum-mechanics/molecular-mechanics of poly-AT and poly-GC A-DNA double strands irradiated by <200 eV carbon ions were performed to investigate the molecular implications of mutation bias. The simulations were focused on the responses of the DNA backbones and nitrogenous bases to irradiation. Analyses of the root mean square displacements of the backbones and non-hydrogen atoms of base rings of the simulated DNA structure after irradiation revealed a potential preference of DNA double strand separation, dependent on the irradiating energy. The results show that for the backbones, the large difference in the displacement between poly-GC and poly-AT in the initial time period could be the reason for the backbone breakage; for the nitrogenous base pairs, A-T is 30% more sensitive or vulnerable to ion irradiation than G-C, demonstrating a preferential, instead of random, effect of irradiation-induced mutation.

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.

Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum.

PubMed

Oyebola, Kolapo M; Idowu, Emmanuel T; Olukosi, Yetunde A; Awolola, Taiwo S; Amambua-Ngwa, Alfred

2017-06-29

The burden of falciparum malaria is especially high in sub-Saharan Africa. Differences in pressure from host immunity and antimalarial drugs lead to adaptive changes responsible for high level of genetic variations within and between the parasite populations. Population-specific genetic studies to survey for genes under positive or balancing selection resulting from drug pressure or host immunity will allow for refinement of interventions. We performed a pooled sequencing (pool-seq) of the genomes of 100 Plasmodium falciparum isolates from Nigeria. We explored allele-frequency based neutrality test (Tajima's D) and integrated haplotype score (iHS) to identify genes under selection. Fourteen shared iHS regions that had at least 2 SNPs with a score > 2.5 were identified. These regions code for genes that were likely to have been under strong directional selection. Two of these genes were the chloroquine resistance transporter (CRT) on chromosome 7 and the multidrug resistance 1 (MDR1) on chromosome 5. There was a weak signature of selection in the dihydrofolate reductase (DHFR) gene on chromosome 4 and MDR5 genes on chromosome 13, with only 2 and 3 SNPs respectively identified within the iHS window. We observed strong selection pressure attributable to continued chloroquine and sulfadoxine-pyrimethamine use despite their official proscription for the treatment of uncomplicated malaria. There was also a major selective sweep on chromosome 6 which had 32 SNPs within the shared iHS region. Tajima's D of circumsporozoite protein (CSP), erythrocyte-binding antigen (EBA-175), merozoite surface proteins - MSP3 and MSP7, merozoite surface protein duffy binding-like (MSPDBL2) and serine repeat antigen (SERA-5) were 1.38, 1.29, 0.73, 0.84 and 0.21, respectively. We have demonstrated the use of pool-seq to understand genomic patterns of selection and variability in P. falciparum from Nigeria, which bears the highest burden of infections. This investigation identified known

Synergy of irofulven in combination with other DNA damaging agents: synergistic interaction with altretamine, alkylating, and platinum-derived agents in the MV522 lung tumor model.

PubMed

Kelner, Michael J; McMorris, Trevor C; Rojas, Rafael J; Estes, Leita A; Suthipinijtham, Pharnuk

2008-12-01

Irofulven (MGI 114, NSC 683863) is a semisynthetic derivative of illudin S, a natural product present in the Omphalotus illudins (Jack O'Lantern) mushroom. This novel agent produces DNA damage, that in contrast to other agents, is predominately ignored by the global genome repair pathway of the nucleotide excision repair (NER)(2) system. The aim of this study was to determine the antitumor activity of irofulven when administered in combination with 44 different DNA damaging agents, whose damage is in general detected and repaired by the genome repair pathway. The human lung carcinoma MV522 cell line and its corresponding xenograft model were used to evaluate the activity of irofulven in combination with different DNA damaging agents. Two main classes of DNA damaging agents, platinum-derived agents, and select bifunctional alkylating agents, demonstrated in vivo synergistic or super-additive interaction with irofulven. DNA helicase inhibiting agents also demonstrated synergy in vitro, but an enhanced interaction with irofulven could not be demonstrated in vivo. There was no detectable synergistic activity between irofulven and agents capable of inducing DNA cleavage or intercalating into DNA. These results indicate that the antitumor activity of irofulven is enhanced when combined with platinum-derived agents, altretamine, and select alkylating agents such as melphalan or chlorambucil. A common factor between these agents appears to be the production of intrastrand DNA crosslinks. The synergistic interaction between irofulven and other agents may stem from the nucleotide excision repair system being selectively overwhelmed at two distinct points in the pathway, resulting in prolonged stalling of transcription forks, and subsequent initiation of apoptosis.

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.

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

[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.

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

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.

Combined immunotherapy of breast cancer with EGF and VEGF vaccines from DNA shuffling in a mouse model.

PubMed

Jin, Dong; Yu, Xin; Chen, Bing; Li, Zhitao; Ding, Jia; Zhao, Xiuyun; Qi, Gaofu

2017-06-01

Development of EGF and VEGF vaccines with high antigenicity for combined immunotherapy of EGF-EGFR signaling-dependent epithelial tumors such as breast cancer. EGF genes from mouse, human and chicken were randomly assembled to chimeric genes by DNA shuffling, then a chimeric EGF was selected out by PCR, SDS-PAGE and immunization for combined immunotherapy of breast cancer with a previously constructed chimeric VEGF vaccine from shuffling. Combined vaccination with chimeric EGF and VEGF from shuffling could induce high titer of antibodies against EGF and VEGF to inhibit tumor growth and angiogenesis, and improve the survival rate of mice with breast cancer. Combined vaccination with EGF and VEGF from shuffling showed better immunotherapy on EGF-EGFR signaling-dependent epithelial tumors such as breast cancer than the single-agent EGF vaccination.

Design of hydrotherapy exercise pools.

PubMed

Edlich, R F; Abidin, M R; Becker, D G; Pavlovich, L J; Dang, M T

1988-01-01

Several hydrotherapy pools have been designed specifically for a variety of aquatic exercise. Aqua-Ark positions the exerciser in the center of the pool for deep-water exercise. Aqua-Trex is a shallow underwater treadmill system for water walking or jogging. Swim-Ex generates an adjustable laminar flow that permits swimming without turning. Musculoskeletal conditioning can be accomplished in the above-ground Arjo shallow-water exercise pool. A hydrotherapy pool also can be custom designed for musculoskeletal conditioning in its shallow part and cardiovascular conditioning in a deeper portion of the pool. Regardless of the type of exercise, there is general agreement that the specific exercise conducted in water requires significantly more energy expenditure than when the same exercise is performed on land.

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.

Swimming pool cleaner poisoning

MedlinePlus

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

DNA Origami: Folded DNA-Nanodevices That Can Direct and Interpret Cell Behavior

PubMed Central

Kearney, Cathal J.; Lucas, Christopher R.; O'Brien, Fergal J.; Castro, Carlos E.

2016-01-01

DNA origami is a DNA-based nanotechnology that utilizes programmed combinations of short complementary oligonucleotides to fold a large single strand of DNA into precise 2-D and 3-D shapes. The exquisite nanoscale shape control of this inherently biocompatible material is combined with the potential to spatially address the origami structures with diverse cargos including drugs, antibodies, nucleic acid sequences, small molecules and inorganic particles. This programmable flexibility enables the fabrication of precise nanoscale devices that have already shown great potential for biomedical applications such as: drug delivery, biosensing and synthetic nanopore formation. In this Progress Report, we will review the advances in the DNA origami field since its inception several years ago and then focus on how these DNA-nanodevices can be designed to interact with cells to direct or probe their behavior. PMID:26840503

Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

NASA Astrophysics Data System (ADS)

Liu, Zhenbao; Zhou, Bo; Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang

2013-09-01

A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 μm in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL-1 to 10 ng mL-1. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.

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.)

Replacing the combined test by cell-free DNA testing in screening for trisomies 21, 18 and 13: impact on the diagnosis of other chromosomal abnormalities.

PubMed

Syngelaki, Argyro; Pergament, Eugene; Homfray, Tessa; Akolekar, Ranjit; Nicolaides, Kypros H

2014-01-01

To estimate the proportion of other chromosomal abnormalities that could be missed if combined testing was replaced by cell-free (cf) DNA testing as the method of screening for trisomies 21, 18 and 13. The prevalence of trisomies 21, 18 or 13, sex chromosome aneuploidies, triploidy and other chromosomal abnormalities was examined in pregnancies undergoing first-trimester combined screening and chorionic villus sampling (CVS). In 1,831 clinically significant chromosomal abnormalities in pregnancies with combined risk for trisomies 21, 18 and 13≥1:100, the contribution of trisomies 21, 18 or 13, sex chromosome aneuploidies, triploidy and other chromosomal abnormalities at high risk of adverse outcome was 82.9, 8.2, 3.9 and 5.0%, respectively. Combined screening followed by CVS for risk≥1:10 and cfDNA testing for risk 1:11-1:2,500 could detect 97% of trisomy 21 and 98% of trisomies 18 and 13. Additionally, 86% of monosomy X, half of 47,XXY, 47,XYY or 47,XXX, half of other chromosomal abnormalities and one third of triploidies, which are currently detected by combined screening and CVS for risk≥1:100, could be detected. Screening by cfDNA testing, contingent on results of combined testing, improves detection of trisomies, but misses a few of the other chromosomal abnormalities detected by screening with the combined test. © 2014 S. Karger AG, Basel.

[Environmental surveillance of a sample of indoor swimming pools from Emilia Romagna region: microclimate characteristics and chemical parameters, particularly disinfection by products, in pool waters].

PubMed

Fantuzzi, G; Righi, E; Predieri, G; Giacobazzi, P; Mastroianni, K; Aggazzotti, G

2010-01-01

The aim of the present study was to investigate the environmental and healthy aspects from a representative sample of indoor swimming pools located in the Emilia Romagna region. During the sampling sessions, the occupational environment was evaluated in terms of microclimate parameters and thermal comfort/discomfort conditions. Moreover the chemical risk was assessed by analyzing from the pool water the presence of disinfection by-products (DBPs), such as: trihalomethanes (THMs), haloacetic acids (HAAs), chlorite, chlorate and bromate. The analytical results are in agreement with the Italian legislation (Accordo Stato-Regioni; 2003) even if in some of the sampled indoor swimming pools, the dosed combined chlorine levels, were greater than the Italian limit. With the regard to the microclimate conditions evaluation, the considered thermal indices, Predicted Mean Vote (PMV) and Predicted Percentage of Dissatisfied (PPD%), described a satisfactory occupational environment. Among DBPs, the THMs mean levels (41.4 +/- 30.0 microg/l) resulted close to the values of the current Italian drinking water legislation, and seem to not represent an health issue. The pool waters chlorate levels (range: 5 - 19537 microg/l) need further investigations as recent epidemiological studies on drinking water hypothesized a potential genotoxicity effect of these compounds which are involved in cellular oxidative processes.

Evaluation of the use of pooled serum, pooled muscle tissue fluid (meat juice) and pooled faeces for monitoring pig herds for Salmonella.

PubMed

Davies, R H; Heath, P J; Coxon, S M; Sayers, A R

2003-01-01

Monitoring for Salmonella in slaughter pigs is important to enable targeted control measures to be applied on problem farms and at the abattoir. The aim of this study was to determine whether pooled serum and meat juice could be used to identify finishing pig herds with a high prevalence of infection. Samples of meat juice, serum, caecal contents, carcase swabs and pooled faeces from pig pens were taken from 20 commercial pig finishing farms and comparisons were made between the results of Salmonella culture, individual ELISA tests on serum and meat juice and pooled samples of serum and meat juice. Salmonella was isolated from samples from 19 of 20 farms. None of the ELISA tests showed a statistically significant correlation with caecal carriage of Salmonella or contamination of carcases. Serum mean optical density (O.D.) from pools of five, 10 or 20 sera showed a significant correlation with the Salmonella status of farm pen faeces. All pooled serum O.D. and sample/positive control ratio results correlated significantly with the results of the conventional individual sample ELISA. There was a statistically significant correlation between the incidence of Salmonella in farm pen pooled faeces and the prevalence of Salmonella in caeca of slaughter pigs. The results show a generally poor correlation between serological and bacteriological results but pooled serum or meat juice samples could be used as a cheaper substitute for serological screening of farms for Salmonella than individual samples. The availability of a cheaper test should allow the costs of Salmonella monitoring of pig farms to be reduced or allow more regular testing to enhance the designation of farm Salmonella risk status.

Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

PubMed

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L; Conrad, Daniel H; Xu, Ping

2010-07-26

Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

Pooled Protein Immunization for Identification of Cell Surface Antigens in Streptococcus sanguinis

PubMed Central

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L.; Conrad, Daniel H.; Xu, Ping

2010-01-01

Background Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. Methods and Findings We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. Conclusions The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases. PMID:20668678

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.

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

An introduction to mid-Atlantic seasonal pools

USGS Publications Warehouse

Brown, L.J.; Jung, R.E.

2005-01-01

Seasonal pools, also known as vernal ponds, provide important ecological services to the mid-Atlantic region. This publication serves as an introduction to seasonal pool ecology and management; it also provides tools for exploring seasonal pools, including a full-color field guide to wildlife. Seasonal pools are defined as having four distinctive features: surface water isolation, periodic drying, small size and shallow depth, and support of a characteristic biological community. Seasonal pools experience regular drying that excludes populations of predatory fish. Thus, pools in the mid-Atlantic region provide critical breeding habitat for amphibian and invertebrate species (e.g., spotted salamander (Ambystoma maculatum), wood frog (Rana sylvatica), and fairy shrimp (Order Anostraca)) that would be at increased risk of predation in more permanent waters. The distinctive features of seasonal pools also make them vulnerable to human disturbance. In the mid-Atlantic region, land-use changes pose the greatest challenges to seasonal pool conservation. Seasonal pools are threatened by direct loss (e.g., filling or draining of the pool) as well as by destruction and fragmentation of adjoining terrestrial habitat. Many of the species that depend on seasonal pools for breeding spend the majority of their lives in the surrounding lands that extend a radius of 1000 feet or more from the pools; these vital habitats are being transected by roads and converted to other land uses. Other threats to seasonal pools include biological introductions and removals, mosquito control practices, amphibian diseases, atmospheric deposition, and climate change. The authors recommend a three-pronged strategy for seasonal pool conservation and management in the mid-Atlantic region: education and research, inventory and monitoring of seasonal pools, and landscape-level planning and management.

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

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.

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

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.

Making the Bend: DNA Tertiary Structure and Protein-DNA Interactions

PubMed Central

Harteis, Sabrina; Schneider, Sabine

2014-01-01

DNA structure functions as an overlapping code to the DNA sequence. Rapid progress in understanding the role of DNA structure in gene regulation, DNA damage recognition and genome stability has been made. The three dimensional structure of both proteins and DNA plays a crucial role for their specific interaction, and proteins can recognise the chemical signature of DNA sequence (“base readout”) as well as the intrinsic DNA structure (“shape recognition”). These recognition mechanisms do not exist in isolation but, depending on the individual interaction partners, are combined to various extents. Driving force for the interaction between protein and DNA remain the unique thermodynamics of each individual DNA-protein pair. In this review we focus on the structures and conformations adopted by DNA, both influenced by and influencing the specific interaction with the corresponding protein binding partner, as well as their underlying thermodynamics. PMID:25026169

Tidal Pools--Miniature Oceans

ERIC Educational Resources Information Center

Plake, Linda Perry

1977-01-01

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

POOL server: machine learning application for functional site prediction in proteins.

PubMed

Somarowthu, Srinivas; Ondrechen, Mary Jo

2012-08-01

We present an automated web server for partial order optimum likelihood (POOL), a machine learning application that combines computed electrostatic and geometric information for high-performance prediction of catalytic residues from 3D structures. Input features consist of THEMATICS electrostatics data and pocket information from ConCavity. THEMATICS measures deviation from typical, sigmoidal titration behavior to identify functionally important residues and ConCavity identifies binding pockets by analyzing the surface geometry of protein structures. Both THEMATICS and ConCavity (structure only) do not require the query protein to have any sequence or structure similarity to other proteins. Hence, POOL is applicable to proteins with novel folds and engineered proteins. As an additional option for cases where sequence homologues are available, users can include evolutionary information from INTREPID for enhanced accuracy in site prediction. The web site is free and open to all users with no login requirements at http://www.pool.neu.edu. m.ondrechen@neu.edu Supplementary data are available at Bioinformatics online.

10 CFR 36.63 - Pool water purity.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 1 2010-01-01 2010-01-01 false Pool water purity. 36.63 Section 36.63 Energy NUCLEAR... § 36.63 Pool water purity. (a) Pool water purification system must be run sufficiently to maintain the conductivity of the pool water below 20 microsiemens per centimeter under normal circumstances. If pool water...

10 CFR 36.63 - Pool water purity.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 1 2011-01-01 2011-01-01 false Pool water purity. 36.63 Section 36.63 Energy NUCLEAR... § 36.63 Pool water purity. (a) Pool water purification system must be run sufficiently to maintain the conductivity of the pool water below 20 microsiemens per centimeter under normal circumstances. If pool water...

10 CFR 36.63 - Pool water purity.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 1 2012-01-01 2012-01-01 false Pool water purity. 36.63 Section 36.63 Energy NUCLEAR... § 36.63 Pool water purity. (a) Pool water purification system must be run sufficiently to maintain the conductivity of the pool water below 20 microsiemens per centimeter under normal circumstances. If pool water...

10 CFR 36.63 - Pool water purity.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 10 Energy 1 2013-01-01 2013-01-01 false Pool water purity. 36.63 Section 36.63 Energy NUCLEAR... § 36.63 Pool water purity. (a) Pool water purification system must be run sufficiently to maintain the conductivity of the pool water below 20 microsiemens per centimeter under normal circumstances. If pool water...

10 CFR 36.63 - Pool water purity.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 1 2014-01-01 2014-01-01 false Pool water purity. 36.63 Section 36.63 Energy NUCLEAR... § 36.63 Pool water purity. (a) Pool water purification system must be run sufficiently to maintain the conductivity of the pool water below 20 microsiemens per centimeter under normal circumstances. If pool water...

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

Model of large pool fires.

PubMed

Fay, J A

2006-08-21

A two zone entrainment model of pool fires is proposed to depict the fluid flow and flame properties of the fire. Consisting of combustion and plume zones, it provides a consistent scheme for developing non-dimensional scaling parameters for correlating and extrapolating pool fire visible flame length, flame tilt, surface emissive power, and fuel evaporation rate. The model is extended to include grey gas thermal radiation from soot particles in the flame zone, accounting for emission and absorption in both optically thin and thick regions. A model of convective heat transfer from the combustion zone to the liquid fuel pool, and from a water substrate to cryogenic fuel pools spreading on water, provides evaporation rates for both adiabatic and non-adiabatic fires. The model is tested against field measurements of large scale pool fires, principally of LNG, and is generally in agreement with experimental values of all variables.

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.

Rank-based pooling for deep convolutional neural networks.

PubMed

Shi, Zenglin; Ye, Yangdong; Wu, Yunpeng

2016-11-01

Pooling is a key mechanism in deep convolutional neural networks (CNNs) which helps to achieve translation invariance. Numerous studies, both empirically and theoretically, show that pooling consistently boosts the performance of the CNNs. The conventional pooling methods are operated on activation values. In this work, we alternatively propose rank-based pooling. It is derived from the observations that ranking list is invariant under changes of activation values in a pooling region, and thus rank-based pooling operation may achieve more robust performance. In addition, the reasonable usage of rank can avoid the scale problems encountered by value-based methods. The novel pooling mechanism can be regarded as an instance of weighted pooling where a weighted sum of activations is used to generate the pooling output. This pooling mechanism can also be realized as rank-based average pooling (RAP), rank-based weighted pooling (RWP) and rank-based stochastic pooling (RSP) according to different weighting strategies. As another major contribution, we present a novel criterion to analyze the discriminant ability of various pooling methods, which is heavily under-researched in machine learning and computer vision community. Experimental results on several image benchmarks show that rank-based pooling outperforms the existing pooling methods in classification performance. We further demonstrate better performance on CIFAR datasets by integrating RSP into Network-in-Network. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Methods for estimating the amount of vernal pool habitat in the northeastern United States

USGS Publications Warehouse

Van Meter, R.; Bailey, L.L.; Grant, E.H.C.

2008-01-01

The loss of small, seasonal wetlands is a major concern for a variety of state, local, and federal organizations in the northeastern U.S. Identifying and estimating the number of vernal pools within a given region is critical to developing long-term conservation and management strategies for these unique habitats and their faunal communities. We use three probabilistic sampling methods (simple random sampling, adaptive cluster sampling, and the dual frame method) to estimate the number of vernal pools on protected, forested lands. Overall, these methods yielded similar values of vernal pool abundance for each study area, and suggest that photographic interpretation alone may grossly underestimate the number of vernal pools in forested habitats. We compare the relative efficiency of each method and discuss ways of improving precision. Acknowledging that the objectives of a study or monitoring program ultimately determine which sampling designs are most appropriate, we recommend that some type of probabilistic sampling method be applied. We view the dual-frame method as an especially useful way of combining incomplete remote sensing methods, such as aerial photograph interpretation, with a probabilistic sample of the entire area of interest to provide more robust estimates of the number of vernal pools and a more representative sample of existing vernal pool habitats.

POOL WATER TREATMENT AND COOLING SYSTEM DESCRIPTION DOCUMENT

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

V. King

2000-06-19

The Pool Water Treatment and Cooling System is located in the Waste Handling Building (WHB), and is comprised of various process subsystems designed to support waste handling operations. This system maintains the pool water temperature within an acceptable range, maintains water quality standards that support remote underwater operations and prevent corrosion, detects leakage from the pool liner, provides the capability to remove debris from the pool, controls the pool water level, and helps limit radiological exposure to personnel. The pool structure and liner, pool lighting, and the fuel staging racks in the pool are not within the scope of themore » Pool Water Treatment and Cooling System. Pool water temperature control is accomplished by circulating the pool water through heat exchangers. Adequate circulation and mixing of the pool water is provided to prevent localized thermal hotspots in the pool. Treatment of the pool water is accomplished by a water treatment system that circulates the pool water through filters, and ion exchange units. These water treatment units remove radioactive and non-radioactive particulate and dissolved solids from the water, thereby providing the water clarity needed to conduct waste handling operations. The system also controls pool water chemistry to prevent advanced corrosion of the pool liner, pool components, and fuel assemblies. Removal of radioactivity from the pool water contributes to the project ALARA (as low as is reasonably achievable) goals. A leak detection system is provided to detect and alarm leaks through the pool liner. The pool level control system monitors the water level to ensure that the minimum water level required for adequate radiological shielding is maintained. Through interface with a demineralized water system, adequate makeup is provided to compensate for loss of water inventory through evaporation and waste handling operations. Interface with the Site Radiological Monitoring System provides

Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays

PubMed Central

2011-01-01

Background The processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP) and methylated DNA (MeDIP) represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription. Results To allow simultaneous measurements of DNA methylation with other genomic processes, we developed and validated a simple and easy-to-use high throughput microplate-based platform for analysis of DNA methylation. Compared to the traditional beads-based MeDIP the microplate MeDIP was more sensitive and had lower non-specific binding. We integrated the MeDIP method with a microplate ChIP assay which allows measurements of both DNA methylation and histone marks at the same time, Matrix ChIP-MeDIP platform. We illustrated several applications of this platform to relate DNA methylation, with chromatin and transcription events at selected genes in cultured cells, human cancer and in a model of diabetic kidney disease. Conclusion The high throughput capacity of Matrix ChIP-MeDIP to profile tens and potentially hundreds of different genomic events at the same time as DNA methylation represents a powerful platform to explore complex genomic mechanism at selected genes in cultured cells and in whole tissues. In this regard, Matrix ChIP-MeDIP should be useful to complement genome-wide studies where the rich chromatin and transcription database resources provide fruitful foundation to pursue mechanistic

Human DNA ligase III recognizes DNA ends by dynamic switching between two DNA-bound states.

PubMed

Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A; Tomkinson, Alan E; Ellenberger, Tom

2010-07-27

Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a "jackknife model" in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.

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

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

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

47 CFR 13.215 - Question pools.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 1 2010-10-01 2010-10-01 false Question pools. 13.215 Section 13.215 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL COMMERCIAL RADIO OPERATORS Examination System § 13.215 Question pools. The question pool for each written examination element will be composed of questions...

Evaluation of forensic DNA mixture evidence: protocol for evaluation, interpretation, and statistical calculations using the combined probability of inclusion.

PubMed

Bieber, Frederick R; Buckleton, John S; Budowle, Bruce; Butler, John M; Coble, Michael D

2016-08-31

The evaluation and interpretation of forensic DNA mixture evidence faces greater interpretational challenges due to increasingly complex mixture evidence. Such challenges include: casework involving low quantity or degraded evidence leading to allele and locus dropout; allele sharing of contributors leading to allele stacking; and differentiation of PCR stutter artifacts from true alleles. There is variation in statistical approaches used to evaluate the strength of the evidence when inclusion of a specific known individual(s) is determined, and the approaches used must be supportable. There are concerns that methods utilized for interpretation of complex forensic DNA mixtures may not be implemented properly in some casework. Similar questions are being raised in a number of U.S. jurisdictions, leading to some confusion about mixture interpretation for current and previous casework. Key elements necessary for the interpretation and statistical evaluation of forensic DNA mixtures are described. Given the most common method for statistical evaluation of DNA mixtures in many parts of the world, including the USA, is the Combined Probability of Inclusion/Exclusion (CPI/CPE). Exposition and elucidation of this method and a protocol for use is the focus of this article. Formulae and other supporting materials are provided. Guidance and details of a DNA mixture interpretation protocol is provided for application of the CPI/CPE method in the analysis of more complex forensic DNA mixtures. This description, in turn, should help reduce the variability of interpretation with application of this methodology and thereby improve the quality of DNA mixture interpretation throughout the forensic community.

HYDROLOGY AND LANDSCAPE CONNECTIVITY OF VERNAL POOLS

EPA Science Inventory

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

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...

Microdosimetry calculations for monoenergetic electrons using Geant4-DNA combined with a weighted track sampling algorithm.

PubMed

Famulari, Gabriel; Pater, Piotr; Enger, Shirin A

2017-07-07

The aim of this study was to calculate microdosimetric distributions for low energy electrons simulated using the Monte Carlo track structure code Geant4-DNA. Tracks for monoenergetic electrons with kinetic energies ranging from 100 eV to 1 MeV were simulated in an infinite spherical water phantom using the Geant4-DNA extension included in Geant4 toolkit version 10.2 (patch 02). The microdosimetric distributions were obtained through random sampling of transfer points and overlaying scoring volumes within the associated volume of the tracks. Relative frequency distributions of energy deposition f(>E)/f(>0) and dose mean lineal energy ([Formula: see text]) values were calculated in nanometer-sized spherical and cylindrical targets. The effects of scoring volume and scoring techniques were examined. The results were compared with published data generated using MOCA8B and KURBUC. Geant4-DNA produces a lower frequency of higher energy deposits than MOCA8B. The [Formula: see text] values calculated with Geant4-DNA are smaller than those calculated using MOCA8B and KURBUC. The differences are mainly due to the lower ionization and excitation cross sections of Geant4-DNA for low energy electrons. To a lesser extent, discrepancies can also be attributed to the implementation in this study of a new and fast scoring technique that differs from that used in previous studies. For the same mean chord length ([Formula: see text]), the [Formula: see text] calculated in cylindrical volumes are larger than those calculated in spherical volumes. The discrepancies due to cross sections and scoring geometries increase with decreasing scoring site dimensions. A new set of [Formula: see text] values has been presented for monoenergetic electrons using a fast track sampling algorithm and the most recent physics models implemented in Geant4-DNA. This dataset can be combined with primary electron spectra to predict the radiation quality of photon and electron beams.

Self-formed waterfall plunge pools in homogeneous rock

NASA Astrophysics Data System (ADS)

Scheingross, Joel S.; Lo, Daniel Y.; Lamb, Michael P.

2017-01-01

Waterfalls are ubiquitous, and their upstream propagation can set the pace of landscape evolution, yet no experimental studies have examined waterfall plunge pool erosion in homogeneous rock. We performed laboratory experiments, using synthetic foam as a bedrock simulant, to produce self-formed waterfall plunge pools via particle impact abrasion. Plunge pool vertical incision exceeded lateral erosion by approximately tenfold until pools deepened to the point that the supplied sediment could not be evacuated and deposition armored the pool bedrock floor. Lateral erosion of plunge pool sidewalls continued after sediment deposition, but primarily at the downstream pool wall, which might lead to undermining of the plunge pool lip, sediment evacuation, and continued vertical pool floor incision in natural streams. Undercutting of the upstream pool wall was absent, and our results suggest that vertical drilling of successive plunge pools is a more efficient waterfall retreat mechanism than the classic model of headwall undercutting and collapse in homogeneous rock.

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

Perceived health problems in swimmers according to the chemical treatment of water in swimming pools.

PubMed

Fernández-Luna, Álvaro; Burillo, Pablo; Felipe, José Luis; del Corral, Julio; García-Unanue, Jorge; Gallardo, Leonor

2016-01-01

The objective of this study was to determine which chemical treatment used for disinfecting water in indoor swimming pools had the least impact on users' perceptions of health problems, and which generated the greatest satisfaction with the quality of the water. A survey on satisfaction and perceived health problems was given to 1001 users at 20 indoor swimming pools which used different water treatment methods [chlorine, bromine, ozone, ultraviolet lamps (UV) and salt electrolysis]. The findings suggest that there is a greater probability of perceived health problems, such as eye and skin irritation, respiratory problems and skin dryness, in swimming pools treated with chlorine than in swimming pools using other chemical treatment methods. Pools treated with bromine have similar, although slightly better, results. Other factors, such as age, gender, time of day of use (morning and afternoon) and type of user (competitive and recreational), can also affect the probability of suffering health problems. For all of the above, using combined treatment methods as ozone and UV, or salt electrolysis produces a lower probability of perceived health problems and greater satisfaction.

47 CFR 97.523 - Question pools.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 5 2010-10-01 2010-10-01 false Question pools. 97.523 Section 97.523 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES AMATEUR RADIO... question pool for each written examination element. Each question pool must contain at least 10 times the...

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.

Enhanced Anti-Tumor Effect of Zoledronic Acid Combined with Temozolomide against Human Malignant Glioma Cell Expressing O6-Methylguanine DNA Methyltransferase

PubMed Central

Fukai, Junya; Koizumi, Fumiaki; Nakao, Naoyuki

2014-01-01

Temozolomide (TMZ), a DNA methylating agent, is widely used in the adjuvant treatment of malignant gliomas. O6-methylguanine-DNA methyltranferase (MGMT), a DNA repair enzyme, is frequently discussed as the main factor that limits the efficacy of TMZ. Zoledronic acid (ZOL), which is clinically applied to treat cancer-induced bone diseases, appears to possess direct anti-tumor activity through apoptosis induction by inhibiting mevalonate pathway and prenylation of intracellular small G proteins. In this study, we evaluated whether ZOL can be effectively used as an adjuvant to TMZ in human malignant glioma cells that express MGMT. Malignant glioma cell lines, in which the expression of MGMT was detected, did not exhibit growth inhibition by TMZ even at a longer exposure. However, combination experiment of TMZ plus ZOL revealed that a supra-additive effect resulted in a significant decrease in cell growth. In combined TMZ/ZOL treatment, an increased apoptotic rate was apparent and significant activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase were observed compared with each single drug exposure. There were decreased amounts of Ras-GTP, MAPK and Akt phosphorylation and MGMT expression in the ZOL-treated cells. Subcutanous xenograft models showed significant decrease of tumor growth with combined TMZ/ZOL treatment. These results suggest that ZOL efficaciously inhibits activity of Ras in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. Based on this work, combination of TMZ with ZOL might be a potential therapy in malignant gliomas that receive less therapeutic effects of TMZ due to cell resistance. PMID:25111384

Impact of ocean acidification and warming on the productivity of a rock pool community.

PubMed

Legrand, Erwann; Riera, Pascal; Bohner, Olivier; Coudret, Jérôme; Schlicklin, Ferdinand; Derrien, Marie; Martin, Sophie

2018-05-01

This study examined experimentally the combined effect of ocean acidification and warming on the productivity of rock pool multi-specific assemblages, composed of coralline algae, fleshy algae, and grazers. Natural rock pool communities experience high environmental fluctuations. This may confer physiological advantage to rock pool communities when facing predicted acidification and warming. The effect of ocean acidification and warming have been assessed at both individual and assemblage level to examine the importance of species interactions in the response of assemblages. We hypothesized that rock pool assemblages have physiological advantage when facing predicted ocean acidification and warming. Species exhibited species-specific responses to increased temperature and pCO 2 . Increased temperature and pCO 2 have no effect on assemblage photosynthesis, which was mostly influenced by fleshy algal primary production. The response of coralline algae to ocean acidification and warming depended on the season, which evidenced the importance of physiological adaptations to their environment in their response to climate change. We suggest that rock pool assemblages are relatively robust to changes in temperature and pCO 2 , in terms of primary production. Copyright © 2018 Elsevier Ltd. All rights reserved.

The combined effects of UV-B radiation and herbicides on photosynthesis, antioxidant enzymes and DNA damage in two bloom-forming cyanobacteria.

PubMed

Chen, Lanzhou; Xie, Mu; Bi, Yonghong; Wang, Gaohong; Deng, Songqiang; Liu, Yongding

2012-06-01

In this study, we investigated the combined effects of UV-B irradiation and herbicides (glyphosate, GPS; 2-Methyl-4-chlorophenoxyacetic acid, MCPA-Na; 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DCMU) and the antioxidant (ascorbic acid, ASC) on photosynthesis, antioxidant enzymes and DNA damage in two bloom-forming cyanobacteria, Anabaena sp. and Microcystis viridis. UV-B irradiance increased reactive oxygen species (ROS) production, which decreased chlorophyll a fluorescence yield, pigment content and superoxide dismutase (SOD) activity, and increased malondialdehyde (MDA) content and caused serious DNA damage. The degree of these damages was aggravated by the addition of DCMU, GPS and MCPA, and was partially mitigated by the addition of ASC. During the recovery process, the degree and mechanism in restoring DNA damage and photosynthesis inhibition were different by the removal of UV-B and herbicides (DCMU, GPS and MCPA) in both cyanobacteria. These results suggest that the combination of UV-B and exogenous herbicides have detrimental effects on cyanobacterial metabolism through either a ROS-mediated process or by affecting the electron transport chain, and may cause the shifts in the phytoplankton community. Copyright © 2012 Elsevier Inc. All rights reserved.

Combinations of chlorocatechols and heavy metals cause DNA degradation in vitro but must not result in increased mutation rates in vivo.

PubMed

Schweigert, N; Belkin, S; Leong-Morgenthaler, P; Zehnder, A J; Eggen, R I

1999-01-01

Chlorocatechols introduced into the environment directly or as a result of degradation processes are highly toxic, particularly when combined with heavy metals. With in vitro DNA degradation assays, the high reactivity of chlorocatechols combined with heavy metals could be shown, whereby copper was shown to be more active than iron. Structure-activity analysis showed that the degradation potential of the chlorocatechols decreased with an increasing number of chloratoms. The addition of reactive oxygen species scavengers allowed the identification of hydrogen peroxide as an important agent leading to DNA damage in this reaction. The potential of other reactive compounds, however, can neither be determined nor excluded with this approach. Exposure of Escherichia coli and Salmonella typhimurium cultures to the same mixtures of chlorocatechols and copper surprisingly did not lead to an enhanced mutation rate. This phenomenon was explained by doing marker gene expression measurements and toxicity tests with E. coli mutants deficient in oxidative stress defense or DNA repair. In catechol-copper-exposed cultures an increased peroxide level could indeed be demonstrated, but the highly efficient defense and repair systems of E. coli avoid the phenotypical establishment of mutations. Increased mutation rates under chronic exposure, however, cannot be excluded.

Forensic DNA Profiling and Database

PubMed Central

Panneerchelvam, S.; Norazmi, M.N.

2003-01-01

The incredible power of DNA technology as an identification tool had brought a tremendous change in crimnal justice . DNA data base is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. This article discusses the essential steps in compilation of COmbined DNA Index System (CODIS) on validated polymerase chain amplified STRs and their use in crime detection. PMID:23386793

Swimming Pools. Managing School Facilities, Guide 2.

ERIC Educational Resources Information Center

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

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

The spatial variability of water chemistry and DOC in bog pools: the importance of slope position, diurnal turnover and pool type

NASA Astrophysics Data System (ADS)

Holden, Joseph; Turner, Ed; Baird, Andy; Beadle, Jeannie; Billett, Mike; Brown, Lee; Chapman, Pippa; Dinsmore, Kerry; Dooling, Gemma; Grayson, Richard; Moody, Catherine; Gee, Clare

2017-04-01

We have previously shown that marine influence is an important factor controlling regional variability of pool water chemistry in blanket peatlands. Here we examine within-site controls on pool water chemistry. We surveyed natural and artificial (restoration sites) bog pools at blanket peatland sites in northern Scotland and Sweden. DOC, pH, conductivity, dissolved oxygen, temperature, cations, anions and absorbance spectra from 220-750nm were sampled. We sampled changes over time but also conducted intensive spatial surveys within individual pools and between pools on the same sampling days at individual study sites. Artificial pools had significantly greater DOC concentrations and different spectral absorbance characteristics when compared to natural pools at all sites studied. Within-pool variability in water chemistry tended to be small, even for very large pools ( 400 m2), except where pools had a layer of loose, mobile detritus on their beds. In these instances rapid changes took place between the overlying water column and the mobile sediment layer wherein dissolved oxygen concentrations dropped from values of around 12-10 mg/L to values less than 0.5 mg/L over just 2-3 cm of the depth profile. Such strong contrasts were not observed for pools which had a hard peat floor and which lacked a significant detritus layer. Strong diurnal turnover occurred within the pools on summer days, including within small, shallow pools (e.g. < 30 cm deep, 1 m2 area). For many pools on these summer days there was an evening spike in dissolved oxygen concentrations which originated at the surface and was then cycled downwards as the pool surface waters cooled. Slope location was a significant control on several pool water chemistry variables including pH and DOC concentration with accumulation (higher concentrations) in pools that were located further downslope in both natural and artificial pool systems. These processes have important implications for our interpretation of

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...

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

13 CFR 120.1705 - Pool formation requirements.

Code of Federal Regulations, 2010 CFR

2010-01-01

... requirements. SBA may adjust the Pool characteristics periodically based on program experience and market... a Pool involving a Pool Loan it does not own, it must purchase the Loan Interest it proposes to pool... purchase the Loan Interest and take it into inventory or settle the purchase of the Loan Interest through...

Method of measuring a liquid pool volume

DOEpatents

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

1991-01-01

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

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

PubMed

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

2013-09-15

Graphene, a single atom thick and two dimensional carbon nano-material, has been proven to possess many unique properties, one of which is the recent discovery that it can interact with single-stranded DNA through noncovalent π-π stacking. In this work, we demonstrate that a new strategy to fabricate many kinds of biosensors can be developed by combining this property with cascade chemical reactions. Taking the fabrication of glucose sensor as an example, while the detection target, glucose, may regulate the graphene-DNA interaction through three cascade chemical reactions, electrochemical techniques are employed to detect the target-regulated graphene-DNA interaction. Experimental results show that in a range from 5μM to 20mM, the glucose concentration is in a natural logarithm with the logarithm of the amperometric response, suggesting a best detection limit and detection range. The proposed biosensor also shows favorable selectivity, and it has the advantage of no need for labeling. What is more, by controlling the cascade chemical reactions, detection of a variety of other targets may be achieved, thus the strategy proposed in this work may have a wide application potential in the future. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

1968 Listing of Swimming Pool Equipment.

ERIC Educational Resources Information Center

National Sanitation Foundation, Ann Arbor, MI. Testing Lab.

An up-to-date listing of swimming pool equipment including--(1) companies authorized to display the National Sanitation Foundation seal of approval, (2) equipment listed as meeting NSF swimming pool equipment standards relating to diatomite type filters, (3) equipment listed as meeting NSF swimming pool equipment standard relating to sand type…

The synergistic effect of combined immunization with a DNA vaccine and chimeric yellow fever/dengue virus leads to strong protection against dengue.

PubMed

Azevedo, Adriana S; Gonçalves, Antônio J S; Archer, Marcia; Freire, Marcos S; Galler, Ricardo; Alves, Ada M B

2013-01-01

The dengue envelope glycoprotein (E) is the major component of virion surface and its ectodomain is composed of domains I, II and III. This protein is the main target for the development of a dengue vaccine with induction of neutralizing antibodies. In the present work, we tested two different vaccination strategies, with combined immunizations in a prime/booster regimen or simultaneous inoculation with a DNA vaccine (pE1D2) and a chimeric yellow fever/dengue 2 virus (YF17D-D2). The pE1D2 DNA vaccine encodes the ectodomain of the envelope DENV2 protein fused to t-PA signal peptide, while the YF17D-D2 was constructed by replacing the prM and E genes from the 17D yellow fever vaccine virus by those from DENV2. Balb/c mice were inoculated with these two vaccines by different prime/booster or simultaneous immunization protocols and most of them induced a synergistic effect on the elicited immune response, mainly in neutralizing antibody production. Furthermore, combined immunization remarkably increased protection against a lethal dose of DENV2, when compared to each vaccine administered alone. Results also revealed that immunization with the DNA vaccine, regardless of the combination with the chimeric virus, induced a robust cell immune response, with production of IFN-γ by CD8+ T lymphocytes.

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-01-01

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

Functional genomics platform for pooled screening and mammalian genetic interaction maps

PubMed Central

Kampmann, Martin; Bassik, Michael C.; Weissman, Jonathan S.

2014-01-01

Systematic genetic interaction maps in microorganisms are powerful tools for identifying functional relationships between genes and defining the function of uncharacterized genes. We have recently implemented this strategy in mammalian cells as a two-stage approach. First, genes of interest are robustly identified in a pooled genome-wide screen using complex shRNA libraries. Second, phenotypes for all pairwise combinations of hit genes are measured in a double-shRNA screen and used to construct a genetic interaction map. Our protocol allows for rapid pooled screening under various conditions without a requirement for robotics, in contrast to arrayed approaches. Each stage of the protocol can be implemented in ~2 weeks, with additional time for analysis and generation of reagents. We discuss considerations for screen design, and present complete experimental procedures as well as a full computational analysis suite for identification of hits in pooled screens and generation of genetic interaction maps. While the protocols outlined here were developed for our original shRNA-based approach, they can be applied more generally, including to CRISPR-based approaches. PMID:24992097

Method of measuring a liquid pool volume

DOEpatents

Garcia, G.V.; Carlson, N.M.; Donaldson, A.D.

1991-03-19

A method of measuring a molten metal liquid pool volume and in particular molten titanium liquid pools is disclosed, including the steps of (a) generating an ultrasonic wave at the surface of the molten metal liquid pool, (b) shining a light on the surface of a molten metal liquid pool, (c) detecting a change in the frequency of light, (d) detecting an ultrasonic wave echo at the surface of the molten metal liquid pool, and (e) computing the volume of the molten metal liquid. 3 figures.

DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants.

PubMed

Sucher, Nikolaus J; Hennell, James R; Carles, Maria C

2012-01-01

DNA fingerprinting of plants has become an invaluable tool in forensic, scientific, and industrial laboratories all over the world. PCR has become part of virtually every variation of the plethora of approaches used for DNA fingerprinting today. DNA sequencing is increasingly used either in combination with or as a replacement for traditional DNA fingerprinting techniques. A prime example is the use of short, standardized regions of the genome as taxon barcodes for biological identification of plants. Rapid advances in "next generation sequencing" (NGS) technology are driving down the cost of sequencing and bringing large-scale sequencing projects into the reach of individual investigators. We present an overview of recent publications that demonstrate the use of "NGS" technology for DNA fingerprinting and DNA barcoding applications.

Mass effects meet species sorting: transformations of microbial assemblages in epiphreatic subsurface karst water pools.

PubMed

Shabarova, Tanja; Widmer, Franco; Pernthaler, Jakob

2013-09-01

We investigated the transformations of the microbial communities in epiphreatic karst cave pools with different flooding frequencies. Fingerprinting of 16S rRNA genes was combined with microscopic and sequence analysis to examine if source water would transport comparable microbial inocula into the pools at consecutive flood events, and to assess possible effects of residence time on the microbial assemblages during stagnant periods. Variability in the concentrations of dissolved organic carbon and conductivity indicated differences between floods and changes of pool water over time. High numbers of Betaproteobacteria affiliated with Methylophilaceae and Comamonadaceae were introduced into the pools during floodings. While the former persisted in the pools, the latter exhibited considerable microdiversification. These Betaproteobacteria might thus represent core microbial groups in karst water. A decrease in the estimated total diversity of the remaining bacterial taxa was apparent after a few weeks of residence: Some were favoured by stagnant conditions, whereas the majority was rapidly outcompeted. Thus, the microbial communities consisted of different components governed by complementary assembly mechanisms (dispersal versus environmental filtering) upon introduction into the pools. High overlap of temporary and persistent community members between samplings from two winters, moreover, reflected the seasonal recurrence of the studied microbial assemblages. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

13 CFR 120.1709 - Transfers of Pool Certificates.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 13 Business Credit and Assistance 1 2012-01-01 2012-01-01 false Transfers of Pool Certificates... Transfers of Pool Certificates. (a) Transfer of Pool Certificates. A Pool Certificate is transferable. A transfer of a Pool Certificate must comply with Article 8 of the Uniform Commercial Code of the State of...

13 CFR 120.1709 - Transfers of Pool Certificates.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Transfers of Pool Certificates... Transfers of Pool Certificates. (a) Transfer of Pool Certificates. A Pool Certificate is transferable. A transfer of a Pool Certificate must comply with Article 8 of the Uniform Commercial Code of the State of...

13 CFR 120.1709 - Transfers of Pool Certificates.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 13 Business Credit and Assistance 1 2014-01-01 2014-01-01 false Transfers of Pool Certificates... Transfers of Pool Certificates. (a) Transfer of Pool Certificates. A Pool Certificate is transferable. A transfer of a Pool Certificate must comply with Article 8 of the Uniform Commercial Code of the State of...

13 CFR 120.1709 - Transfers of Pool Certificates.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 13 Business Credit and Assistance 1 2011-01-01 2011-01-01 false Transfers of Pool Certificates... Transfers of Pool Certificates. (a) Transfer of Pool Certificates. A Pool Certificate is transferable. A transfer of a Pool Certificate must comply with Article 8 of the Uniform Commercial Code of the State of...

7 CFR 1006.7 - Pool plant.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1006.7 Section 1006.7 Agriculture... Handling Definitions § 1006.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1131.7 - Pool plant.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1131.7 Section 1131.7 Agriculture... Handling Definitions § 1131.7 Pool plant. Pool Plant means a plant or unit of plants specified in paragraphs (a) through (e) of this section, but excluding a plant specified in paragraph (g) of this section...

7 CFR 1007.7 - Pool plant.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1007.7 Section 1007.7 Agriculture... Handling Definitions § 1007.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1006.7 - Pool plant.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1006.7 Section 1006.7 Agriculture... Handling Definitions § 1006.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1124.7 - Pool plant.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1124.7 Section 1124.7 Agriculture... Regulating Handling Definitions § 1124.7 Pool plant. Pool plant means a plant, unit of plants, or a system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified in...

7 CFR 1124.7 - Pool plant.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1124.7 Section 1124.7 Agriculture... Regulating Handling Definitions § 1124.7 Pool plant. Pool plant means a plant, unit of plants, or a system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified in...

7 CFR 1124.7 - Pool plant.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1124.7 Section 1124.7 Agriculture... Regulating Handling Definitions § 1124.7 Pool plant. Pool plant means a plant, unit of plants, or a system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified in...

7 CFR 1007.7 - Pool plant.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1007.7 Section 1007.7 Agriculture... Handling Definitions § 1007.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1005.7 - Pool plant.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1005.7 Section 1005.7 Agriculture... Handling Definitions § 1005.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1007.7 - Pool plant.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1007.7 Section 1007.7 Agriculture... Handling Definitions § 1007.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1124.7 - Pool plant.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1124.7 Section 1124.7 Agriculture... Regulating Handling Definitions § 1124.7 Pool plant. Pool plant means a plant, unit of plants, or a system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified in...

7 CFR 1126.7 - Pool plant.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1126.7 Section 1126.7 Agriculture... Handling Definitions § 1126.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1126.7 - Pool plant.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1126.7 Section 1126.7 Agriculture... Handling Definitions § 1126.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1005.7 - Pool plant.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1005.7 Section 1005.7 Agriculture... Handling Definitions § 1005.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1131.7 - Pool plant.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1131.7 Section 1131.7 Agriculture... Handling Definitions § 1131.7 Pool plant. Pool Plant means a plant or unit of plants specified in paragraphs (a) through (e) of this section, but excluding a plant specified in paragraph (g) of this section...

7 CFR 1124.7 - Pool plant.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1124.7 Section 1124.7 Agriculture... Regulating Handling Definitions § 1124.7 Pool plant. Pool plant means a plant, unit of plants, or a system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified in...

7 CFR 1006.7 - Pool plant.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1006.7 Section 1006.7 Agriculture... Handling Definitions § 1006.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1126.7 - Pool plant.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1126.7 Section 1126.7 Agriculture... Handling Definitions § 1126.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1006.7 - Pool plant.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1006.7 Section 1006.7 Agriculture... Handling Definitions § 1006.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1005.7 - Pool plant.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1005.7 Section 1005.7 Agriculture... Handling Definitions § 1005.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1005.7 - Pool plant.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1005.7 Section 1005.7 Agriculture... Handling Definitions § 1005.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1131.7 - Pool plant.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1131.7 Section 1131.7 Agriculture... Handling Definitions § 1131.7 Pool plant. Pool Plant means a plant or unit of plants specified in paragraphs (a) through (e) of this section, but excluding a plant specified in paragraph (g) of this section...

7 CFR 1006.7 - Pool plant.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1006.7 Section 1006.7 Agriculture... Handling Definitions § 1006.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1126.7 - Pool plant.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1126.7 Section 1126.7 Agriculture... Handling Definitions § 1126.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1005.7 - Pool plant.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1005.7 Section 1005.7 Agriculture... Handling Definitions § 1005.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1131.7 - Pool plant.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1131.7 Section 1131.7 Agriculture... Handling Definitions § 1131.7 Pool plant. Pool Plant means a plant or unit of plants specified in paragraphs (a) through (e) of this section, but excluding a plant specified in paragraph (g) of this section...

7 CFR 1007.7 - Pool plant.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1007.7 Section 1007.7 Agriculture... Handling Definitions § 1007.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1007.7 - Pool plant.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1007.7 Section 1007.7 Agriculture... Handling Definitions § 1007.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1126.7 - Pool plant.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1126.7 Section 1126.7 Agriculture... Handling Definitions § 1126.7 Pool plant. Pool plant means a plant specified in paragraphs (a) through (d) of this section, a unit of plants as specified in paragraph (e) of this section, or a plant specified...

7 CFR 1131.7 - Pool plant.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1131.7 Section 1131.7 Agriculture... Handling Definitions § 1131.7 Pool plant. Pool Plant means a plant or unit of plants specified in paragraphs (a) through (e) of this section, but excluding a plant specified in paragraph (g) of this section...

A Three-Pool Model Dissecting Readily Releasable Pool Replenishment at the Calyx of Held

PubMed Central

Guo, Jun; Ge, Jian-long; Hao, Mei; Sun, Zhi-cheng; Wu, Xin-sheng; Zhu, Jian-bing; Wang, Wei; Yao, Pan-tong; Lin, Wei; Xue, Lei

2015-01-01

Although vesicle replenishment is critical in maintaining exo-endocytosis recycling, the underlying mechanisms are not well understood. Previous studies have shown that both rapid and slow endocytosis recycle into a very large recycling pool instead of within the readily releasable pool (RRP), and the time course of RRP replenishment is slowed down by more intense stimulation. This finding contradicts the calcium/calmodulin-dependence of RRP replenishment. Here we address this issue and report a three-pool model for RRP replenishment at a central synapse. Both rapid and slow endocytosis provide vesicles to a large reserve pool (RP) ~42.3 times the RRP size. When moving from the RP to the RRP, vesicles entered an intermediate pool (IP) ~2.7 times the RRP size with slow RP-IP kinetics and fast IP-RRP kinetics, which was responsible for the well-established slow and rapid components of RRP replenishment. Depletion of the IP caused the slower RRP replenishment observed after intense stimulation. These results establish, for the first time, a realistic cycling model with all parameters measured, revealing the contribution of each cycling step in synaptic transmission. The results call for modification of the current view of the vesicle recycling steps and their roles. PMID:25825223

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

Recent progress in DNA origami technology.

PubMed

Endo, Masayuki; Sugiyama, Hiroshi

2011-06-01

DNA origami is an emerging technology for designing defined two-dimensional DNA nanostructures. In this review, we focus on and describe several types of DNA origami-related studies, as follows: (1) programmed DNA origami assembly, (2) DNA origami-templated molecular assembly, (3) design and construction of various three-dimensional DNA origami structures, (4) programmed functionalization of DNA origami and combination with top-down nanotechnology, (5) single molecular observation on a designed DNA origami, and (6) DNA nanomachines working on a DNA origami. © 2011 by John Wiley & Sons, Inc.

HIV DNA in CD14+ reservoirs is associated with regional brain atrophy in patients naive to combination antiretroviral therapy.

PubMed

Kallianpur, Kalpana J; Valcour, Victor G; Lerdlum, Sukalaya; Busovaca, Edgar; Agsalda, Melissa; Sithinamsuwan, Pasiri; Chalermchai, Thep; Fletcher, James L K; Tipsuk, Somporn; Shikuma, Cecilia M; Shiramizu, Bruce T; Ananworanich, Jintanat

2014-07-17

To examine associations between regional brain volumes and HIV DNA in peripheral CD14 cells (monocytes) among HIV-infected individuals naive to combination antiretroviral therapy (cART). A prospective study of HIV-infected Thai individuals who met Thai national criteria for cART initiation. Enrolment was stratified by HIV DNA in a blinded fashion. CD14 cells were isolated from peripheral mononuclear cells to high purity (median 91.4% monocytes by flow cytometry), and HIV DNA was quantified by multiplex real-time PCR. Baseline regional brain volumes obtained by T1-weighted 1.5-Tesla MRI were compared between HIV DNA groups using analysis of covariance (ANCOVA). We studied 60 individuals with mean (SD) age of 34.7 (7.0) years, CD4 T-lymphocyte count of 232 (137) cells/μl and log10 plasma HIV RNA of 4.8 (0.73). Median (interquartile range, IQR) HIV DNA copy number per 10 CD14 cells was 54 (102). Using our previously determined optimal cut-point of 45 copies/10 cells for this cohort, a threshold value above which CD14 HIV DNA identified HIV-associated neurocognitive disorders (HANDs), we found that CD14 HIV DNA  ≥ 45 copies/10 cells was associated with reduced volumes of the nucleus accumbens (P=0.021), brainstem (P=0.033) and total gray matter (P=0.045) independently of age, CD4 cell count and intracranial volume. HIV DNA burden in CD14 monocytes is directly linked to brain volumetric loss. Our findings implicate peripheral viral reservoirs in HIV-associated brain atrophy and support their involvement in the neuropathogenesis of HAND, underscoring the need for therapies that target these cells.

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

PubMed

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

2016-08-17

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

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).

DNA replication stress restricts ribosomal DNA copy number

PubMed Central

Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

2017-01-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

DNA replication stress restricts ribosomal DNA copy number.

PubMed

Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

2017-09-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

7 CFR 1030.7 - Pool plant.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1030.7 Section 1030.7 Agriculture... Handling Definitions § 1030.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified in paragraph (h) of...

7 CFR 1033.7 - Pool plant.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1033.7 Section 1033.7 Agriculture... Handling Definitions § 1033.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (j) of this...

7 CFR 1032.7 - Pool plant.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1032.7 Section 1032.7 Agriculture... Handling Definitions § 1032.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (i) of this...

7 CFR 1030.7 - Pool plant.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1030.7 Section 1030.7 Agriculture... Handling Definitions § 1030.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified in paragraph (h) of...

7 CFR 1033.7 - Pool plant.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1033.7 Section 1033.7 Agriculture... Handling Definitions § 1033.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (j) of this...

7 CFR 1032.7 - Pool plant.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1032.7 Section 1032.7 Agriculture... Handling Definitions § 1032.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (i) of this...

7 CFR 1033.7 - Pool plant.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1033.7 Section 1033.7 Agriculture... Handling Definitions § 1033.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (j) of this...

7 CFR 1033.7 - Pool plant.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1033.7 Section 1033.7 Agriculture... Handling Definitions § 1033.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (j) of this...

7 CFR 1001.7 - Pool plant.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 9 2011-01-01 2011-01-01 false Pool plant. 1001.7 Section 1001.7 Agriculture... Handling Definitions § 1001.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant described in paragraph (h) of...

7 CFR 1030.7 - Pool plant.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1030.7 Section 1030.7 Agriculture... Handling Definitions § 1030.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified in paragraph (h) of...

7 CFR 1032.7 - Pool plant.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1032.7 Section 1032.7 Agriculture... Handling Definitions § 1032.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (i) of this...

7 CFR 1001.7 - Pool plant.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1001.7 Section 1001.7 Agriculture... Handling Definitions § 1001.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant described in paragraph (h) of...

7 CFR 1030.7 - Pool plant.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1030.7 Section 1030.7 Agriculture... Handling Definitions § 1030.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified in paragraph (h) of...

7 CFR 1032.7 - Pool plant.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1032.7 Section 1032.7 Agriculture... Handling Definitions § 1032.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (i) of this...

7 CFR 1033.7 - Pool plant.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 9 2012-01-01 2012-01-01 false Pool plant. 1033.7 Section 1033.7 Agriculture... Handling Definitions § 1033.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (j) of this...

7 CFR 1001.7 - Pool plant.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1001.7 Section 1001.7 Agriculture... Handling Definitions § 1001.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant described in paragraph (h) of...

7 CFR 1032.7 - Pool plant.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 9 2013-01-01 2013-01-01 false Pool plant. 1032.7 Section 1032.7 Agriculture... Handling Definitions § 1032.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, or a plant specified in paragraph (i) of this...

7 CFR 1001.7 - Pool plant.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1001.7 Section 1001.7 Agriculture... Handling Definitions § 1001.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant described in paragraph (h) of...

7 CFR 1001.7 - Pool plant.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 9 2014-01-01 2013-01-01 true Pool plant. 1001.7 Section 1001.7 Agriculture... Handling Definitions § 1001.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant described in paragraph (h) of...

7 CFR 1030.7 - Pool plant.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 9 2010-01-01 2009-01-01 true Pool plant. 1030.7 Section 1030.7 Agriculture... Handling Definitions § 1030.7 Pool plant. Pool plant means a plant, unit of plants, or system of plants as specified in paragraphs (a) through (f) of this section, but excluding a plant specified in paragraph (h) of...

Mechanism for priming DNA synthesis by yeast DNA Polymerase α

PubMed Central

Perera, Rajika L; Torella, Rubben; Klinge, Sebastian; Kilkenny, Mairi L; Maman, Joseph D; Pellegrini, Luca

2013-01-01

The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment. DOI: http://dx.doi.org/10.7554/eLife.00482.001 PMID:23599895

Keyhole behavior and liquid flow in molten pool during laser-arc hybrid welding

NASA Astrophysics Data System (ADS)

Naito, Yasuaki; Katayama, Seiji; Matsunawa, Akira

2003-03-01

Hybrid welding was carried out on Type 304 stainless steel plate under various conditions using YAG laser combined with TIG arc. During arc and laser-arc hybrid welding, arc voltage variation was measured, and arc plasma, laser-induced plume and evaporation spots as well as keyhole behavior and liquid flow in the molten pool were observed through CCD camera and X-ray real-time transmission apparatus. It was consequently found that hybrid welding possessed many features in comparison with YAG laser welding. The deepest weld bead could be produced when the YAG laser beam of high power density was shot on the molten pool made beforehand stably with TIG arc. A keyhole was long and narrow, and its behavior was rather stable inside the molten pool. It was also confirmed that porosity was reduced by the suppression of bubble formation in hybrid welding utilizing a laser of a moderate power density.

Salt-Dependent DNA-DNA Spacings in Intact Bacteriophage lambda Reflect Relative Importance of DNA Self-Repulsion and Bending Energies

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

X Qiu; D Rau; V Parsegian

2011-12-31

Using solution synchrotron x-ray scattering, we measure the variation of DNA-DNA d spacings in bacteriophage {lambda} with mono-, di-, and polyvalent salt concentrations, for wild-type [48.5 x 10{sup 3} base pairs (bp)] and short-genome-mutant (37.8 kbp) strains. From the decrease in d spacings with increasing salt, we deduce the relative contributions of DNA self-repulsion and bending to the energetics of packaged phage genomes. We quantify the DNA-DNA interaction energies within the intact phage by combining the measured d spacings in the capsid with measurements of osmotic pressure in DNA assemblies under the same salt conditions in bulk solution. In themore » commonly used Tris-Mg buffer, the DNA-DNA interaction energies inside the phage capsids are shown to be about 1 kT/bp, an order of magnitude larger than the bending energies.« less

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.

Enhanced antitumor effects by combining an IL-12/anti-DNA fusion protein with avelumab, an anti-PD-L1 antibody

PubMed Central

Fallon, Jonathan K.; Vandeveer, Amanda J.

2017-01-01

The combined therapeutic potential of an immunocytokine designed to deliver IL-12 to the necrotic regions of solid tumors with an anti-PD-L1 antibody that disrupts the immunosuppressive PD-1/PD-L1 axis yielded a combinatorial benefit in multiple murine tumor models. The murine version of the immunocytokine, NHS-muIL12, consists of an antibody (NHS76) recognizing DNA/DNA-histone complexes, fused with two molecules of murine IL-12 (NHS-muIL12). By its recognition of exposed DNA, NHS-muIL12 targets IL-12 to the necrotic portions of tumors; it has a longer plasma half-life and better antitumor efficacy against murine tumors than recombinant murine IL-12. It is shown here that NHS-muIL12, in an IFN-γ‒dependent mechanism, upregulates mPD-L1 expression on mouse tumors, which could be construed as an immunosuppressive action. Yet concurrent therapy with NHS-muIL12 and an anti-PD-L1 antibody resulted in additive/synergistic antitumor effects in PD-L1‒expressing subcutaneously transplanted tumors (MC38, MB49) and in an intravesical bladder tumor model (MB49). Antitumor efficacy correlated with (a) with a higher frequency of tumor antigen-specific splenic CD8+ T cells and (b) enhanced T cell activation over a wide range of NHS-muIL12 concentrations. These findings suggest that combining NHS-muIL12 and an anti-PD-L1 antibody enhances T cell activation and T cell effector functions within the tumor microenvironment, significantly improving overall tumor regression. These results should provide the rationale to examine the combination of these agents in clinical studies. PMID:28423552

Life cycle environmental implications of residential swimming pools.

PubMed

Forrest, Nigel; Williams, Eric

2010-07-15

Ownership of private swimming pools in the U.S. grew 2 to 4% per annum from 1997 to 2007. The environmental implications of pool ownership are analyzed by hybrid life cycle assessment (LCA) for nine U.S. cities. An operational model is constructed estimating consumption of chemicals, water, and energy for a typical residential pool. The model incorporates geographical climatic variations and upstream water and energy use from electricity and water supply networks. Results vary considerably by city: a factor of 5-6 for both water and energy use. Water use is driven by aridness and length of the swimming season, while energy use is mainly driven by length of the swimming season. Water and energy impacts of pools are significant, particularly in arid climates. In Phoenix for example pools account for 22% and 13% of a household's electricity and water use, respectively. Measures to reduce water and energy use in pools such as optimizing the pump schedule and covering the pool in winter can realize greater savings than many common household efficiency improvements. Private versus community pools are also compared. Community pools in Phoenix use 60% less swimming pool water and energy per household than subdivisions without community pools.

Site-directed DNA crosslinking of large multisubunit protein-DNA complexes.

PubMed

Persinger, Jim; Bartholomew, Blaine

2009-01-01

Several methods have been developed to site-specifically incorporate photoreactive nucleotide analogs into DNA for the purpose of identifying the proteins and their domains that are in contact with particular regions of DNA. The synthesis of several deoxynucleotide analogs that have a photoreactive group tethered to the nucleotide base and the incorporation of these analogs into DNA are described. In a second approach, oligonucleotide with a photoreactive group attached to the phosphate backbone is chemically synthesized. The photoreactive oligonucleotide is then enzymatically incorporated into DNA by annealing it to a complementary DNA template and extending with DNA polymerase. Both approaches have been effectively used to map protein-DNA interactions in large multisubunit complexes such as the eukaryotic transcription or ATP-dependent chromatin remodeling complexes. Not only do these techniques map the binding sites of the various subunits in these complexes, but when coupled with peptide mapping also determine the protein domain that is in close proximity to the different DNA sites. The strength of these techniques is the ability to scan a large number of potential sites by making combinations of different DNA probes and is facilitated by using an immobilized DNA template for synthesis.

Characterisation of the Permafrost Carbon Pool

USGS Publications Warehouse

Kuhry, P.; Grosse, G.; Harden, J.W.; Hugelius, G.; Koven, C.D.; Ping, C.-L.; Schirrmeister, L.; Tarnocai, C.

2013-01-01

The current estimate of the soil organic carbon (SOC) pool in the northern permafrost region of 1672 Petagrams (Pg) C is much larger than previously reported and needs to be incorporated in global soil carbon (C) inventories. The Northern Circumpolar Soil Carbon Database (NCSCD), extended to include the range 0–300 cm, is now available online for wider use by the scientific community. An important future aim is to provide quantitative uncertainty ranges for C pool estimates. Recent studies have greatly improved understanding of the regional patterns, landscape distribution and vertical (soil horizon) partitioning of the permafrost C pool in the upper 3 m of soils. However, the deeper C pools in unconsolidated Quaternary deposits need to be better constrained. A general lability classification of the permafrost C pool should be developed to address potential C release upon thaw. The permafrost C pool and its dynamics are beginning to be incorporated into Earth System models, although key periglacial processes such as thermokarst still need to be properly represented to obtain a better quantification of the full permafrost C feedback on global climate change.

Genome-wide inference of transcription factor-DNA binding specificity in cell regeneration using a combination strategy.

PubMed

Wang, Xiaofeng; Zhang, Aiqun; Ren, Weizheng; Chen, Caiyu; Dong, Jiahong

2012-11-01

The cell growth, development, and regeneration of tissue and organ are associated with a large number of gene regulation events, which are mediated in part by transcription factors (TFs) binding to cis-regulatory elements involved in the genome. Predicting the binding affinity and inferring the binding specificity of TF-DNA interactions at the genomic level would be fundamentally helpful for our understanding of the molecular mechanism and biological implication underlying sequence-specific TF-DNA recognition. In this study, we report the development of a combination method to characterize the interaction behavior of a 11-mer oligonucleotide segment and its mutations with the Gcn4p protein, a homodimeric, basic leucine zipper TF, and to predict the binding affinity and specificity of potential Gcn4p binders in the genome-wide scale. In this procedure, a position-mutated energy matrix is created based on molecular modeling analysis of native and mutated Gcn4p-DNA complex structures to describe the position-independent interaction energy profile of Gcn4p with different nucleotide types at each position of the oligonucleotide, and the energy terms extracted from the matrix and their interactives are then correlated with experimentally measured affinities of 19268 distinct oligonucleotides using statistical modeling methodology. Subsequently, the best one of built regression models is successfully applied to screen those of potential high-affinity Gcn4p binders from the complete genome. The findings arising from this study are briefly listed below: (i) The 11 positions of oligonucleotides are highly interactive and non-additive in contribution to Gcn4p-DNA binding affinity; (ii) Indirect conformational effects upon nucleotide mutations as well as associated subtle changes in interfacial atomic contacts, but not the direct nonbonded interactions, are primarily responsible for the sequence-specific recognition; (iii) The intrinsic synergistic effects among the sequence

Modelling the sensitivity of soil mercury storage to climate-induced changes in soil carbon pools

NASA Astrophysics Data System (ADS)

Hararuk, O.; Obrist, D.; Luo, Y.

2013-04-01

Substantial amounts of mercury (Hg) in the terrestrial environment reside in soils and are associated with soil organic carbon (C) pools, where they accumulated due to increased atmospheric deposition resulting from anthropogenic activities. The purpose of this study was to examine potential sensitivity of surface soil Hg pools to global change variables, particularly affected by predicted changes in soil C pools, in the contiguous US. To investigate, we included a soil Hg component in the Community Land Model based on empirical statistical relationships between soil Hg / C ratios and precipitation, latitude, and clay; and subsequently explored the sensitivity of soil C and soil Hg densities (i.e., areal-mass) to climate scenarios in which we altered annual precipitation, carbon dioxide (CO2) concentrations and temperature. Our model simulations showed that current sequestration of Hg in the contiguous US accounted for 15 230 metric tons of Hg in the top 0-40 cm of soils, or for over 300 000 metric tons when extrapolated globally. In the simulations, US soil Hg pools were most sensitive to changes in precipitation because of strong effects on soil C pools, plus a direct effect of precipitation on soil Hg / C ratios. Soil Hg pools were predicted to increase beyond present-day values following an increase in precipitation amounts and decrease following a reduction in precipitation. We found pronounced regional differences in sensitivity of soil Hg to precipitation, which were particularly high along high-precipitation areas along the West and East Coasts. Modelled increases in CO2 concentrations to 700 ppm stimulated soil C and Hg accrual, while increased air temperatures had small negative effects on soil C and Hg densities. The combined effects of increased CO2, increased temperature and increased or decreased precipitation were strongly governed by precipitation and CO2 showing pronounced regional patterns. Based on these results, we conclude that the combination

[The Russian gene pool: gene geography of Alu-insertions (ACE, APOA1, B65, PV92 TPA25)].