Dynamic protein assembly by programmable DNA strand displacement

Science.gov (United States)

Chen, Rebecca P.; Blackstock, Daniel; Sun, Qing; Chen, Wilfred

2018-03-01

Inspired by the remarkable ability of natural protein switches to sense and respond to a wide range of environmental queues, here we report a strategy to engineer synthetic protein switches by using DNA strand displacement to dynamically organize proteins with highly diverse and complex logic gate architectures. We show that DNA strand displacement can be used to dynamically control the spatial proximity and the corresponding fluorescence resonance energy transfer between two fluorescent proteins. Performing Boolean logic operations enabled the explicit control of protein proximity using multi-input, reversible and amplification architectures. We further demonstrate the power of this technology beyond sensing by achieving dynamic control of an enzyme cascade. Finally, we establish the utility of the approach as a synthetic computing platform that drives the dynamic reconstitution of a split enzyme for targeted prodrug activation based on the sensing of cancer-specific miRNAs.

Design and Characterization of DNA Strand-Displacement Circuits in Serum-Supplemented Cell Medium.

Science.gov (United States)

Fern, Joshua; Schulman, Rebecca

2017-09-15

The functional stability and lifetimes of synthetic molecular circuits in biological environments are important for long-term, stable sensors or controllers of cell or tissue behavior. DNA-based molecular circuits, in particular DNA strand-displacement circuits, provide simple and effective biocompatible control mechanisms and sensors, but are vulnerable to digestion by nucleases present in living tissues and serum-supplemented cell culture. The stability of double-stranded and single-stranded DNA circuit components in serum-supplemented cell medium and the corresponding effect of nuclease-mediated degradation on circuit performance were characterized to determine the major routes of degradation and DNA strand-displacement circuit failure. Simple circuit design choices, such as the use of 5' toeholds within the DNA complexes used as reactants in the strand-displacement reactions and the termination of single-stranded components with DNA hairpin domains at the 3' termini, significantly increase the functional lifetime of the circuit components in the presence of nucleases. Simulations of multireaction circuits, guided by the experimentally measured operation of single-reaction circuits, enable predictive realization of multilayer and competitive-reaction circuit behavior. Together, these results provide a basic route to increased DNA circuit stability in cell culture environments.

Solving probability reasoning based on DNA strand displacement and probability modules.

Science.gov (United States)

Zhang, Qiang; Wang, Xiaobiao; Wang, Xiaojun; Zhou, Changjun

2017-12-01

In computation biology, DNA strand displacement technology is used to simulate the computation process and has shown strong computing ability. Most researchers use it to solve logic problems, but it is only rarely used in probabilistic reasoning. To process probabilistic reasoning, a conditional probability derivation model and total probability model based on DNA strand displacement were established in this paper. The models were assessed through the game "read your mind." It has been shown to enable the application of probabilistic reasoning in genetic diagnosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

C-terminal phenylalanine of bacteriophage T7 single-stranded DNA-binding protein is essential for strand displacement synthesis by T7 DNA polymerase at a nick in DNA.

Science.gov (United States)

Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C

2009-10-30

Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5'-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.

C-terminal Phenylalanine of Bacteriophage T7 Single-stranded DNA-binding Protein Is Essential for Strand Displacement Synthesis by T7 DNA Polymerase at a Nick in DNA*

Science.gov (United States)

Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C.

2009-01-01

Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5′-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations. PMID:19726688

Second-strand cDNA synthesis: classical method

International Nuclear Information System (INIS)

Gubler, U.

1987-01-01

The classical scheme for the synthesis of double-stranded cDNA as it was reported in 1976 is described. Reverse transcription of mRNA with oligo(dT) as the primer generates first strands with a small loop at the 3' end of the cDNA (the end that corresponds to the 5' end of the mRNA). Subsequent removal of the mRNA by alkaline hydrolysis leaves single-stranded cDNA molecules again with a small 3' loop. This loop can be used by either reverse transcriptase or Klenow fragment of DNA polymerase I as a primer for second-strand synthesis. The resulting products are double-stranded cDNA molecules that are covalently closed at the end corresponding to the 5' end of the original mRNA. Subsequent cleavage of the short piece of single-stranded cDNA within the loop with the single-strand-specific S 1 nuclease generate open double-stranded molecules that can be used for molecular cloning in plasmids or in phage. Useful variations of this scheme have been described

Identification of cis-acting elements on positive-strand subgenomic mRNA required for the synthesis of negative-strand counterpart in bovine coronavirus.

Science.gov (United States)

Yeh, Po-Yuan; Wu, Hung-Yi

2014-07-30

It has been demonstrated that, in addition to genomic RNA, sgmRNA is able to serve as a template for the synthesis of the negative-strand [(-)-strand] complement. However, the cis-acting elements on the positive-strand [(+)-strand] sgmRNA required for (-)-strand sgmRNA synthesis have not yet been systematically identified. In this study, we employed real-time quantitative reverse transcription polymerase chain reaction to analyze the cis-acting elements on bovine coronavirus (BCoV) sgmRNA 7 required for the synthesis of its (-)-strand counterpart by deletion mutagenesis. The major findings are as follows. (1) Deletion of the 5'-terminal leader sequence on sgmRNA 7 decreased the synthesis of the (-)-strand sgmRNA complement. (2) Deletions of the 3' untranslated region (UTR) bulged stem-loop showed no effect on (-)-strand sgmRNA synthesis; however, deletion of the 3' UTR pseudoknot decreased the yield of (-)-strand sgmRNA. (3) Nucleotides positioned from -15 to -34 of the sgmRNA 7 3'-terminal region are required for efficient (-)-strand sgmRNA synthesis. (4) Nucleotide species at the 3'-most position (-1) of sgmRNA 7 is correlated to the efficiency of (-)-strand sgmRNA synthesis. These results together suggest, in principle, that the 5'- and 3'-terminal sequences on sgmRNA 7 harbor cis-acting elements are critical for efficient (-)-strand sgmRNA synthesis in BCoV.

Protected DNA strand displacement for enhanced single nucleotide discrimination in double-stranded DNA

OpenAIRE

Khodakov, Dmitriy A.; Khodakova, Anastasia S.; Huang, David M.; Linacre, Adrian; Ellis, Amanda V.

2015-01-01

Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within doubl...

Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus

Directory of Open Access Journals (Sweden)

Po-Yuan Yeh

2014-07-01

Full Text Available It has been demonstrated that, in addition to genomic RNA, sgmRNA is able to serve as a template for the synthesis of the negative-strand [(−-strand] complement. However, the cis-acting elements on the positive-strand [(+-strand] sgmRNA required for (−-strand sgmRNA synthesis have not yet been systematically identified. In this study, we employed real-time quantitative reverse transcription polymerase chain reaction to analyze the cis-acting elements on bovine coronavirus (BCoV sgmRNA 7 required for the synthesis of its (−-strand counterpart by deletion mutagenesis. The major findings are as follows. (1 Deletion of the 5'-terminal leader sequence on sgmRNA 7 decreased the synthesis of the (−-strand sgmRNA complement. (2 Deletions of the 3' untranslated region (UTR bulged stem-loop showed no effect on (−-strand sgmRNA synthesis; however, deletion of the 3' UTR pseudoknot decreased the yield of (−-strand sgmRNA. (3 Nucleotides positioned from −15 to −34 of the sgmRNA 7 3'-terminal region are required for efficient (−-strand sgmRNA synthesis. (4 Nucleotide species at the 3'-most position (−1 of sgmRNA 7 is correlated to the efficiency of (−-strand sgmRNA synthesis. These results together suggest, in principle, that the 5'- and 3'-terminal sequences on sgmRNA 7 harbor cis-acting elements are critical for efficient (−-strand sgmRNA synthesis in BCoV.

Fluorometric determination of nucleic acids based on the use of polydopamine nanotubes and target-induced strand displacement amplification.

Science.gov (United States)

Ge, Jia; Bai, Dong-Mei; -Geng, Xin; Hu, Ya-Lei; Cai, Qi-Yong; Xing, Ke; Zhang, Lin; Li, Zhao-Hui

2018-01-10

The authors describe a fluorometric method for the quantitation of nucleic acids by combining (a) cycled strand displacement amplification, (b) the unique features of the DNA probe SYBR Green, and (c) polydopamine nanotubes. SYBR Green undergoes strong fluorescence enhancement upon intercalation into double-stranded DNA (dsDNA). The polydopamine nanotubes selectively adsorb single-stranded DNA (ssDNA) and molecular beacons. In the absence of target DNA, the molecular beacon, primer and SYBR Green are adsorbed on the surface of polydopamine nanotubes. This results in quenching of the fluorescence of SYBR Green, typically measured at excitation/emission wavelengths of 488/518 nm. Upon addition of analyte (target DNA) and polymerase, the stem of the molecular beacon is opened so that it can bind to the primer. This triggers target strand displacement polymerization, during which dsDNA is synthesized. The hybridized target is then displaced due to the strand displacement activity of the polymerase. The displaced target hybridizes with another molecular beacon. This triggers the next round of polymerization. Consequently, a large amount of dsDNA is formed which is detected by addition of SYBR Green. Thus, sensitive and selective fluorometric detection is realized. The fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 0.05 to 25 nM with a low limit of detection of 20 pM. Graphical abstract Schematic of a fluorometric strategy for highly sensitive and selective determination of nucleic acids by combining strand displacement amplification and the unique features of SYBR Green I (SG) and polydopamine nanotubes.

Topology synthesis of large-displacement compliant mechanisms

DEFF Research Database (Denmark)

Pedersen, Claus B. Wittendorf; Buhl, Thomas; Sigmund, Ole

2001-01-01

This paper describes the use of topology optimization as a synthesis tool for the design of large-displacement compliant mechanisms. An objective function for the synthesis of large-displacement mechanisms is proposed together with a formulation for synthesis of path-generating compliant mechanisms...

A cascade autocatalytic strand displacement amplification and hybridization chain reaction event for label-free and ultrasensitive electrochemical nucleic acid biosensing.

Science.gov (United States)

Chen, Zhiqiang; Liu, Ying; Xin, Chen; Zhao, Jikuan; Liu, Shufeng

2018-04-23

Herein, an autocatalytic strand displacement amplification (ASDA) strategy was proposed for the first time, which was further ingeniously coupled with hybridization chain reaction (HCR) event for the isothermal, label-free and multiple amplification toward nucleic acid detection. During the ASDA module, the target recognition opens the immobilized hairpin probe (IP) and initiates the annealing of the auxiliary DNA strand (AS) with the opened IP for the successive polymerization and nicking reaction in the presence of DNA polymerase and nicking endonuclease. This induces the target recycling and generation of a large amount of intermediate DNA sequences, which can be used as target analogy to execute the autocatalytic strand displacement amplification. Simultaneously, the introduced AS strand can propagate the HCR between two hairpins (H1 and H2) to form a linear DNA concatamer with cytosine (C)-rich loop region, which can facilitate the in-situ synthesis of silver nanoclusters (AgNCs) as electrochemical tags for further amplification toward target responses. With current cascade ASDA and HCR strategy, the detection of target DNA could be achieved with a low detection limit of about 0.16 fM and a good selectivity. The developed biosensor also exhibits the distinct advantages of flexibility and simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus opens a promising avenue for the detection of nucleic acid with low abundance in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier B.V. All rights reserved.

Programming chemical kinetics: engineering dynamic reaction networks with DNA strand displacement

Science.gov (United States)

Srinivas, Niranjan

Over the last century, the silicon revolution has enabled us to build faster, smaller and more sophisticated computers. Today, these computers control phones, cars, satellites, assembly lines, and other electromechanical devices. Just as electrical wiring controls electromechanical devices, living organisms employ "chemical wiring" to make decisions about their environment and control physical processes. Currently, the big difference between these two substrates is that while we have the abstractions, design principles, verification and fabrication techniques in place for programming with silicon, we have no comparable understanding or expertise for programming chemistry. In this thesis we take a small step towards the goal of learning how to systematically engineer prescribed non-equilibrium dynamical behaviors in chemical systems. We use the formalism of chemical reaction networks (CRNs), combined with mass-action kinetics, as our programming language for specifying dynamical behaviors. Leveraging the tools of nucleic acid nanotechnology (introduced in Chapter 1), we employ synthetic DNA molecules as our molecular architecture and toehold-mediated DNA strand displacement as our reaction primitive. Abstraction, modular design and systematic fabrication can work only with well-understood and quantitatively characterized tools. Therefore, we embark on a detailed study of the "device physics" of DNA strand displacement (Chapter 2). We present a unified view of strand displacement biophysics and kinetics by studying the process at multiple levels of detail, using an intuitive model of a random walk on a 1-dimensional energy landscape, a secondary structure kinetics model with single base-pair steps, and a coarse-grained molecular model that incorporates three-dimensional geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Our findings are consistent with previously measured or inferred rates for

Enzyme-free colorimetric detection systems based on the DNA strand displacement competition reaction

Science.gov (United States)

Zhang, Z.; Birkedal, V.; Gothelf, K. V.

2016-05-01

The strand displacement competition assay is based on the dynamic equilibrium of the competitive hybridization of two oligonucleotides (A and B) to a third oligonucleotide (S). In the presence of an analyte that binds to a specific affinity-moiety conjugated to strand B, the equilibrium shifts, which can be detected by a shift in the fluorescence resonance energy transfer signal between dyes attached to the DNA strands. In the present study we have integrated an ATP aptamer in the strand B and demonstrated the optical detection of ATP. Furthermore we explore a new readout method using a split G-quadruplex DNAzyme for colorimetric readout of the detection of streptavidin by the naked eye. Finally, we integrate the whole G-quadruplex DNAzyme system in a single DNA strand and show that it is applicable to colorimetric detection.

Enzyme-free colorimetric detection systems based on the DNA strand displacement competition reaction

DEFF Research Database (Denmark)

Zhang, Zhao; Birkedal, Victoria; Gothelf, Kurt Vesterager

2016-01-01

The strand displacement competition assay is based on the dynamic equilibrium of the competitive hybridization of two oligonucleotides (A and B) to a third oligonucleotide (S). In the presence of an analyte that binds to a specific affinity-moiety conjugated to strand B, the equilibrium shifts, w...... G-quadruplex DNAzyme for colorimetric readout of the detection of streptavidin by the naked eye. Finally, we integrate the whole G-quadruplex DNAzyme system in a single DNA strand and show that it is applicable to colorimetric detection......., which can be detected by a shift in the fluorescence resonance energy transfer signal between dyes attached to the DNA strands. In the present study we have integrated an ATP aptamer in the strand B and demonstrated the optical detection of ATP. Furthermore we explore a new readout method using a split...

Highly sensitive chemiluminescent point mutation detection by circular strand-displacement amplification reaction.

Science.gov (United States)

Shi, Chao; Ge, Yujie; Gu, Hongxi; Ma, Cuiping

2011-08-15

Single nucleotide polymorphism (SNP) genotyping is attracting extensive attentions owing to its direct connections with human diseases including cancers. Here, we have developed a highly sensitive chemiluminescence biosensor based on circular strand-displacement amplification and the separation by magnetic beads reducing the background signal for point mutation detection at room temperature. This method took advantage of both the T4 DNA ligase recognizing single-base mismatch with high selectivity and the strand-displacement reaction of polymerase to perform signal amplification. The detection limit of this method was 1.3 × 10(-16)M, which showed better sensitivity than that of most of those reported detection methods of SNP. Additionally, the magnetic beads as carrier of immobility was not only to reduce the background signal, but also may have potential apply in high through-put screening of SNP detection in human genome. Copyright © 2011 Elsevier B.V. All rights reserved.

Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress.

Science.gov (United States)

Gan, Haiyun; Yu, Chuanhe; Devbhandari, Sujan; Sharma, Sushma; Han, Junhong; Chabes, Andrei; Remus, Dirk; Zhang, Zhiguo

2017-10-19

The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs in vivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations in vitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel single fluorophore-labeled double-stranded oligonucleotide probe for fluorescence-enhanced nucleic acid detection based on the inherent quenching ability of deoxyguanosine bases and competitive strand-displacement reaction.

Science.gov (United States)

Zhang, Yingwei; Tian, Jingqi; Li, Hailong; Wang, Lei; Sun, Xuping

2012-01-01

We develop a novel single fluorophore-labeled double-stranded oligonucleotide (OND) probe for rapid, nanostructure-free, fluorescence-enhanced nucleic acid detection for the first time. We further demonstrate such probe is able to well discriminate single-base mutation in nucleic acid. The design takes advantage of an inherent quenching ability of guanine bases. The short strand of the probe is designed with an end-labeled fluorophore that is placed adjacent to two guanines as the quencher located on the long opposite strand, resulting in great quenching of dye fluorescence. In the presence of a target complementary to the long strand of the probe, a competitive strand-displacement reaction occurs and the long strand forms a more stable duplex with the target, resulting in the two strands of the probe being separated from each other. As a consequence of this displacement, the fluorophore and the quencher are no longer in close proximity and dye fluorescence increases, signaling the presence of target.

DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

Science.gov (United States)

Dou, Baoting; Yang, Jianmei; Shi, Kai; Yuan, Ruo; Xiang, Yun

2016-09-15

We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Gener: a minimal programming module for chemical controllers based on DNA strand displacement.

Science.gov (United States)

Kahramanoğulları, Ozan; Cardelli, Luca

2015-09-01

: Gener is a development module for programming chemical controllers based on DNA strand displacement. Gener is developed with the aim of providing a simple interface that minimizes the opportunities for programming errors: Gener allows the user to test the computations of the DNA programs based on a simple two-domain strand displacement algebra, the minimal available so far. The tool allows the user to perform stepwise computations with respect to the rules of the algebra as well as exhaustive search of the computation space with different options for exploration and visualization. Gener can be used in combination with existing tools, and in particular, its programs can be exported to Microsoft Research's DSD tool as well as to LaTeX. Gener is available for download at the Cosbi website at http://www.cosbi.eu/research/prototypes/gener as a windows executable that can be run on Mac OS X and Linux by using Mono. ozan@cosbi.eu. © The Author 2015. Published by Oxford University Press.

Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction.

Science.gov (United States)

Wang, Xinyi; Zou, Mingjian; Huang, Hongduan; Ren, Yuqian; Li, Limei; Yang, Xiaoda; Li, Na

2013-03-15

We developed a highly differentiating, homogeneous gold nanoparticle (AuNP) enhanced fluorescence anisotropic method for single nucleotide polymorphism (SNP) detection at nanomolar level using toehold-mediated strand-displacement reaction. The template strand, containing a toehold domain with an allele-specific site, was immobilized on the surface of AuNPs, and the solution fluorescence anisotropy was markedly enhanced when the fluorescein-labeled blocking DNA was attached to the AuNP via hybridization. Strand-displacement by the target ssDNA strand resulted in detachment of fluorescein-labeled DNA from AuNPs, and thus decreased fluorescence anisotropy. The drastic kinetic difference in strand-displacement from toehold design was used to distinguish between the perfectly matched and the single-base mismatched strands. Free energy changes were calculated to elucidate the dependence of the differentiation ability on the mutation site in the toehold region. A solid negative signal change can be obtained for single-base mismatched strand in the dynamic range of the calibration curve, and a more than 10-fold signal difference can still be observed in a mixed solution containing 100 times the single-base mismatched strand, indicating the good specificity of the method. This proposed method can be performed with a standard spectrofluorimeter in a homogeneous and cost-effective manner, and has the potential to be extended to the application of fluorescence anisotropy method of SNP detection. Copyright © 2012 Elsevier B.V. All rights reserved.

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks.

Science.gov (United States)

Chen, Yuan-Jyue; Rao, Sundipta D; Seelig, Georg

2015-11-25

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

The importance of becoming double-stranded: Innate immunity and the kinetic model of HIV-1 central plus strand synthesis

International Nuclear Information System (INIS)

Poeschla, Eric

2013-01-01

Central initiation of plus strand synthesis is a conserved feature of lentiviruses and certain other retroelements. This complication of the standard reverse transcription mechanism produces a transient “central DNA flap” in the viral cDNA, which has been proposed to mediate its subsequent nuclear import. This model has assumed that the important feature is the flapped DNA structure itself rather than the process that produces it. Recently, an alternative kinetic model was proposed. It posits that central plus strand synthesis functions to accelerate conversion to the double-stranded state, thereby helping HIV-1 to evade single-strand DNA-targeting antiviral restrictions such as APOBEC3 proteins, and perhaps to avoid innate immune sensor mechanisms. The model is consistent with evidence that lentiviruses must often synthesize their cDNAs when dNTP concentrations are limiting and with data linking reverse transcription and uncoating. There may be additional kinetic advantages for the artificial genomes of lentiviral gene therapy vectors. - Highlights: • Two main functional models for HIV central plus strand synthesis have been proposed. • In one, a transient central DNA flap in the viral cDNA mediates HIV-1 nuclear import. • In the other, multiple kinetic consequences are emphasized. • One is defense against APOBEC3G, which deaminates single-stranded DNA. • Future questions pertain to antiviral restriction, uncoating and nuclear import

Strand-Specific Analysis of DNA Synthesis and Proteins Association with DNA Replication Forks in Budding Yeast.

Science.gov (United States)

Yu, Chuanhe; Gan, Haiyun; Zhang, Zhiguo

2018-01-01

DNA replication initiates at DNA replication origins after unwinding of double-strand DNA(dsDNA) by replicative helicase to generate single-stranded DNA (ssDNA) templates for the continuous synthesis of leading-strand and the discontinuous synthesis of lagging-strand. Therefore, methods capable of detecting strand-specific information will likely yield insight into the association of proteins at leading and lagging strand of DNA replication forks and the regulation of leading and lagging strand synthesis during DNA replication. The enrichment and Sequencing of Protein-Associated Nascent DNA (eSPAN), which measure the relative amounts of proteins at nascent leading and lagging strands of DNA replication forks, is a step-wise procedure involving the chromatin immunoprecipitation (ChIP) of a protein of interest followed by the enrichment of protein-associated nascent DNA through BrdU immunoprecipitation. The isolated ssDNA is then subjected to strand-specific sequencing. This method can detect whether a protein is enriched at leading or lagging strand of DNA replication forks. In addition to eSPAN, two other strand-specific methods, (ChIP-ssSeq), which detects potential protein-ssDNA binding and BrdU-IP-ssSeq, which can measure synthesis of both leading and lagging strand, were developed along the way. These methods can provide strand-specific and complementary information about the association of the target protein with DNA replication forks as well as synthesis of leading and lagging strands genome wide. Below, we describe the detailed eSPAN, ChIP-ssSeq, and BrdU-IP-ssSeq protocols.

In-stem labelling allows visualization of DNA strand displacements by distinct fluorescent colour change.

Science.gov (United States)

Barrois, Sebastian; Wagenknecht, Hans-Achim

2013-05-21

The combination of thiazole orange (TO) and thiazole red (TR) as an internal pair of fluorescent DNA base surrogates ("DNA traffic lights") allows us to follow at least two consecutive DNA strand displacements in real time through a distinct fluorescence colour change from green to red and vice versa.

Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates.

Science.gov (United States)

Stengel, Gudrun; Kuchta, Robert D

2011-01-01

The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not

Design of a sensitive aptasensor based on magnetic microbeads-assisted strand displacement amplification and target recycling.

Science.gov (United States)

Li, Ying; Ji, Xiaoting; Song, Weiling; Guo, Yingshu

2013-04-03

A cross-circular amplification system for sensitive detection of adenosine triphosphate (ATP) in cancer cells was developed based on aptamer-target interaction, magnetic microbeads (MBs)-assisted strand displacement amplification and target recycling. Here we described a new recognition probe possessing two parts, the ATP aptamer and the extension part. The recognition probe was firstly immobilized on the surface of MBs and hybridized with its complementary sequence to form a duplex. When combined with ATP, the probe changed its conformation, revealing the extension part in single-strand form, which further served as a toehold for subsequent target recycling. The released complementary sequence of the probe acted as the catalyst of the MB-assisted strand displacement reaction. Incorporated with target recycling, a large amount of biotin-tagged MB complexes were formed to stimulate the generation of chemiluminescence (CL) signal in the presence of luminol and H2O2 by incorporating with streptavidin-HRP, reaching a detection limit of ATP as low as 6.1×10(-10)M. Moreover, sample assays of ATP in Ramos Burkitt's lymphoma B cells were performed, which confirmed the reliability and practicality of the protocol. Copyright © 2013 Elsevier B.V. All rights reserved.

Toehold strand displacement-driven assembly of G-quadruplex DNA for enzyme-free and non-label sensitive fluorescent detection of thrombin.

Science.gov (United States)

Xu, Yunying; Zhou, Wenjiao; Zhou, Ming; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

2015-02-15

Based on a new signal amplification strategy by the toehold strand displacement-driven cyclic assembly of G-quadruplex DNA, the development of an enzyme-free and non-label aptamer sensing approach for sensitive fluorescent detection of thrombin is described. The target thrombin associates with the corresponding aptamer of the partial dsDNA probes and liberates single stranded initiation sequences, which trigger the toehold strand displacement assembly of two G-quadruplex containing hairpin DNAs. This toehold strand displacement reaction leads to the cyclic reuse of the initiation sequences and the production of DNA assemblies with numerous G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binds to these G-quadruplex structures and generates significantly amplified fluorescent signals to achieve highly sensitive detection of thrombin down to 5 pM. Besides, this method shows high selectivity towards the target thrombin against other control proteins. The developed thrombin sensing method herein avoids the modification of the probes and the involvement of any enzyme or nanomaterial labels for signal amplification. With the successful demonstration for thrombin detection, our approach can be easily adopted to monitor other target molecules in a simple, low-cost, sensitive and selective way by choosing appropriate aptamer/ligand pairs. Copyright © 2014 Elsevier B.V. All rights reserved.

Programmable autonomous synthesis of single-stranded DNA

Science.gov (United States)

Kishi, Jocelyn Y.; Schaus, Thomas E.; Gopalkrishnan, Nikhil; Xuan, Feng; Yin, Peng

2018-02-01

DNA performs diverse functional roles in biology, nanotechnology and biotechnology, but current methods for autonomously synthesizing arbitrary single-stranded DNA are limited. Here, we introduce the concept of primer exchange reaction (PER) cascades, which grow nascent single-stranded DNA with user-specified sequences following prescribed reaction pathways. PER synthesis happens in a programmable, autonomous, in situ and environmentally responsive fashion, providing a platform for engineering molecular circuits and devices with a wide range of sensing, monitoring, recording, signal-processing and actuation capabilities. We experimentally demonstrate a nanodevice that transduces the detection of a trigger RNA into the production of a DNAzyme that degrades an independent RNA substrate, a signal amplifier that conditionally synthesizes long fluorescent strands only in the presence of a particular RNA signal, molecular computing circuits that evaluate logic (AND, OR, NOT) combinations of RNA inputs, and a temporal molecular event recorder that records in the PER transcript the order in which distinct RNA inputs are sequentially detected.

A lateral flow biosensor for detection of single nucleotide polymorphism by circular strand displacement reaction.

Science.gov (United States)

Xiao, Zhuo; Lie, Puchang; Fang, Zhiyuan; Yu, Luxin; Chen, Junhua; Liu, Jie; Ge, Chenchen; Zhou, Xuemeng; Zeng, Lingwen

2012-09-04

A lateral flow biosensor for detection of single nucleotide polymorphism based on circular strand displacement reaction (CSDPR) has been developed. Taking advantage of high fidelity of T4 DNA ligase, signal amplification by CSDPR, and the optical properties of gold nanoparticles, this assay has reached a detection limit of 0.01 fM.

Design and Analysis of Compact DNA Strand Displacement Circuits for Analog Computation Using Autocatalytic Amplifiers.

Science.gov (United States)

Song, Tianqi; Garg, Sudhanshu; Mokhtar, Reem; Bui, Hieu; Reif, John

2018-01-19

A main goal in DNA computing is to build DNA circuits to compute designated functions using a minimal number of DNA strands. Here, we propose a novel architecture to build compact DNA strand displacement circuits to compute a broad scope of functions in an analog fashion. A circuit by this architecture is composed of three autocatalytic amplifiers, and the amplifiers interact to perform computation. We show DNA circuits to compute functions sqrt(x), ln(x) and exp(x) for x in tunable ranges with simulation results. A key innovation in our architecture, inspired by Napier's use of logarithm transforms to compute square roots on a slide rule, is to make use of autocatalytic amplifiers to do logarithmic and exponential transforms in concentration and time. In particular, we convert from the input that is encoded by the initial concentration of the input DNA strand, to time, and then back again to the output encoded by the concentration of the output DNA strand at equilibrium. This combined use of strand-concentration and time encoding of computational values may have impact on other forms of molecular computation.

An amplified graphene oxide-based fluorescence aptasensor based on target-triggered aptamer hairpin switch and strand-displacement polymerization recycling for bioassays.

Science.gov (United States)

Hu, Kun; Liu, Jinwen; Chen, Jia; Huang, Yong; Zhao, Shulin; Tian, Jianniao; Zhang, Guohai

2013-04-15

An amplified graphene oxide (GO) based fluorescence aptasensor based on target-triggered aptamer hairpin switch and strand-displacement polymerization recycling is developed for bioassays. The dye-labeled single-strand DNA (aptamer hairpin) was adsorbed on the surface of GO, which result in the fluorescence quenching of dye, and exhibiting minimal background fluorescence. Upon the target, primer and polymerase, the stem of the aptamer hairpin was opened, and binds with the primer to triggers the circular target strand-displacement polymerization reaction, which produces huge amounts of duplex helixes DNA and lead to strong fluorescence emission due to shielding of nucelobases within its double-helix structure. During the polymerization reaction, the primer was extended, and target was displaced. And the displaced target recognizes and hybridizes with another hairpin probe, triggering the next round of polymerization reaction, and the circle process induces fluorescence signal amplification for the detection of analyte. To test the feasibility of the aptasensor systems, interferon-gamma (IFN-γ) was employed as a model analyte. A detection limit as low as 1.5 fM is obtained based on the GO aptasensor with a linear range of three orders of magnitude. The present method was successfully applied for the detection of IFN-γ in human plasma. Copyright © 2012 Elsevier B.V. All rights reserved.

Integrating DNA strand-displacement circuitry with DNA tile self-assembly

Science.gov (United States)

Zhang, David Yu; Hariadi, Rizal F.; Choi, Harry M.T.; Winfree, Erik

2013-01-01

DNA nanotechnology has emerged as a reliable and programmable way of controlling matter at the nanoscale through the specificity of Watson–Crick base pairing, allowing both complex self-assembled structures with nanometer precision and complex reaction networks implementing digital and analog behaviors. Here we show how two well-developed frameworks, DNA tile self-assembly and DNA strand-displacement circuits, can be systematically integrated to provide programmable kinetic control of self-assembly. We demonstrate the triggered and catalytic isothermal self-assembly of DNA nanotubes over 10 μm long from precursor DNA double-crossover tiles activated by an upstream DNA catalyst network. Integrating more sophisticated control circuits and tile systems could enable precise spatial and temporal organization of dynamic molecular structures. PMID:23756381

Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification.

Science.gov (United States)

Wang, Yi; Wang, Yan; Ma, Ai-Jing; Li, Dong-Xun; Luo, Li-Juan; Liu, Dong-Xin; Jin, Dong; Liu, Kai; Ye, Chang-Yun

2015-07-08

We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61-65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.

Regulatory RNA design through evolutionary computation and strand displacement.

Science.gov (United States)

Rostain, William; Landrain, Thomas E; Rodrigo, Guillermo; Jaramillo, Alfonso

2015-01-01

The discovery and study of a vast number of regulatory RNAs in all kingdoms of life over the past decades has allowed the design of new synthetic RNAs that can regulate gene expression in vivo. Riboregulators, in particular, have been used to activate or repress gene expression. However, to accelerate and scale up the design process, synthetic biologists require computer-assisted design tools, without which riboregulator engineering will remain a case-by-case design process requiring expert attention. Recently, the design of RNA circuits by evolutionary computation and adapting strand displacement techniques from nanotechnology has proven to be suited to the automated generation of DNA sequences implementing regulatory RNA systems in bacteria. Herein, we present our method to carry out such evolutionary design and how to use it to create various types of riboregulators, allowing the systematic de novo design of genetic control systems in synthetic biology.

A Co-Opted DEAD-Box RNA helicase enhances tombusvirus plus-strand synthesis.

Directory of Open Access Journals (Sweden)

Nikolay Kovalev

2012-02-01

Full Text Available Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV. To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3'-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3'-end of the TBSV (-RNA, rendering the RNA compatible for initiation of (+-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, which is another host factor for TBSV, play non-overlapping functions to enhance (+-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (-RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV, a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells.

Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair.

Science.gov (United States)

Zapotoczny, Grzegorz; Sekelsky, Jeff

2017-04-03

DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate) can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells. Copyright © 2017 Zapotoczny and Sekelsky.

Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair

Directory of Open Access Journals (Sweden)

Grzegorz Zapotoczny

2017-04-01

Full Text Available DNA double-strand breaks (DSBs are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila. To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells.

The 3'-terminal 55 nucleotides of bovine coronavirus defective interfering RNA harbor cis-acting elements required for both negative- and positive-strand RNA synthesis.

Directory of Open Access Journals (Sweden)

Wei-Yu Liao

Full Text Available The synthesis of the negative-strand [(--strand] complement of the ∼30 kilobase, positive-strand [(+-strand] coronaviral genome is a necessary early step for genome replication. The identification of cis-acting elements required for (--strand RNA synthesis in coronaviruses, however, has been hampered due to insufficiencies in the techniques used to detect the (--strand RNA species. Here, we employed a method of head-to-tail ligation and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR to detect and quantitate the synthesis of bovine coronavirus (BCoV defective interfering (DI RNA (- strands. Furthermore, using the aforementioned techniques along with Northern blot assay, we specifically defined the cis-acting RNA elements within the 3'-terminal 55 nucleotides (nts which function in the synthesis of (-- or (+-strand BCoV DI RNA. The major findings are as follows: (i nts from -5 to -39 within the 3'-terminal 55 nts are the cis-acting elements responsible for (--strand BCoV DI RNA synthesis, (ii nts from -3 to -34 within the 3'-terminal 55 nts are cis-acting elements required for (+-strand BCoV DI RNA synthesis, and (iii the nucleotide species at the 3'-most position (-1 is important, but not critical, for both (-- and (+-strand BCoV DI RNA synthesis. These results demonstrate that the 3'-terminal 55 nts in BCoV DI RNA harbor cis-acting RNA elements required for both (-- and (+-strand DI RNA synthesis and extend our knowledge on the mechanisms of coronavirus replication. The method of head-to-tail ligation and qRT-PCR employed in the study may also be applied to identify other cis-acting elements required for (--strand RNA synthesis in coronaviruses.

The RNA synthesis machinery of negative-stranded RNA viruses

International Nuclear Information System (INIS)

Ortín, Juan; Martín-Benito, Jaime

2015-01-01

The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes

The RNA synthesis machinery of negative-stranded RNA viruses

Energy Technology Data Exchange (ETDEWEB)

Ortín, Juan, E-mail: jortin@cnb.csic.es [Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CSIC) and CIBER de Enfermedades Respiratorias (ISCIII), Madrid (Spain); Martín-Benito, Jaime, E-mail: jmartinb@cnb.csic.es [Department of Macromolecular Structures, Centro Nacional de Biotecnología (CSIC), Madrid (Spain)

2015-05-15

The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.

Five-Strand versus Four-Strand Hamstring Tendon Graft Technique for Anterior Cruciate Ligament Reconstruction: A Biomechanical Comparison.

Science.gov (United States)

Vaillant, Eric R; Parks, Brent G; Camire, Lyn M; Hinton, Richard Y

2017-11-01

The aim of this article is to compare diameter and stiffness, displacement, and strain in a five-strand versus four-strand hamstring graft for anterior cruciate ligament reconstruction. Eight matched pairs of lower extremities underwent four-strand or five-strand hamstring graft reconstruction. Diameter was significantly higher in the five-strand versus the four-strand construct ( p  = 0.002). No significant difference was found between the groups in construct displacement or stiffness. Significantly higher strain was observed in the inner limb versus the outer limb in the four-strand construct ( p  = 0.001) and in the inner limb versus the fifth limb in the 5-strand construct ( p  = 0.004). A fifth limb added to a four-strand hamstring graft significantly increased graft diameter but did not significantly change stiffness or displacement, suggesting that attachment of additional graft material via suture did not provide for full incorporation of the added limb into the graft at time zero. The inner limb in both constructs absorbed significantly greater load than did other limbs. The use of suture to attach additional material to a four-strand hamstring graft may not contribute to improved biomechanical qualities of the graft at time zero. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Signals for the initiation and termination of synthesis of the viral strand of bacteriophage f1

International Nuclear Information System (INIS)

Dotto, G.P.; Horiuchi, K.; Jakes, K.S.; Zinder, N.D.

1983-01-01

In this paper the sequence around the plus origin that is required for efficient plus-strand synthesis as well as that necessary for gene-II-protein recognition is described. Results which demonstrate that the nucleotide sequence of the f1 plus origin contains two overlapping but distinct signals, one for initiation and the other for termination of plus-strand synthesis is presented. 29 references, 6 figures, 1 table

Strand displacement amplification for ultrasensitive detection of human pluripotent stem cells.

Science.gov (United States)

Wu, Wei; Mao, Yiping; Zhao, Shiming; Lu, Xuewen; Liang, Xingguo; Zeng, Lingwen

2015-06-30

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a powerful model system for studies of cellular identity and early mammalian development, which hold great promise for regenerative medicine. It is necessary to develop a convenient method to discriminate hPSCs from other cells in clinics and basic research. Herein, a simple and reliable biosensor for stem cell detection was established. In this biosensor system, stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4) were used to mark human pluripotent stem cells (hPSCs). Antibody specific for SSEA-3 was coated onto magnetic beads for hPSCs enrichment, and antibody specific for SSEA-4 was conjugated with carboxyl-modified tDNA sequence which was used as template for strand displacement amplification (SDA). The amplified single strand DNA (ssDNA) was detected with a lateral flow biosensor (LFB). This biosensor is capable of detecting a minimum of 19 human embryonic stem cells by a strip reader and 100 human embryonic stem cells by the naked eye within 80min. This approach has also shown excellent specificity to distinguish hPSCs from other types of cells, showing that it is promising for specific and handy detection of human pluripotent stem cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Highly sensitive "signal-on" electrochemiluminescent biosensor for the detection of DNA based on dual quenching and strand displacement reaction.

Science.gov (United States)

Lou, Jing; Wang, Zhaoyin; Wang, Xiao; Bao, Jianchun; Tu, Wenwen; Dai, Zhihui

2015-10-07

A "signal-on" electrochemiluminescent DNA biosensing platform was proposed based on the dual quenching and strand displacement reaction. This novel "signal-on" detection strategy revealed its sensitivity in achieving a detection limit of 2.4 aM and its selectivity in distinguishing single nucleotide polymorphism of target DNA.

Ultraselective electrochemiluminescence biosensor based on locked nucleic acid modified toehold-mediated strand displacement reaction and junction-probe.

Science.gov (United States)

Zhang, Xi; Zhang, Jing; Wu, Dongzhi; Liu, Zhijing; Cai, Shuxian; Chen, Mei; Zhao, Yanping; Li, Chunyan; Yang, Huanghao; Chen, Jinghua

2014-12-07

Locked nucleic acid (LNA) is applied in toehold-mediated strand displacement reaction (TMSDR) to develop a junction-probe electrochemiluminescence (ECL) biosensor for single-nucleotide polymorphism (SNP) detection in the BRCA1 gene related to breast cancer. More than 65-fold signal difference can be observed with perfectly matched target sequence to single-base mismatched sequence under the same conditions, indicating good selectivity of the ECL biosensor.

Detection of lead(II) ions with a DNAzyme and isothermal strand displacement signal amplification.

Science.gov (United States)

Li, Wenying; Yang, Yue; Chen, Jian; Zhang, Qingfeng; Wang, Yan; Wang, Fangyuan; Yu, Cong

2014-03-15

A DNAzyme based method for the sensitive and selective quantification of lead(II) ions has been developed. A DNAzyme that requires Pb(2+) for activation was selected. An RNA containing DNA substrate was cleaved by the DNAzyme in the presence of Pb(2+). The 2',3'-cyclic phosphate of the cleaved 5'-part of the substrate was efficiently removed by Exonuclease III. The remaining part of the single stranded DNA (9 or 13 base long) was subsequently used as the primer for the strand displacement amplification reaction (SDAR). The method is highly sensitive, 200 pM lead(II) could be easily detected. A number of interference ions were tested, and the sensor showed good selectivity. Underground water samples were also tested, which demonstrated the feasibility of the current approach for real sample applications. It is feasible that our method could be used for DNAzyme or aptazyme based new sensing method developments for the quantification of other target analytes with high sensitivity and selectivity. © 2013 Elsevier B.V. All rights reserved.

Universal, colorimetric microRNA detection strategy based on target-catalyzed toehold-mediated strand displacement reaction

Science.gov (United States)

Park, Yeonkyung; Lee, Chang Yeol; Kang, Shinyoung; Kim, Hansol; Park, Ki Soo; Park, Hyun Gyu

2018-02-01

In this work, we developed a novel, label-free, and enzyme-free strategy for the colorimetric detection of microRNA (miRNA), which relies on a target-catalyzed toehold-mediated strand displacement (TMSD) reaction. The system employs a detection probe that specifically binds to the target miRNA and sequentially releases a catalyst strand (CS) intended to trigger the subsequent TMSD reaction. Thus, the presence of target miRNA releases the CS that mediates the formation of an active G-quadruplex DNAzyme which is initially caged and inactivated by a blocker strand. In addition, a fuel strand that is supplemented for the recycling of the CS promotes another TMSD reaction, consequently generating a large number of active G-quadruplex DNAzymes. As a result, a distinct colorimetric signal is produced by the ABTS oxidation promoted by the peroxidase mimicking activity of the released G-quadruplex DNAzymes. Based on this novel strategy, we successfully detected miR-141, a promising biomarker for human prostate cancer, with high selectivity. The diagnostic capability of this system was also demonstrated by reliably determining target miR-141 in human serum, showing its great potential towards real clinical applications. Importantly, the proposed approach is composed of separate target recognition and signal transduction modules. Thus, it could be extended to analyze different target miRNAs by simply redesigning the detection probe while keeping the same signal transduction module as a universal signal amplification unit, which was successfully demonstrated by analyzing another target miRNA, let-7d.

Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate.

Science.gov (United States)

Li, Wei; Jiang, Wei; Ding, Yongshun; Wang, Lei

2015-09-15

MicroRNAs (miRNAs) play important roles in a variety of biological processes and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5 fM, which displayed an excellent sensitivity. Moreover, the proposed method which can accurately be identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

Cascaded strand displacement for non-enzymatic target recycling amplification and label-free electronic detection of microRNA from tumor cells

Energy Technology Data Exchange (ETDEWEB)

Shi, Kai; Dou, Baoting; Yang, Jianmei; Yuan, Ruo; Xiang, Yun, E-mail: yunatswu@swu.edu.cn

2016-04-15

The monitoring of microRNA (miRNA) expression levels is of great importance in cancer diagnosis. In the present work, based on two cascaded toehold-mediated strand displacement reactions (TSDRs), we have developed a label- and enzyme-free target recycling signal amplification approach for sensitive electronic detection of miRNA-21 from human breast cancer cells. The junction probes containing the locked G-quadruplex forming sequences are self-assembled on the senor surface. The presence of the target miRNA-21 initiates the first TSDR and results in the disassembly of the junction probes and the release of the active G-quadruplex forming sequences. Subsequently, the DNA fuel strand triggers the second TSDR and leads to cyclic reuse of the target miRNA-21. The cascaded TSDRs thus generate many active G-quadruplex forming sequences on the sensor surface, which associate with hemin to produce significantly amplified current response for sensitive detection of miRNA-21 at 1.15 fM. The sensor is also selective and can be employed to monitor miRNA-21 from human breast cancer cells. - Highlights: • Amplified and sensitive detection of microRNA from tumor cells is achieved. • Signal amplification is realized by two cascaded strand displacement reactions. • The developed sensor is selective and label-free without involving any enzymes.

Dynamics of Leading-strand Lesion Skipping by the Replisome

Science.gov (United States)

Yeeles, Joseph T.P.; Marians, Kenneth J.

2013-01-01

SUMMARY The E. coli replisome stalls transiently when it encounters a lesion in the leading-strand template, skipping over the damage by reinitiating replication at a new primer synthesized downstream by the primase. We report here that template unwinding and lagging-strand synthesis continue downstream of the lesion at a reduced rate after replisome stalling, that one replisome is capable of skipping multiple lesions, and that the rate limiting steps of replication restart involve the synthesis and activation of the new primer downstream. We also find little support for the concept that polymerase uncoupling, where extensive lagging-strand synthesis proceeds downstream in the absence of leading-strand synthesis, involves physical separation of the leading-strand polymerase from the replisome. Instead, our data indicate that extensive uncoupled replication likely results from a failure of the leading-strand polymerase still associated with the DNA helicase and the lagging-strand polymerase that are proceeding downstream to reinitiate synthesis. PMID:24268579

Accurate quantification of microRNA via single strand displacement reaction on DNA origami motif.

Directory of Open Access Journals (Sweden)

Jie Zhu

Full Text Available DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs.

Accurate Quantification of microRNA via Single Strand Displacement Reaction on DNA Origami Motif

Science.gov (United States)

Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

2013-01-01

DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs. PMID:23990889

Accurate quantification of microRNA via single strand displacement reaction on DNA origami motif.

Science.gov (United States)

Zhu, Jie; Feng, Xiaolu; Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

2013-01-01

DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs.

Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

Science.gov (United States)

Cheng, Rui; Tao, Mangjuan; Shi, Zhilu; Zhang, Xiafei; Jin, Yan; Li, Baoxin

2015-11-15

Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research. Copyright © 2015 Elsevier B.V. All rights reserved.

Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA

Science.gov (United States)

Yang, Zhiyu; Price, Nathan E.; Johnson, Kevin M.

2017-01-01

Abstract Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3’ddR5p) at the 3’-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,β-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3’ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3’ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems. PMID:28531327

Rotavirus NSP2 interferes with the core lattice protein VP2 in initiation of minus-strand synthesis

International Nuclear Information System (INIS)

Vende, Patrice; Tortorici, M. Alejandra; Taraporewala, Zenobia F.; Patton, John T.

2003-01-01

The rotavirus nonstructural protein NSP2 self-assembles into stable octameric structures that possess nonspecific affinity for single-stranded (ss)RNA and RNA-RNA helix-destabilizing and NTPase activities. Furthermore, NSP2 is a component of replication intermediates with replicase activity and plays a critical role in the packaging and replication of the segmented dsRNA genome of rotavirus. To better understand the function of the protein in genome replication, we examined the effect that purified recombinant NSP2 had on the synthesis of dsRNA by the open core replication system. The results showed that NSP2 inhibited the synthesis of dsRNA from viral mRNA in vitro, in a concentration-dependent manner. The inhibition was overcome by adding increasing amounts of viral mRNA or nonviral ssRNA to the system, indicating that the inhibition was mediated by the nonspecific RNA-binding activity of NSP2. Further analysis revealed that NSP2 interfered with the ability of the open core proteins, GTP, and viral mRNA to form the initiation complex for (-) strand synthesis. Additional experiments indicated that NSP2 did not perturb recognition of viral mRNA by the viral RNA polymerase VP1, but rather interfered with the function of VP2, a protein that is essential for (-) strand initiation and dsRNA synthesis and that forms the T = 1 lattice of the virion core. In contrast to initiation, NSP2 did not inhibit (-) strand elongation. Collectively, the findings provide evidence that the temporal order of interaction of RNA-binding proteins with viral mRNA is a crucial factor impacting the formation of replication intermediates

A Universal Fast Colorimetric Method for DNA Signal Detection with DNA Strand Displacement and Gold Nanoparticles

Directory of Open Access Journals (Sweden)

Xin Li

2015-01-01

Full Text Available DNA or gene signal detection is of great significance in many fields including medical examination, intracellular molecular monitoring, and gene disease signal diagnosis, but detection of DNA or gene signals in a low concentration with instant visual results remains a challenge. In this work, a universal fast and visual colorimetric detection method for DNA signals is proposed. Specifically, a DNA signal amplification “circuit” based on DNA strand displacement is firstly designed to amplify the target DNA signals, and then thiol modified hairpin DNA strands and gold nanoparticles are used to make signal detection results visualized in a colorimetric manner. If the target DNA signal exists, the gold nanoparticles aggregate and settle down with color changing from dark red to grey quickly; otherwise, the gold nanoparticles’ colloids remain stable in dark red. The proposed method provides a novel way to detect quickly DNA or gene signals in low concentrations with instant visual results. When applied in real-life, it may provide a universal colorimetric method for gene disease signal diagnosis.

Detection of single-nucleotide polymorphisms using an ON-OFF switching of regenerated biosensor based on a locked nucleic acid-integrated and toehold-mediated strand displacement reaction.

Science.gov (United States)

Gao, Zhong Feng; Ling, Yu; Lu, Lu; Chen, Ning Yu; Luo, Hong Qun; Li, Nian Bing

2014-03-04

Although various strategies have been reported for single-nucleotide polymorphisms (SNPs) detection, development of a time-saving, specific, and regenerated electrochemical sensing platform still remains a realistic goal. In this study, an ON-OFF switching of a regenerated biosensor based on a locked nucleic acid (LNA)-integrated and toehold-mediated strand displacement reaction technique is constructed for detection of SNPs. The LNA-integrated and methylene blue-labeled capture probe with an external toehold is designed to switch on the sensing system. The mutant-type DNA probe completes complementary with the capture probe to trigger the strand displacement reaction, which switches off the sensing system. However, when the single-base mismatched wild-type DNA probe is presented, the strand displacement reaction cannot be achieved; therefore, the sensing system still keeps the ON state. This DNA sensor is stable over five reuses. We further testify that the LNA-integrated sequence has better recognition ability for SNPs detection compared to the DNA-integrated sequence. Moreover, this DNA senor exhibits a remarkable discrimination capability of SNPs among abundant wild-type targets and 6000-fold (m/m) excess of genomic DNA. In addition, it is selective enough in complex and contaminant-ridden samples, such as human urine, soil, saliva, and beer. Overall, these results demonstrate that this reliable DNA sensor is easy to be fabricated, simple to operate, and stable enough to be readily regenerated.

Preparation of molybdenum borides by combustion synthesis involving solid-phase displacement reactions

International Nuclear Information System (INIS)

Yeh, C.L.; Hsu, W.S.

2008-01-01

Preparation of molybdenum borides of five different phases in the Mo-B binary system (including Mo 2 B, MoB, MoB 2 , Mo 2 B 5 , and MoB 4 ) was performed by self-propagating high-temperature synthesis (SHS) with two kinds of the reactant samples. When elemental powder compacts with an exact stoichiometry corresponding to the boride phase were employed, self-sustaining reaction was only achieved in the sample with Mo:B = 1:1 and nearly single-phase MoB was yielded. Therefore, the other four boride compounds were prepared from the reactant compacts composed of MoO 3 , Mo, and B powders, within which the displacement reaction of MoO 3 with boron was involved in combustion synthesis. Experimental evidence shows that the extent of displacement reaction in the overall reaction has a significant impact on sustainability of the synthesis reaction, combustion temperature, reaction front velocity, and composition of the end product. An increase in the solid-phase displacement reaction taking place during the SHS process contributes more heat flux to the synthesis reaction, thus resulting in the increase of combustion temperature and enhancement of the reaction front velocity. Based upon the XRD analysis, formation of Mo 2 B, MoB 2 , and Mo 2 B 5 as the dominant boride phase in the end product was successful through the SHS reaction with powder compacts under appropriate stoichiometries between MoO 3 , Mo, and B. However, a poor conversion was observed in the synthesis of MoB 4 . The powder compact prepared for the production of MoB 4 yielded mostly Mo 2 B 5

Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA.

Science.gov (United States)

Yang, Zhiyu; Price, Nathan E; Johnson, Kevin M; Wang, Yinsheng; Gates, Kent S

2017-06-20

Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3'ddR5p) at the 3'-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,β-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3'ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3'ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Role of the Pepino mosaic virus 3'-untranslated region elements in negative-strand RNA synthesis in vitro.

Science.gov (United States)

Osman, Toba A M; Olsthoorn, René C L; Livieratos, Ioannis C

2014-09-22

Pepino mosaic virus (PepMV) is a mechanically-transmitted positive-strand RNA potexvirus, with a 6410 nt long single-stranded (ss) RNA genome flanked by a 5'-methylguanosine cap and a 3' poly-A tail. Computer-assisted folding of the 64 nt long PepMV 3'-untranslated region (UTR) resulted in the prediction of three stem-loop structures (hp1, hp2, and hp3 in the 3'-5' direction). The importance of these structures and/or sequences for promotion of negative-strand RNA synthesis and binding to the RNA dependent RNA polymerase (RdRp) was tested in vitro using a specific RdRp assay. Hp1, which is highly variable among different PepMV isolates, appeared dispensable for negative-strand synthesis. Hp2, which is characterized by a large U-rich loop, tolerated base-pair changes in its stem as long as they maintained the stem integrity but was very sensitive to changes in the U-rich loop. Hp3, which harbours the conserved potexvirus ACUUAA hexamer motif, was essential for template activity. Template-RNA polymerase binding competition experiments showed that the ACUUAA sequence represents a high-affinity RdRp binding element. Copyright © 2014 Elsevier B.V. All rights reserved.

Yield of DNA strand breaks and their relationship to DNA polymerase I-dependent repair synthesis and ligation following x-ray exposure of toluene-treated Escherichia coli

International Nuclear Information System (INIS)

Billen, D.

1981-01-01

In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is observed. This is an exaggerated repair synthesis which can be abruptly terminated by the addition of the DNA ligase cofactor, nicotinamide adenine dinucleotide. This communication describes experiments which bear on the relationship between measurable strand breaks, DNA polymerase I-directed, exaggerated repair synthesis, and strand-break repair

Contribution of gold nanoparticles to the catalytic DNA strand displacement in leakage reduction and signal amplification.

Science.gov (United States)

Wang, Bei; Zhou, Xiang; Yao, Dongbao; Sun, Xianbao; He, Miao; Wang, Xiaojing; Yin, Xue; Liang, Haojun

2017-10-03

A new model using a gold nanoparticle (AuNP)-DNA system to constrain leakage and improve efficiency of catalytic toehold-mediated strand displacement reactions was outlined. A 10-bp spacer on AuNPs and fourfold amount of fuels were determined for good performance of this model with an optimized toehold strategy. After the reaction at 25 °C for 10 h, a 258 pM target could be identified, which is a remarkable improvement compared with the traditional AuNP-DNA system without fuel. Moreover, this model was also studied to differentiate specific single nucleotide polymorphism on target with superior selection factors. This model may help by introducing a proposition of target detection to guide further investigation.

Large-scale synthesis of Tellurium nanostructures via galvanic displacement of metals

Science.gov (United States)

Kok, Kuan-Ying; Choo, Thye-Foo; Ubaidah Saidin, Nur; Rahman, Che Zuraini Che Ab

2018-01-01

Tellurium (Te) is an attractive semiconductor material for a wide range of applications in various functional devices including, radiation dosimeters, optical storage materials, thermoelectric or piezoelectric generators. In this work, large scale synthesis of tellurium (Te) nanostructures have been successfully carried out in different concentrations of aqueous solutions containing TeO2 and NaOH, by galvanic displacements of Zn and Al which served as the sacrificial materials. Galvanic displacement process is cost-effective and it requires no template or surfactant for the synthesis of nanostructures. By varying the concentrations of TeO2 and NaOH, etching temperatures and etching times, Te nanostructures of various forms of nanostructures were successfully obtained, ranging from one-dimensional needles and rod-like structures to more complex hierarchical structures. Microscopy examinations on the nanostructures obtained have shown that both the diameters and lengths of the Te nanostructures increased with increasing etching temperature and etching time.

A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots

Science.gov (United States)

Gliddon, H. D.; Howes, P. D.; Kaforou, M.; Levin, M.; Stevens, M. M.

2016-05-01

The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar

Signal-off Electrochemiluminescence Biosensor Based on Phi29 DNA Polymerase Mediated Strand Displacement Amplification for MicroRNA Detection.

Science.gov (United States)

Chen, Anyi; Gui, Guo-Feng; Zhuo, Ying; Chai, Ya-Qin; Xiang, Yun; Yuan, Ruo

2015-06-16

A target induced cycling strand displacement amplification (SDA) mediated by phi29 DNA polymerase (phi29) was first investigated and applied in a signal-off electrochemiluminescence (ECL) biosensor for microRNA (miRNA) detection. Herein, the target miRNA triggered the phi29-mediated SDA which could produce amounts of single-stranded DNA (assistant probe) with accurate and comprehensive nucleotide sequence. Then, the assistant probe hybridized with the capture probe and the ferrocene-labeled probe (Fc-probe) to form a ternary "Y" structure for ECL signal quenching by ferrocene. Therefore, the ECL intensity would decrease with increasing concentration of the target miRNA, and the sensitivity of biosensor would be promoted on account of the efficient signal amplification of the target induced cycling reaction. Besides, a self-enhanced Ru(II) ECL system was designed to obtain a stable and strong initial signal to further improve the sensitivity. The ECL assay for miRNA-21 detection is developed with excellent sensitivity of a concentration variation from 10 aM to 1.0 pM and limit of detection down to 3.3 aM.

Voltammetric determination of attomolar levels of a sequence derived from the genom of hepatitis B virus by using molecular beacon mediated circular strand displacement and rolling circle amplification.

Science.gov (United States)

Huang, Shan; Feng, Mengmeng; Li, Jiawen; Liu, Yi; Xiao, Qi

2018-03-03

The authors describe an electrochemical method for the determination of the single-stranded DNA (ssDNA) oligonucleotide with a sequence derived from the genom of hepatitis B virus (HBV). It is making use of circular strand displacement (CSD) and rolling circle amplification (RCA) strategies mediated by a molecular beacon (MB). This ssDNA hybridizes with the loop portion of the MB immobilized on the surface of a gold electrode, while primer DNA also hybridizes with the rest of partial DNA sequences of MB. This triggers the MB-mediated CSD. The RCA is then initiated to produce a long DNA strand with multiple tandem-repeat sequences, and this results in a significant increase of the differential pulse voltammetric response of the electrochemical probe Methylene Blue at a rather low working potential of -0.24 V (vs. Ag/AgCl). Under optimal experimental conditions, the assay displays an ultrahigh sensitivity (with a 2.6 aM detection limit) and excellent selectivity. Response is linear in the 10 to 700 aM DNA concentration range. Graphical abstract Schematic of a voltammetric method for the determination of attomolar levels of target DNA. It is based on molecular beacon mediated circular strand displacement and rolling circle amplification strategies. Under optimal experimental conditions, the assay displays an ultrahigh sensitivity with a 2.6 aM detection limit and excellent selectivity.

Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices.

Science.gov (United States)

Giuffrida, Maria Chiara; Zanoli, Laura Maria; D'Agata, Roberta; Finotti, Alessia; Gambari, Roberto; Spoto, Giuseppe

2015-02-01

Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells.

Replisome-mediated Translesion Synthesis and Leading Strand Template Lesion Skipping Are Competing Bypass Mechanisms*

Science.gov (United States)

Gabbai, Carolina B.; Yeeles, Joseph T. P.; Marians, Kenneth J.

2014-01-01

A number of different enzymatic pathways have evolved to ensure that DNA replication can proceed past template base damage. These pathways include lesion skipping by the replisome, replication fork regression followed by either correction of the damage and origin-independent replication restart or homologous recombination-mediated restart of replication downstream of the lesion, and bypass of the damage by a translesion synthesis DNA polymerase. We report here that of two translesion synthesis polymerases tested, only DNA polymerase IV, not DNA polymerase II, could engage productively with the Escherichia coli replisome to bypass leading strand template damage, despite the fact that both enzymes are shown to be interacting with the replicase. Inactivation of the 3′ → 5′ proofreading exonuclease of DNA polymerase II did not enable bypass. Bypass by DNA polymerase IV required its ability to interact with the β clamp and act as a translesion polymerase but did not require its “little finger” domain, a secondary region of interaction with the β clamp. Bypass by DNA polymerase IV came at the expense of the inherent leading strand lesion skipping activity of the replisome, indicating that they are competing reactions. PMID:25301949

Sites of termination of in vitro DNA synthesis on psoralen phototreated single-stranded templates

International Nuclear Information System (INIS)

Piette, J.; Hearst, J.

1985-01-01

Single-stranded DNA has been photochemically induced to react with 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and used as substrate for DNA replication with E. coli DNA polymerase I large fragment. By using the dideoxy sequencing procedure, it is possible to map the termination sites on the template photoreacted with HMT. These sites occur at the nucleotides preceding each thymine residue (and a few cytosine residues), emphasizing the fact that in a single-stranded stretch of DNA, HMT reacts with each thymine residue without any specificity regarding the flanking base sequence of the thymine residues. In addition, termination of DNA synthesis due to psoralen-adducted thymine is not influenced by the efficiency of the 3'-5' exonuclease proof-reading activity of the DNA polymerase. (author)

Detection of DNA damage based on metal-mediated molecular beacon and DNA strands displacement reaction

Science.gov (United States)

Xiong, Yanxiang; Wei, Min; Wei, Wei; Yin, Lihong; Pu, Yuepu; Liu, Songqin

2014-01-01

DNA hairpin structure probes are usually designed by forming intra-molecular duplex based on Watson-Crick hydrogen bonds. In this paper, a molecular beacon based on silver ions-mediated cytosine-Ag+-cytosine base pairs was used to detect DNA. The inherent characteristic of the metal ligation facilitated the design of functional probe and the adjustment of its binding strength compared to traditional DNA hairpin structure probes, which make it be used to detect DNA in a simple, rapid and easy way with the help of DNA strands displacement reaction. The method was sensitive and also possesses the good specificity to differentiate the single base mismatched DNA from the complementary DNA. It was also successfully applied to study the damage effect of classic genotoxicity chemicals such as styrene oxide and sodium arsenite on DNA, which was significant in food science, environmental science and pharmaceutical science.

[Investigation of RNA viral genome amplification by multiple displacement amplification technique].

Science.gov (United States)

Pang, Zheng; Li, Jian-Dong; Li, Chuan; Liang, Mi-Fang; Li, De-Xin

2013-06-01

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.

CdS nanowires formed by chemical synthesis using conjugated single-stranded DNA molecules

Science.gov (United States)

Sarangi, S. N.; Sahu, S. N.; Nozaki, S.

2018-03-01

CdS nanowires were successfully grown by chemical synthesis using two conjugated single-stranded (ss) DNA molecules, poly G (30) and poly C (30), as templates. During the early stage of the synthesis with the DNA molecules, the Cd 2+ interacts with Poly G and Poly C and produces the (Cd 2+)-Poly GC complex. As the growth proceeds, it results in nanowires. The structural analysis by grazing angle x-ray diffraction and transmission electron microscopy confirmed the zinc-blende CdS nanowires with the growth direction of . Although the nanowires are well surface-passivated with the DNA molecules, the photoluminescence quenching was caused by the electron transfer from the nanowires to the DNA molecules. The quenching can be used to detect and label the DNAs.

In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication.

Science.gov (United States)

Schauer, Grant; Finkelstein, Jeff; O'Donnell, Mike

2017-09-20

The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other proteins. We have previously established the mechanisms by which the polymerases ε and δ are targeted to their 'correct' strands, as well as quality control mechanisms that evict polymerases when they associate with an 'incorrect' strand. Here, we provide a practical guide to differentially assay leading and lagging strand replication in vitro using pure proteins.

A label-free ultrasensitive fluorescence detection of viable Salmonella enteritidis using enzyme-induced cascade two-stage toehold strand-displacement-driven assembly of G-quadruplex DNA.

Science.gov (United States)

Zhang, Peng; Liu, Hui; Ma, Suzhen; Men, Shuai; Li, Qingzhou; Yang, Xin; Wang, Hongning; Zhang, Anyun

2016-06-15

The harm of Salmonella enteritidis (S. enteritidis ) to public health mainly by contaminating fresh food and water emphasizes the urgent need for rapid detection techniques to help control the spread of the pathogen. In this assay, an newly designed capture probe complex that contained specific S. enteritidis-aptamer and hybridized signal target sequence was used for viable S. enteritidis recognition directly. In the presence of the target S. enteritidis, single-stranded target sequences were liberated and initiated the replication-cleavage reaction, producing numerous G-quadruplex structures with a linker on the 3'-end. And then, the sensing system took innovative advantage of quadratic linker-induced strand-displacement for the first time to release target sequence in succession, leading to the cyclic reuse of the target sequences and cascade signal amplification, thereby achieving the successive production of G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binded to these G-quadruplex structures and generated significantly enhanced fluorescent signals to achieve highly sensitive detection of S. enteritidis down to 60 CFU/mL with a linear range from 10(2) to 10(7)CFU/mL. By coupling the cascade two-stage target sequences-recyclable toehold strand-displacement with aptamer-based target recognition successfully, it is the first report on a novel non-label, modification-free and DNA extraction-free ultrasensitive fluorescence biosensor for detecting viable S. enteritidis directly, which can discriminate from dead S. enteritidis. Copyright © 2016 Elsevier B.V. All rights reserved.

Attomolar detection of proteins via cascade strand-displacement amplification and polystyrene nanoparticle enhancement in fluorescence polarization aptasensors.

Science.gov (United States)

Huang, Yong; Liu, Xiaoqian; Huang, Huakui; Qin, Jian; Zhang, Liangliang; Zhao, Shulin; Chen, Zhen-Feng; Liang, Hong

2015-08-18

Extremely sensitive and accurate measurements of protein markers for early detection and monitoring of diseases pose a formidable challenge. Herein, we develop a new type of amplified fluorescence polarization (FP) aptasensor based on allostery-triggered cascade strand-displacement amplification (CSDA) and polystyrene nanoparticle (PS NP) enhancement for ultrasensitive detection of proteins. The assay system consists of a fluorescent dye-labeled aptamer hairpin probe and a PS NP-modified DNA duplex (assistant DNA/trigger DNA duplex) probe with a single-stranded part and DNA polymerase. Two probes coexist stably in the absence of target, and the dye exhibits relatively low FP background. Upon recognition and binding with a target protein, the stem of the aptamer hairpin probe is opened, after which the opened hairpin probe hybridizes with the single-stranded part in the PS NP-modified DNA duplex probe and triggers the CSDA reaction through the polymerase-catalyzed recycling of both target protein and trigger DNA. Throughout this CSDA process, numerous massive dyes are assembled onto PS NPs, which results in a substantial FP increase that provides a readout signal for the amplified sensing process. Our newly proposed amplified FP aptasensor enables the quantitative measurement of proteins with the detection limit in attomolar range, which is about 6 orders of magnitude lower than that of traditional homogeneous aptasensors. Moreover, this sensing method also exhibits high specificity for target proteins and can be performed in homogeneous solutions. In addition, the suitability of this method for the quantification of target protein in biological samples has also been shown. Considering these distinct advantages, the proposed sensing method can be expected to provide an ultrasensitive platform for the analysis of various types of target molecules.

Detection of bacteriophage phi 6 minus-strand RNA and novel mRNA isoconformers synthesized in vivo and in vitro, by strand-separating agarose gels

International Nuclear Information System (INIS)

Pagratis, N.; Revel, H.R.

1990-01-01

Two urea-free agarose gel protocols that resolve the six individual strands of bacteriophage phi 6 dsRNA were developed and used to analyze phage RNA synthesis in vivo and in vitro. Citrate gels separate strands of the large and medium chromosomes while Tris-borate-EDTA (TBE) gels resolve the medium and small dsRNA segments. Minus strands migrate faster than plus strands on citrate gels but are retarded on TBE gels. A study of electrophoretic conditions showed that pH affects strand resolution on citrate gels, and that voltage gradient, agarose concentration, and ethidium bromide significantly alter strand migration on TBE gels. Analysis of native phi 6 RNA synthesized in vivo and in vitro showed that the large and medium message RNAs comigrate with the corresponding plus strands of denatured virion dsRNA. The small messenger RNA is exceptional. Native small mRNA was detected as three isoconformers in vivo and in vitro. The isoconformers were converted by heat denaturation to a single RNA species that comigrates with the virion s+ strand. Minus strands labeled in vivo were detected only after heat denaturation. Minus strand synthesis was detected also in heat-denatured samples from in vitro phi 6 nucleocapsid RNA polymerase reactions at pH values suboptimal for transcription

RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand.

Science.gov (United States)

Chloé, Jaubert; Amina, Bedrat; Laura, Bartolucci; Carmelo, Di Primo; Michel, Ventura; Jean-Louis, Mergny; Samir, Amrane; Marie-Line, Andreola

2018-05-25

DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or "G4" that are based on the stacking of G-quartets. The role of DNA and RNA G4 is documented in eukaryotic cells and in pathogens such as viruses. Yet, G4 have been identified only in a few RNA viruses, including the Flaviviridae family. In this study, we analysed the last 157 nucleotides at the 3'end of the HCV (-) strand. This sequence is known to be the minimal sequence required for an efficient RNA replication. Using bioinformatics and biophysics, we identified a highly conserved G4-prone sequence located in the stem-loop IIy' of the negative strand. We also showed that the formation of this G-quadruplex inhibits the in vitro RNA synthesis by the RdRp. Furthermore, Phen-DC3, a specific G-quadruplex binder, is able to inhibit HCV viral replication in cells in conditions where no cytotoxicity was measured. Considering that this domain of the negative RNA strand is well conserved among HCV genotypes, G4 ligands could be of interest for new antiviral therapies.

Signal-on electrochemiluminescence biosensor for microRNA-319a detection based on two-stage isothermal strand-displacement polymerase reaction.

Science.gov (United States)

Wang, Minghui; Zhou, Yunlei; Yin, Huanshun; Jiang, Wenjing; Wang, Haiyan; Ai, Shiyun

2018-06-01

MicroRNAs play crucial role in regulating gene expression in organism, thus it is very necessary to exploit an efficient method for the sensitive and specific detection of microRNA. Herein, a signal-on electrochemiluminescence biosensor was fabricated for microRNA-319a detection based on two-stage isothermal strand-displacement polymerase reaction (ISDPR). In the presence of target microRNA, amounts of trigger DNA could be generated by the first ISDPR. Then, the trigger DNA and the primer hybridized simultaneously with the hairpin probe to open the stem of the probe, and then the ECL signal will be emitted. In the presence of phi29 DNA polymerase and dNTPs, the trigger DNA could be displaced to initiate a new cycle which was the second ISDPR. Due to the two-stage amplification, this method presented excellent detection sensitivity with a low detection limit of 0.14 fM. Moreover, the applicability of the developed method was demonstrated by detecting the change of microRNA-319a content in the leaves of rice seedlings after the rice seeds were incubated with chemical mutagen of ethyl methanesulfonate. Copyright © 2018 Elsevier B.V. All rights reserved.

Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis.

Science.gov (United States)

Thormar, Hans G; Gudmundsson, Bjarki; Eiriksdottir, Freyja; Kil, Siyoen; Gunnarsson, Gudmundur H; Magnusson, Magnus Karl; Hsu, Jason C; Jonsson, Jon J

2013-04-01

The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids. We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis. The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. © 2013 American Association for Clinical Chemistry

A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification

Energy Technology Data Exchange (ETDEWEB)

Wu, Wei [College of Food Science and Engineering, Ocean University of China, Qingdao 266003 (China); Zhao, Shiming [Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530 (China); Mao, Yiping [Yueyang Institute for Food and Drug Control, Yueyang 430198 (China); Fang, Zhiyuan [Affiliated Tumor Hospital of Guangzhou Medical University, Guangzhou 510095 (China); Lu, Xuewen [Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530 (China); Zeng, Lingwen, E-mail: zeng6@yahoo.com [Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530 (China)

2015-02-25

Highlights: • Limit of detection as low as 10 CFU mL{sup −1}Escherichia coli O157:H7. • No need of antibodies and substituted with aptamers. • Isothermal strand displacement amplification for signal amplification. • Results observed by the naked eye. • Great potential application in the area of food control. - Abstract: Foodborne diseases caused by pathogens are one of the major problems in food safety. Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Commonly used methods for pathogen detection rely on conventional culture-based tests, antibody-based assays and polymerase chain reaction (PCR)-based techniques. These methods are costly, laborious and time-consuming. Herein, we present a simple and sensitive aptamer based biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). In this assay, two different aptamers specific for the outmembrane of E. coli O157:H7 were used. One of the aptamers was used for magnetic bead enrichment, and the other was used as a signal reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. Only the captured aptamers on cell membrane were amplified, limitations of conventional DNA amplification based method such as false-positive can be largely reduced. The generated signals (red bands on the test zone of a lateral flow strip) can be unambiguously read out by the naked eye. As low as 10 colony forming units (CFU) of E. coli O157:H7 were detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.

A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification

International Nuclear Information System (INIS)

Wu, Wei; Zhao, Shiming; Mao, Yiping; Fang, Zhiyuan; Lu, Xuewen; Zeng, Lingwen

2015-01-01

Highlights: • Limit of detection as low as 10 CFU mL −1 Escherichia coli O157:H7. • No need of antibodies and substituted with aptamers. • Isothermal strand displacement amplification for signal amplification. • Results observed by the naked eye. • Great potential application in the area of food control. - Abstract: Foodborne diseases caused by pathogens are one of the major problems in food safety. Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Commonly used methods for pathogen detection rely on conventional culture-based tests, antibody-based assays and polymerase chain reaction (PCR)-based techniques. These methods are costly, laborious and time-consuming. Herein, we present a simple and sensitive aptamer based biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). In this assay, two different aptamers specific for the outmembrane of E. coli O157:H7 were used. One of the aptamers was used for magnetic bead enrichment, and the other was used as a signal reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. Only the captured aptamers on cell membrane were amplified, limitations of conventional DNA amplification based method such as false-positive can be largely reduced. The generated signals (red bands on the test zone of a lateral flow strip) can be unambiguously read out by the naked eye. As low as 10 colony forming units (CFU) of E. coli O157:H7 were detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods

Signal amplification of microRNAs with modified strand displacement-based cycling probe technology.

Science.gov (United States)

Jia, Huning; Bu, Ying; Zou, Bingjie; Wang, Jianping; Kumar, Shalen; Pitman, Janet L; Zhou, Guohua; Song, Qinxin

2016-10-24

Micro ribose nucleic acids (miRNAs) play an important role in biological processes such as cell differentiation, proliferation and apoptosis. Therefore, miRNAs are potentially a powerful marker for monitoring cancer and diagnosis. Here, we present sensitive signal amplification for miRNAs based on modified cycling probe technology with strand displacement amplification. miRNA was captured by the template coupled with beads, and then the first cycle based on SDA was repeatedly extended to the nicking end, which was produced by the extension reaction of miRNA. The products generated by SDA are captured by a molecular beacon (MB), which is designed to initiate the second amplification cycle, with a similar principle to the cycling probe technology (CPT), which is based on repeated digestion of the DNA-RNA hybrid by the RNase H. After one sample enrichment and two steps of signal amplification, 0.1 pM of let-7a can be detected. The miRNA assay exhibits a great dynamic range of over 100 orders of magnitude and high specificity to clearly discriminate a single base difference in miRNA sequences. This isothermal amplification does not require any special temperature control instrument. The assay is also about signal amplification rather than template amplification, therefore minimising contamination issues. In addition, there is no need for the reverse transcription (RT) process. Thus the amplification is suitable for miRNA detection.

CTC1-STN1 coordinates G- and C-strand synthesis to regulate telomere length.

Science.gov (United States)

Gu, Peili; Jia, Shuting; Takasugi, Taylor; Smith, Eric; Nandakumar, Jayakrishnan; Hendrickson, Eric; Chang, Sandy

2018-05-17

Coats plus (CP) is a rare autosomal recessive disorder caused by mutations in CTC1, a component of the CST (CTC1, STN1, and TEN1) complex important for telomere length maintenance. The molecular basis of how CP mutations impact upon telomere length remains unclear. The CP CTC1 L1142H mutation has been previously shown to disrupt telomere maintenance. In this study, we used CRISPR/Cas9 to engineer this mutation into both alleles of HCT116 and RPE cells to demonstrate that CTC1:STN1 interaction is required to repress telomerase activity. CTC1 L1142H interacts poorly with STN1, leading to telomerase-mediated telomere elongation. Impaired interaction between CTC1 L1142H :STN1 and DNA Pol-α results in increased telomerase recruitment to telomeres and further telomere elongation, revealing that C:S binding to DNA Pol-α is required to fully repress telomerase activity. CP CTC1 mutants that fail to interact with DNA Pol-α resulted in loss of C-strand maintenance and catastrophic telomere shortening. Our findings place the CST complex as an important regulator of both G-strand extensions by telomerase and C-strand synthesis by DNA Pol-α. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Toehold-mediated strand displacement reaction-dependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity.

Science.gov (United States)

Wu, Yushu; Wang, Lei; Jiang, Wei

2017-03-15

Sensitive detection of uracil-DNA glycosylase (UDG) activity is beneficial for evaluating the repairing process of DNA lesions. Here, toehold-mediated strand displacement reaction (TSDR)-dependent fluorescent strategy was constructed for sensitive detection of UDG activity. A single-stranded DNA (ssDNA) probe with two uracil bases and a trigger sequence were designed. A hairpin probe with toehold domain was designed, and a reporter probe was also designed. Under the action of UDG, two uracil bases were removed from ssDNA probe, generating apurinic/apyrimidinic (AP) sites. Then, the AP sites could inhibit the TSDR between ssDNA probe and hairpin probe, leaving the trigger sequence in ssDNA probe still free. Subsequently, the trigger sequence was annealed with the reporter probe, initiating the polymerization and nicking amplification reaction. As a result, numerous G-quadruplex (G4) structures were formed, which could bind with N-methyl-mesoporphyrin IX (NMM) to generate enhanced fluorescent signal. In the absence of UDG, the ssDNA probe could hybridize with the toehold domain of the hairpin probe to initiate TSDR, blocking the trigger sequence, and then the subsequent amplification reaction would not occur. The proposed strategy was successfully implemented for detecting UDG activity with a detection limit of 2.7×10 -5 U/mL. Moreover, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Natural minus-strand RNAs of alfalfa mosaic virus as in vitro templates for viral RNA polymerase. 3'-Terminal non-coded guanosine and coat protein are insufficient factors for full-size plus-strand synthesis

NARCIS (Netherlands)

Houwing, C.J.; Huis in 't Veld, M.; Zuidema, D.; Graaff, de M.; Jaspars, E.M.J.

2001-01-01

Replication complexes of alfalfa mosaic virus produce in vivo large quantities of plus-strand RNAs, but this production is fully dependent on the presence of coat protein. In order to study this process of RNA-dependent and coat protein-regulated RNA synthesis we have isolated the three natural

Single-molecule visualization of Saccharomyces cerevisiae leading-strand synthesis reveals dynamic interaction between MTC and the replisome.

Science.gov (United States)

Lewis, Jacob S; Spenkelink, Lisanne M; Schauer, Grant D; Hill, Flynn R; Georgescu, Roxanna E; O'Donnell, Michael E; van Oijen, Antoine M

2017-10-03

The replisome, the multiprotein system responsible for genome duplication, is a highly dynamic complex displaying a large number of different enzyme activities. Recently, the Saccharomyces cerevisiae minimal replication reaction has been successfully reconstituted in vitro. This provided an opportunity to uncover the enzymatic activities of many of the components in a eukaryotic system. Their dynamic behavior and interactions in the context of the replisome, however, remain unclear. We use a tethered-bead assay to provide real-time visualization of leading-strand synthesis by the S. cerevisiae replisome at the single-molecule level. The minimal reconstituted leading-strand replisome requires 24 proteins, forming the CMG helicase, the Pol ε DNA polymerase, the RFC clamp loader, the PCNA sliding clamp, and the RPA single-stranded DNA binding protein. We observe rates and product lengths similar to those obtained from ensemble biochemical experiments. At the single-molecule level, we probe the behavior of two components of the replication progression complex and characterize their interaction with active leading-strand replisomes. The Minichromosome maintenance protein 10 (Mcm10), an important player in CMG activation, increases the number of productive replication events in our assay. Furthermore, we show that the fork protection complex Mrc1-Tof1-Csm3 (MTC) enhances the rate of the leading-strand replisome threefold. The introduction of periods of fast replication by MTC leads to an average rate enhancement of a factor of 2, similar to observations in cellular studies. We observe that the MTC complex acts in a dynamic fashion with the moving replisome, leading to alternating phases of slow and fast replication.

Seed displacements after permanent brachytherapy for prostate cancer in dependence on the prostate level

International Nuclear Information System (INIS)

Pinkawa, M.; Gagel, B.; Asadpour, B.; Piroth, M.D.; Klotz, J.; Eble, M.J.; Borchers, H.; Jakse, G.

2008-01-01

Purpose: to evaluate seed displacements after permanent prostate brachytherapy considering different prostate levels. Patients and methods: in 61 patients, postimplant CT scans were performed 1 day and 1 month after an implant with stranded seeds. Seed and prostate surface displacements were determined relative to pelvic bones. Four groups of seed locations were selected: seeds at the base (n = 305; B), at the apex (n = 305; A), close to the urethra (n = 306; U), and close to the rectal wall (n = 204; R). The length of two strands (always containing four seeds) per patient was measured in all CT scans and compared. Results: the largest inferior seed displacements were found at the base: mean 5.3 mm (B), 2.2 mm (A), 2.7 mm (U), 3.3 mm (R; p 3 vs. 41 cm 3 ; p < 0.001), a mean caudal prostate base displacement of 3.9 mm was found, whereas the mean inward displacement ranged from 1.2 to 1.6 mm at the remaining borders (lateral, anterior, posterior, apical). The analysis of the strand lengths revealed an implant compression between day 1 and 30 (mean 1.7 mm; p < 0.001). Conclusion: the largest prostate tissue and seed displacements were observed at the prostate base, associated with an implant compression. Predominantly inferior and posterior displacements implicate consequential smaller preplanning margins at the apex and the posterior prostate. (orig.)

Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells

International Nuclear Information System (INIS)

Takeda, N.; Kuhn, R.J.; Yang, C.F.; Takegami, T.; Wimmer, E.

1986-01-01

An in vitro poliovirus RNA-synthesizing system derived from a crude membrance fraction of infected HeLa cells was used to analyze the mechanism of initiation of poliovirus plus-strand RNA synthesis. This system contains an activity that synthesizes the nucleotidyl proteins VPg-pU and VPg-pUpU. These molecules represent the 5'-terminal structure of nascent RNA molecules and of virion RNA. The membranous replication complex is also capable of synthesizing mucleotidyl proteins containing nine or more of the poliovirus 5'-proximal nucleotides as assayed by the formation of the RNase T 1 -resistant oligonucleotide VPg-pUUAAAACAGp or by fingerprint analysis of the in vitro-synthesized 32 P-RNA. Incubation of preformed VPg-pUpU with unlabeled nucleoside triphosphates resulted in the formation of VPg-pUUAAAACAGp. This reaction, which appeared to be an elongation of VPg-pUpU, was stimulated by the addition of a soluble fraction (S-10) obtained from uninfected HeLa cells. Preformed VPg-pU could be chased into VPg-pUpU in the presence of UTP. The data are consistent with a model that VPg-pU can function as a primer for poliovirus plus-strand RNA synthesis in the membranous replication complex and that the elongation reaction may be stimulated by a host cellular factor

Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification

OpenAIRE

Yi Wang; Yan Wang; Ai-Jing Ma; Dong-Xun Li; Li-Juan Luo; Dong-Xin Liu; Dong Jin; Kai Liu; Chang-Yun Ye

2015-01-01

We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61?65??C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primer...

Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

Science.gov (United States)

Kurat, Christoph F; Yeeles, Joseph T P; Patel, Harshil; Early, Anne; Diffley, John F X

2017-01-05

The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Toehold-Mediated Displacement of an Adenosine-Binding Aptamer from a DNA Duplex by its Ligand.

Science.gov (United States)

Monserud, Jon H; Macri, Katherine M; Schwartz, Daniel K

2016-10-24

DNA is increasingly used to engineer dynamic nanoscale circuits, structures, and motors, many of which rely on DNA strand-displacement reactions. The use of functional DNA sequences (e.g., aptamers, which bind to a wide range of ligands) in these reactions would potentially confer responsiveness on such devices, and integrate DNA computation with highly varied molecular stimuli. By using high-throughput single-molecule FRET methods, we compared the kinetics of a putative aptamer-ligand and aptamer-complement strand-displacement reaction. We found that the ligands actively disrupted the DNA duplex in the presence of a DNA toehold in a similar manner to complementary DNA, with kinetic details specific to the aptamer structure, thus suggesting that the DNA strand-displacement concept can be extended to functional DNA-ligand systems. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Equilibrious Strand Exchange Promoted by DNA Conformational Switching

Science.gov (United States)

Wu, Zhiguo; Xie, Xiao; Li, Puzhen; Zhao, Jiayi; Huang, Lili; Zhou, Xiang

2013-01-01

Most of DNA strand exchange reactions in vitro are based on toehold strategy which is generally nonequilibrium, and intracellular strand exchange mediated by proteins shows little sequence specificity. Herein, a new strand exchange promoted by equilibrious DNA conformational switching is verified. Duplexes containing c-myc sequence which is potentially converted into G-quadruplex are designed in this strategy. The dynamic equilibrium between duplex and G4-DNA is response to the specific exchange of homologous single-stranded DNA (ssDNA). The SER is enzyme free and sequence specific. No ATP is needed and the displaced ssDNAs are identical to the homologous ssDNAs. The SER products and exchange kenetics are analyzed by PAGE and the RecA mediated SER is performed as the contrast. This SER is a new feature of G4-DNAs and a novel strategy to utilize the dynamic equilibrium of DNA conformations.

Mass strandings of various ommastrephid squid species have been ...

African Journals Online (AJOL)

spamer

escape reaction, to jet backwards at speed, is commonly observed. Appearing to ... on the occasion of a single mass stranding (La Pylaie, as cited in Lane 1957). ..... have also been associated with the displacement of major water masses ...

Asymmetric strand segregation: epigenetic costs of genetic fidelity?

Directory of Open Access Journals (Sweden)

Diane P Genereux

2009-06-01

Full Text Available Asymmetric strand segregation has been proposed as a mechanism to minimize effective mutation rates in epithelial tissues. Under asymmetric strand segregation, the double-stranded molecule that contains the oldest DNA strand is preferentially targeted to the somatic stem cell after each round of DNA replication. This oldest DNA strand is expected to have fewer errors than younger strands because some of the errors that arise on daughter strands during their synthesis fail to be repaired. Empirical findings suggest the possibility of asymmetric strand segregation in a subset of mammalian cell lineages, indicating that it may indeed function to increase genetic fidelity. However, the implications of asymmetric strand segregation for the fidelity of epigenetic information remain unexplored. Here, I explore the impact of strand-segregation dynamics on epigenetic fidelity using a mathematical-modelling approach that draws on the known molecular mechanisms of DNA methylation and existing rate estimates from empirical methylation data. I find that, for a wide range of starting methylation densities, asymmetric -- but not symmetric -- strand segregation leads to systematic increases in methylation levels if parent strands are subject to de novo methylation events. I found that epigenetic fidelity can be compromised when enhanced genetic fidelity is achieved through asymmetric strand segregation. Strand segregation dynamics could thus explain the increased DNA methylation densities that are observed in structured cellular populations during aging and in disease.

Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication

Science.gov (United States)

Seco, Elena M.

2017-01-01

Abstract Firmicutes have two distinct replicative DNA polymerases, the PolC leading strand polymerase, and PolC and DnaE synthesizing the lagging strand. We have reconstituted in vitro Bacillus subtilis bacteriophage SPP1 θ-type DNA replication, which initiates unidirectionally at oriL. With this system we show that DnaE is not only restricted to lagging strand synthesis as previously suggested. DnaG primase and DnaE polymerase are required for initiation of DNA replication on both strands. DnaE and DnaG synthesize in concert a hybrid RNA/DNA ‘initiation primer’ on both leading and lagging strands at the SPP1 oriL region, as it does the eukaryotic Pol α complex. DnaE, as a RNA-primed DNA polymerase, extends this initial primer in a reaction modulated by DnaG and one single-strand binding protein (SSB, SsbA or G36P), and hands off the initiation primer to PolC, a DNA-primed DNA polymerase. Then, PolC, stimulated by DnaG and the SSBs, performs the bulk of DNA chain elongation at both leading and lagging strands. Overall, these modulations by the SSBs and DnaG may contribute to the mechanism of polymerase switch at Firmicutes replisomes. PMID:28575448

Seed displacements after permanent brachytherapy for prostate cancer in dependence on the prostate level

Energy Technology Data Exchange (ETDEWEB)

Pinkawa, M.; Gagel, B.; Asadpour, B.; Piroth, M.D.; Klotz, J.; Eble, M.J. [Dept. of Radiotherapy, RWTH Aachen (Germany); Borchers, H.; Jakse, G. [Dept. of Urology, RWTH Aachen (Germany)

2008-10-15

Purpose: to evaluate seed displacements after permanent prostate brachytherapy considering different prostate levels. Patients and methods: in 61 patients, postimplant CT scans were performed 1 day and 1 month after an implant with stranded seeds. Seed and prostate surface displacements were determined relative to pelvic bones. Four groups of seed locations were selected: seeds at the base (n = 305; B), at the apex (n = 305; A), close to the urethra (n = 306; U), and close to the rectal wall (n = 204; R). The length of two strands (always containing four seeds) per patient was measured in all CT scans and compared. Results: the largest inferior seed displacements were found at the base: mean 5.3 mm (B), 2.2 mm (A), 2.7 mm (U), 3.3 mm (R; p < 0.001). Posterior displacements predominated both at the base and the central region: mean 2.2 mm (B), 2.0 mm (U), 0.8 mm (A), -0.6 mm (R; p < 0.001). With a decreasing edema between day 1 and 30 (mean prostate volume of 51 cm{sup 3} vs. 41 cm{sup 3}; p < 0.001), a mean caudal prostate base displacement of 3.9 mm was found, whereas the mean inward displacement ranged from 1.2 to 1.6 mm at the remaining borders (lateral, anterior, posterior, apical). The analysis of the strand lengths revealed an implant compression between day 1 and 30 (mean 1.7 mm; p < 0.001). Conclusion: the largest prostate tissue and seed displacements were observed at the prostate base, associated with an implant compression. Predominantly inferior and posterior displacements implicate consequential smaller preplanning margins at the apex and the posterior prostate. (orig.)

Increased rate of repair of ultraviolet-induced DNA strand breaks in mitogen stimulated lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Hamlet, S.M.; Lavin, M.F.; Jennings, P.A. (Queensland Univ., St. Lucia (Australia). Dept. of Biochemistry; Queensland Univ., St. Lucia (Australia). Dept. of Veterinary Pathology; Queensland Univ. St. Lucia (Australia). Dept. of Public Health)

1982-05-01

Previous results have shown that phytohaemagglutinin-stimulated bovine lymphocytes exhibit a peak of ultraviolet-induced DNA repair synthesis 3 to 4 days after addition of mitogen. The level of repair synthesis was approximately tenfold higher than that in unstimulated lymphocytes. These studies have been extended to examine the rate of repair of strand breaks in U.V.-irradiated bovine lymphocytes. The extent of breakage of DNA was shown to be the same in mitogen-stimulated and unstimulated lymphocytes from two breeds of cattle, when determined by sedimentation of nucleoids on sucrose gradients. However, in mitogen-stimulated cells the time taken to repair DNA strand breaks was 6 hours compared with 12 hours in stationary phase lymphocytes after a U.V. dose of 5 J/m/sup 2/. These results suggest that the increased rate of repair of strand breaks is due to the induction of enzymes involved at the post-incision stage of DNA repair. Thus the increased level of repair synthesis observed in earlier work correlates with an increased rate of repair of DNA strand breaks in phytohaemagglutinin-stimulated bovine lymphocytes.

Increased rate of repair of ultraviolet-induced DNA strand breaks in mitogen stimulated lymphocytes

International Nuclear Information System (INIS)

Hamlet, S.M.; Lavin, M.F.; Jennings, P.A.; Queensland Univ., St. Lucia; Queensland Univ. St. Lucia

1982-01-01

Previous results have shown that phytohaemagglutinin-stimulated bovine lymphocytes exhibit a peak of ultraviolet-induced DNA repair synthesis 3 to 4 days after addition of mitogen. The level of repair synthesis was approximately tenfold higher than that in unstimulated lymphocytes. These studies have been extended to examine the rate of repair of strand breaks in U.V.-irradiated bovine lymphocytes. The extent of breakage of DNA was shown to be the same in mitogen-stimulated and unstimulated lymphocytes from two breeds of cattle, when determined by sedimentation of nucleoids on sucrose gradients. However, in mitogen-stimulated cells the time taken to repair DNA strand breaks was 6 hours compared with 12 hours in stationary phase lymphocytes after a U.V. dose of 5 J/m 2 . These results suggest that the increased rate of repair of strand breaks is due to the induction of enzymes involved at the post-incision stage of DNA repair. Thus the increased level of repair synthesis observed in earlier work correlates with an increased rate of repair of DNA strand breaks in phytohaemagglutinin-stimulated bovine lymphocytes. (author)

Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation.

Science.gov (United States)

Zhu, Jing; Ding, Yongshun; Liu, Xingti; Wang, Lei; Jiang, Wei

2014-09-15

Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8 pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA.

Science.gov (United States)

Osman, Toba A M; Coutts, Robert H A; Buck, Kenneth W

2006-11-01

Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.

A Selective Surface-Enhanced Raman Scattering Sensor for Mercury(II) Based on a Porous Polymer Material and the Target-Mediated Displacement of a T-Rich Strand

Science.gov (United States)

Kang, Y.; Zhang, L.; Zhang, H.; Wu, T.; Du, Y.

2017-05-01

A sensitive and selective surface-enhanced Raman scattering (SERS) sensor for mercury(II) was fabricated based on the target-mediated displacement of a T-rich oligonucleotide strand. A DNA/aptamer duplex was prepared by the hybridization between a tetramethylrhodamine(TMR)-labeled thymine(T)-rich Hg2+-specific aptamer (denoted as TMR-aptamer) and a thiolated adenine-rich capturing DNA. The duplex can be immobilized onto the SERS substrate of the Ag-moiety modified glycidyl methacrylate-ethylene dimethacrylate (denoted as Ag-GMA-EDMA) via self-assembly by the thiol anchor, in which the TMR-aptamer exists in a double-stranded chain. In this case, the label of the TMR moiety approaches the substrate surface and produces a strong SERS signal. Upon the addition of the target, a pair of TMR-aptamers could cooperatively coordinate with Hg2+ to form a stable duplex-like structure mediated by the T-Hg2+-T complex between two adjacent strands, which triggers the release of the TMR-aptamer from the SERS substrate surface, thus drawing the TMR tags away from the substrate with a significant decrease in the SERS signal. This optical sensor shows a sensitive response to Hg2+ in a concentration from 5 nM to 2.0 μM with a detection limit of 2.5 nM. The prepared sensor is negligibly responsive to other metal ions, can be easily regenerated, and shows good performance in real sample analysis.

Selection and application of strand displacement probes for a fumonisin B1 aptamer

Science.gov (United States)

Fumonisin B1 (FB1) is a toxin produced by Fusarium moniliforme, mainly on contaminated maize and maize products. In this study a solid surface chain displacement strategy was used to isolate oligonucleotide displacement probes for a FB1 aptamer. The probes were used as the basis for the development ...

MDA5 Detects the Double-Stranded RNA Replicative Form in Picornavirus-Infected Cells

Directory of Open Access Journals (Sweden)

Qian Feng

2012-11-01

Full Text Available RIG-I and MDA5 are cytosolic RNA sensors that play a critical role in innate antiviral responses. Major advances have been made in identifying RIG-I ligands, but our knowledge of the ligands for MDA5 remains restricted to data from transfection experiments mostly using poly(I:C, a synthetic dsRNA mimic. Here, we dissected the IFN-α/β-stimulatory activity of different viral RNA species produced during picornavirus infection, both by RNA transfection and in infected cells in which specific steps of viral RNA replication were inhibited. Our results show that the incoming genomic plus-strand RNA does not activate MDA5, but minus-strand RNA synthesis and production of the 7.5 kbp replicative form trigger a strong IFN-α/β response. IFN-α/β production does not rely on plus-strand RNA synthesis and thus generation of the partially double-stranded replicative intermediate. This study reports MDA5 activation by a natural RNA ligand under physiological conditions.

CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

Science.gov (United States)

Langston, Lance D; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E; Finkelstein, Jeff; Yao, Nina Y; Indiani, Chiara; O'Donnell, Mike E

2014-10-28

DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

Nucleic Acid Analogue Induced Transcription of Double Stranded DNA

DEFF Research Database (Denmark)

1998-01-01

RNA is transcribed from a double stranded DNA template by forming a complex by hybridizing to the template at a desired transcription initiation site one or more oligonucleic acid analogues of the PNA type capable of forming a transcription initiation site with the DNA and exposing the complex...... to the action of a DNA dependant RNA polymerase in the presence of nucleoside triphosphates. Equal length transcripts may be obtained by placing a block to transcription downstream from the initiation site or by cutting the template at such a selected location. The initiation site is formed by displacement...... of one strand of the DNA locally by the PNA hybridization....

Mechanism of chimera formation during the Multiple Displacement Amplification reaction

Directory of Open Access Journals (Sweden)

Stockwell Timothy B

2007-04-01

Full Text Available Abstract Background Multiple Displacement Amplification (MDA is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them. Results Here, we characterize the major types of chimeras formed by carrying out an MDA whole genome amplification from a single E. coli cell and sequencing by the 454 Life Sciences method. Analysis of 475 chimeras revealed the predominant reaction mechanisms that create the DNA rearrangements. The highly branched DNA synthesized in MDA can assume many alternative secondary structures. DNA strands extended on an initial template can be displaced becoming available to prime on a second template creating the chimeras. Evidence supports a model in which branch migration can displace 3'-ends freeing them to prime on the new templates. More than 85% of the resulting DNA rearrangements were inverted sequences with intervening deletions that the model predicts. Intramolecular rearrangements were favored, with displaced 3'-ends reannealing to single stranded 5'-strands contained within the same branched DNA molecule. In over 70% of the chimeric junctions, the 3' termini had initiated priming at complimentary sequences of 2–21 nucleotides (nts in the new templates. Conclusion Formation of chimeras is an important limitation to the MDA method, particularly for whole genome sequencing. Identification of the mechanism for chimera formation provides new insight into the MDA reaction and suggests methods to reduce chimeras. The 454 sequencing approach used here will provide a rapid method to assess the utility of reaction modifications.

Ultrasensitive electrochemical biosensor for detection of DNA from Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification.

Science.gov (United States)

Hu, Yuhua; Xu, Xueqin; Liu, Qionghua; Wang, Ling; Lin, Zhenyu; Chen, Guonan

2014-09-02

A simple, ultrasensitive, and specific electrochemical biosensor was designed to determine the given DNA sequence of Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification. The target DNA (TD, the DNA sequence from the hypervarient region of 16S rDNA of Bacillus subtilis) could be detected by the differential pulse voltammetry (DPV) in a range from 0.1 fM to 20 fM with the detection limit down to 0.08 fM at the 3s(blank) level. This electrochemical biosensor exhibits high distinction ability to single-base mismatch, double-bases mismatch, and noncomplementary DNA sequence, which may be expected to detect single-base mismatch and single nucleotide polymorphisms (SNPs). Moreover, the applicability of the designed biosensor for detecting the given DNA sequence from Bacillus subtilis was investigated. The result obtained by electrochemical method is approximately consistent with that by a real-time quantitative polymerase chain reaction detecting system (QPCR) with SYBR Green.

AgNPs-3D nanostructure enhanced electrochemiluminescence of CdSe quantum dot coupled with strand displacement amplification for sensitive biosensing of DNA

International Nuclear Information System (INIS)

Jiao, Meng; Jie, Guifen; Tan, Lu; Niu, Shuyan

2017-01-01

A novel strategy using Ag nanoparticles-3D (AgNPs-3D) nanostructure enhanced electrochemiluminescence (ECL) of CdSe quantum dots (QDs) coupled with strand displacement amplification (SDA) for sensitive biosensing of DNA was successfully designed. The prepared CdSe QDs with intense ECL were assembled on the poly (diallyldimethylammonium chloride) (PDDA) graphene oxide (GO) nanocomposites modified electrode, then gold nanoparticles (NPs) as the quenching probe was conjugated to the QDs, ECL signal was efficiently quenched. The target DNA induced cycling SDA and generated a large number of DNA s1. The released DNA s1 could open the hairpin DNA with quenching probe. So the presence of low levels of target DNA can potentially result in a significant enhancement of ECL signal. Furthermore, large number of AgNPs were then in situ reduced in the 3D DNA skeleton on the electrode, which dramaticlly enhanced ECL signal of QDs owing to the excellent electrical conductivity, and the much amplified ECL signal change has a quantitative relation with the target DNA. So by combining the AgNPs-3D nanostructure and cycling SDA to achieve greatly amplified detection of DNA, the promising ECL strategy could provide a highly sensitive platform for various biomolecules and has a good prospect for clinical diagnosis in the future. - Graphical abstract: A novel strategy using AgNPs-3D nanostructure enhanced electrochemiluminescence of CdSe quantum dot coupled with DNA strand displacement amplification for sensitive biosensing of DNA was successfully designed, the proposed biosensor can be expected to be an emerging alternative for straightforward nucleic acid detection in complex samples with an easy and rapid way. - Highlights: • AgNPs-3D nanostructure for enhancing ECL signal of CdSe QDs was successfully designed. • A new dual amplification strategy for detection of DNA by using AgNPs-3D nanostructure coupled with SDA was developed. • It is for the first time AgNPs-3D nanostructure

Interaction between the cellular protein eEF1A and the 3'-terminal stem-loop of West Nile virus genomic RNA facilitates viral minus-strand RNA synthesis.

Science.gov (United States)

Davis, William G; Blackwell, Jerry L; Shi, Pei-Yong; Brinton, Margo A

2007-09-01

RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3'(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3'(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3' SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3' SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3' SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3' end of the genome and the RC. eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

Time-Resolved Nucleic Acid Hybridization Beacons Utilizing Unimolecular and Toehold-Mediated Strand Displacement Designs.

Science.gov (United States)

Massey, Melissa; Ancona, Mario G; Medintz, Igor L; Algar, W Russ

2015-12-01

Nucleic acid hybridization probes are sought after for numerous assay and imaging applications. These probes are often limited by the properties of fluorescent dyes, prompting the development of new probes where dyes are paired with novel or nontraditional luminescent materials. Luminescent terbium complexes are an example of such a material, and these complexes offer several unique spectroscopic advantages. Here, we demonstrate two nonstem-loop designs for light-up nucleic acid hybridization beacons that utilize time-resolved Förster resonance energy transfer (TR-FRET) between a luminescent Lumi4-Tb cryptate (Tb) donor and a fluorescent reporter dye, where time-resolved emission from the dye provides an analytical signal. Both designs are based on probe oligonucleotides that are labeled at their opposite termini with Tb and a fluorescent reporter dye. In one design, a probe is partially blocked with a quencher dye-labeled oligonucleotide, and target hybridization is signaled through toehold-mediated strand displacement and loss of a competitive FRET pathway. In the other design, the intrinsic folding properties of an unblocked probe are utilized in combination with a temporal mechanism for signaling target hybridization. This temporal mechanism is based on a recently elucidated "sweet spot" for TR-FRET measurements and exploits distance control over FRET efficiencies to shift the Tb lifetime within or outside the time-gated detection window for measurements. Both the blocked and unblocked beacons offer nanomolar (femtomole) detection limits, response times on the order of minutes, multiplexing through the use of different reporter dyes, and detection in complex matrices such as serum and blood. The blocked beacons offer better mismatch selectivity, whereas the unblocked beacons are simpler in design. The temporal mechanism of signaling utilized with the unblocked beacons also plays a significant role with the blocked beacons and represents a new and effective

Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

NARCIS (Netherlands)

Hamdan, Samir M.; Loparo, Joseph J.; Takahashi, Masateru; Richardson, Charles C.; Oijen, Antoine M. van

2009-01-01

In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to

Potent Inhibitors of the Hepatitis C Virus NS3 Protease: Design and Synthesis of Macrocyclic Substrate-Based [beta]-Strand Mimics

Energy Technology Data Exchange (ETDEWEB)

Goudreau, Nathalie; Brochu, Christian; Cameron, Dale R.; Duceppe, Jean-Simon; Faucher, Anne-Marie; Ferland, Jean-Marie; Grand-Maître, Chantal; Poirier, Martin; Simoneau, Bruno; Tsantrizos, Youla S. (Boehringer)

2008-06-30

The virally encoded NS3 protease is essential to the life cycle of the hepatitis C virus (HCV), an important human pathogen causing chronic hepatitis, cirrhosis of the liver, and hepatocellular carcinoma. The design and synthesis of 15-membered ring {beta}-strand mimics which are capable of inhibiting the interactions between the HCV NS3 protease enzyme and its polyprotein substrate will be described. The binding interactions between a macrocyclic ligand and the enzyme were explored by NMR and molecular dynamics, and a model of the ligand/enzyme complex was developed.

Visual detection of glial cell line-derived neurotrophic factor based on a molecular translator and isothermal strand-displacement polymerization reaction.

Science.gov (United States)

Zhang, Li-Yong; Xing, Tao; Du, Li-Xin; Li, Qing-Min; Liu, Wei-Dong; Wang, Ji-Yue; Cai, Jing

2015-01-01

Glial cell line-derived neurotrophic factor (GDNF) is a small protein that potently promotes the survival of many types of neurons. Detection of GDNF is vital to monitoring the survival of sympathetic and sensory neurons. However, the specific method for GDNF detection is also un-discovered. The purpose of this study is to explore the method for protein detection of GDNF. A novel visual detection method based on a molecular translator and isothermal strand-displacement polymerization reaction (ISDPR) has been proposed for the detection of GDNF. In this study, a molecular translator was employed to convert the input protein to output deoxyribonucleic acid signal, which was further amplified by ISDPR. The product of ISDPR was detected by a lateral flow biosensor within 30 minutes. This novel visual detection method based on a molecular translator and ISDPR has very high sensitivity and selectivity, with a dynamic response ranging from 1 pg/mL to 10 ng/mL, and the detection limit was 1 pg/mL of GDNF. This novel visual detection method exhibits high sensitivity and selectivity, which is very simple and universal for GDNF detection to help disease therapy in clinical practice.

Homogeneous and label-free detection of microRNAs using bifunctional strand displacement amplification-mediated hyperbranched rolling circle amplification.

Science.gov (United States)

Zhang, Li-rong; Zhu, Guichi; Zhang, Chun-yang

2014-07-01

MicroRNAs (miRNAs) are an emerging class of biomarkers and therapeutic targets for various diseases including cancers. Here, we develop a homogeneous and label-free method for sensitive detection of let-7a miRNA based on bifunctional strand displacement amplification (SDA)-mediated hyperbranched rolling circle amplification (HRCA). The binding of target miRNA with the linear template initiates the bifunctional SDA reaction, generating two different kinds of triggers which can hybridize with the linear template to initiate new rounds of SDA reaction for the production of more and more triggers. In the meantime, the released two different kinds of triggers can function as the first and the second primers, respectively, to initiate the HRCA reaction whose products can be simply monitored by a standard fluorometer with SYBR Green I as the fluorescent indicator. The proposed method exhibits high sensitivity with a detection limit of as low as 1.8 × 10(-13) M and a large dynamic range of 5 orders of magnitude from 0.1 pM to 10 nM, and it can even discriminate the single-base difference among the miRNA family members. Moreover, this method can be used to analyze the total RNA samples from the human lung tissues and might be further applied for sensitive detection of various proteins, small molecules, and metal ions in combination with specific aptamers.

Time-lapse crystallography snapshots of a double-strand break repair polymerase in action.

Science.gov (United States)

Jamsen, Joonas A; Beard, William A; Pedersen, Lars C; Shock, David D; Moon, Andrea F; Krahn, Juno M; Bebenek, Katarzyna; Kunkel, Thomas A; Wilson, Samuel H

2017-08-15

DNA polymerase (pol) μ is a DNA-dependent polymerase that incorporates nucleotides during gap-filling synthesis in the non-homologous end-joining pathway of double-strand break repair. Here we report time-lapse X-ray crystallography snapshots of catalytic events during gap-filling DNA synthesis by pol μ. Unique catalytic intermediates and active site conformational changes that underlie catalysis are uncovered, and a transient third (product) metal ion is observed in the product state. The product manganese coordinates phosphate oxygens of the inserted nucleotide and PP i . The product metal is not observed during DNA synthesis in the presence of magnesium. Kinetic analyses indicate that manganese increases the rate constant for deoxynucleoside 5'-triphosphate insertion compared to magnesium. The likely product stabilization role of the manganese product metal in pol μ is discussed. These observations provide insight on structural attributes of this X-family double-strand break repair polymerase that impact its biological function in genome maintenance.DNA polymerase (pol) μ functions in DNA double-strand break repair. Here the authors use time-lapse X-ray crystallography to capture the states of pol µ during the conversion from pre-catalytic to product complex and observe a third transiently bound metal ion in the product state.

An oligonucleotide complementary to the SL-B1 domain in the 3'-end of the minus-strand RNA of the hepatitis C virus inhibits in vitro initiation of RNA synthesis by the viral polymerase

International Nuclear Information System (INIS)

Reigadas, Sandrine; Ventura, Michel; Andreola, Marie-Line; Michel, Justine; Gryaznov, Sergei; Tarrago-Litvak, Laura; Litvak, Simon; Astier-Gin, Therese

2003-01-01

We describe oligonucleotides (ODNs) that inhibit hepatitis C virus (HCV) RNA synthesis in vitro. From a series of 13 ODNs complementary to the 3'-end of the minus-strand HCV RNA, only 4 inhibited RNA synthesis with IC 50 values lower than 1 μM. The inhibition was sequence-specific, since no effect was observed when the ODNs were used with a noncomplementary template. The introduction of a 2'-O-methyl modification increased the inhibitor activity 11-fold (IC 50 = 50 nM) in just 1 (ODN7) of the 4 inhibitory ODNs. ODNs did not inhibit RNA synthesis by interfering with the elongation process as no short RNAs products were detected. We also show that ODN7 did not prevent binding of NS5B to the template or cause polymerase trapping by the duplex RNA/ODN. Our data demonstrate that ODN7 inhibits the initiation process, most probably by modifying structural features present at the 3'-end of the minus-strand RNA

Postreplicational formation and repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB cells

International Nuclear Information System (INIS)

Wang, Tzuchien V.; Smith, K.C.

1986-01-01

The number of DNA double-strand breaks formed in UV-irradiated uvrB recF recB cells correlates with the number of unrepaired DNA daughter-strand gaps, and is dependent on DNA synthesis after UV-irradiation. These results are consistent with the model that the DNA double-strand breaks that are produced in UV-irradiated excision-deficient cells occur as the result of breaks in the parental DNA opposite unrepaired DNA daughter-strand gaps. By employing a temperature-sensitive recA200 mutation, we have devised an improved assay for studying the formation and repair of these DNA double-strand breaks. Possible mechanisms for the postreplication repair of DNA double-strand breaks are discussed. (Auth.)

A single-strand specific lesion drives MMS-induced hyper-mutability at a double-strand break in yeast.

Science.gov (United States)

Yang, Yong; Gordenin, Dmitry A; Resnick, Michael A

2010-08-05

Localized hyper-mutability (LHM) can be important in evolution, immunity, and genetic diseases. We previously reported that single-strand DNA (ssDNA) can be an important source of damage-induced LHM in yeast. Here, we establish that the generation of LHM by methyl methanesulfonate (MMS) during repair of a chromosomal double-strand break (DSB) can result in over 0.2 mutations/kb, which is approximately 20,000-fold higher than the MMS-induced mutation density without a DSB. The MMS-induced mutations associated with DSB repair were primarily due to substitutions via translesion DNA synthesis at damaged cytosines, even though there are nearly 10 times more MMS-induced lesions at other bases. Based on this mutation bias, the promutagenic lesion dominating LHM is likely 3-methylcytosine, which is single-strand specific. Thus, the dramatic increase in mutagenesis at a DSB is concluded to result primarily from the generation of non-repairable lesions in ssDNA associated with DSB repair along with efficient induction of highly mutagenic ssDNA-specific lesions. These findings with MMS-induced LHM have broad biological implications for unrepaired damage generated in ssDNA and possibly ssRNA. Published by Elsevier B.V.

Hairpin DNA-Templated Silver Nanoclusters as Novel Beacons in Strand Displacement Amplification for MicroRNA Detection.

Science.gov (United States)

Zhang, Jingpu; Li, Chao; Zhi, Xiao; Ramón, Gabriel Alfranca; Liu, Yanlei; Zhang, Chunlei; Pan, Fei; Cui, Daxiang

2016-01-19

MicroRNA (miRNA) biomarkers display great potential for cancer diagnosis and prognosis. The development of rapid and specific methods for miRNA detection has become a hotspot. Herein, hairpin DNA-templated silver nanoclusters (AgNCs/HpDNA) were prepared and integrated into strand-displacement amplification (SDA) as a novel beacon for miRNA detection. The light-up platform was established based on guanine (G)-rich fluorescence enhancement that essentially converted the excitation/emission pair of AgNCs/HpDNAs from a shorter wavelength to a longer wavelength, and then achieved fluorescent enhancement at longer wavelength. On the basis of the validation of the method, the single and duplex detection were conducted in two plasma biomarkers (miR-16-5p and miR-19b-3p) for the diagnosis of gastric cancer. The probe (AgNCs/RED 16(7s)C) utilized for miR-16-5p detection adopted a better conformation with high specificity to recognize single-base mismatches by producing dramatically opposite signals (increase or decrease at 580 nm ex/640 nm em) while the probe (AgNCs/GRE 19b(5s)C) for miR-19b-3p generated dual signals (increase at 490 nm ex/570 nm em and decrease at 430 nm ex/530 nm em) with bright fluorescence in one reaction during the amplification, but unexpectedly was partially digested. This is for the first time to allow the generation of enhanced fluorescent AgNCs and the target recognition integrated into a single process, which offers great opportunity for specific miRNA detection in an easy and rapid way.

Towards quantitative viromics for both double-stranded and single-stranded DNA viruses

Directory of Open Access Journals (Sweden)

Simon Roux

2016-12-01

Full Text Available Background Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA viral genomes captured in quantitative viral metagenomes (viromes. This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation. Methods Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. Results Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5% of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. Discussion Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6

DEFF Research Database (Denmark)

Devert, Anthony; Fabre, Nicolas; Floris, Maina Huguette Joséphine

2015-01-01

) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer......Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA......-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer...

Motion laws synthesis for cam mechanisms with multiple follower displacement

Science.gov (United States)

Podgornyj, Yu I.; Skeeba, V. Yu; Kirillov, A. V.; Martynova, T. G.; Skeeba, P. Yu

2018-03-01

The research discusses the cam mechanisms design. The analysis of specialized literature indicates that the synthesis of the cam mechanisms laws of motion is currently done mainly by a standard set of acceleration curves. In some cases, the designer needs to synthesize a new acceleration law which should be task-specific and enforce a certain production step. The values of the technological loads and inertia forces loads generated by the mechanism are calculated to analyze the slay mechanism behavior in the production of closely woven fabrics. Mathematical packages MathCad and SolidWorks are used in calculations. As a result of the research, the authors propose the methodology for synthesizing the slay mechanism with multiple follower displacements for the point of contact between the reed and the fabric edge. Theoretical studies have been tested on a specific machine model (STB loom). The authors have synthesized the motion law of the filling threads beat-up mechanism for the production of strong fabrics. New basic and closing cam profiles are proposed. The results are designed to enhance the possibilities of the looms and to recommend the most efficient equipment operation modes for the producers.

Synthesis of double-stranded RNA in a virus-enriched fraction from Agaricus bisporus

International Nuclear Information System (INIS)

Sriskantha, A.; Wach, P.; Schlagnhaufer, B.; Romaine, C.P.

1986-01-01

Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from health sporophores. Enzyme activity was dependent upon the presence of Mg 2+ and the four nucleoside triphosphates and was insensitive to actinomycin D, α-amanitin, and rifampin. The 3 H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 /times/ 10 6 and 1.4 /times/ 10 6 . Cs 2 SO 4 equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 /times/ 10 6 and 1.4 /times/ 10 6 . The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs

Late Quaternary slip history of the Mill Creek strand of the San Andreas fault in San Gorgonio Pass, southern California: The role of a subsidiary left-lateral fault in strand switching

Science.gov (United States)

Kendrick, Katherine J.; Matti, Jonathan; Mahan, Shannon

2015-01-01

The fault history of the Mill Creek strand of the San Andreas fault (SAF) in the San Gorgonio Pass region, along with the reconstructed geomorphology surrounding this fault strand, reveals the important role of the left-lateral Pinto Mountain fault in the regional fault strand switching. The Mill Creek strand has 7.1–8.7 km total slip. Following this displacement, the Pinto Mountain fault offset the Mill Creek strand 1–1.25 km, as SAF slip transferred to the San Bernardino, Banning, and Garnet Hill strands. An alluvial complex within the Mission Creek watershed can be linked to palinspastic reconstruction of drainage segments to constrain slip history of the Mill Creek strand. We investigated surface remnants through detailed geologic mapping, morphometric and stratigraphic analysis, geochronology, and pedogenic analysis. The degree of soil development constrains the duration of surface stability when correlated to other regional, independently dated pedons. This correlation indicates that the oldest surfaces are significantly older than 500 ka. Luminescence dates of 106 ka and 95 ka from (respectively) 5 and 4 m beneath a younger fan surface are consistent with age estimates based on soil-profile development. Offset of the Mill Creek strand by the Pinto Mountain fault suggests a short-term slip rate of ∼10–12.5 mm/yr for the Pinto Mountain fault, and a lower long-term slip rate. Uplift of the Yucaipa Ridge block during the period of Mill Creek strand activity is consistent with thermochronologic modeled uplift estimates.

Dual-cyclical nucleic acid strand-displacement polymerization based signal amplification system for highly sensitive determination of p53 gene.

Science.gov (United States)

Xu, Jianguo; Wu, Zai-Sheng; Li, Hongling; Wang, Zhenmeng; Le, Jingqing; Zheng, Tingting; Jia, Lee

2016-12-15

In the present study, we proposed a novel dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) based signal amplification system for highly sensitive determination of tumor suppressor genes. The system primarily consisted of a signaling hairpin probe (SHP), a label-free hairpin probe (LHP) and an initiating primer (IP). The presence of target DNA was able to induce one CNDP through continuous process of ligation, polymerization and nicking, leading to extensively accumulation of two nicked triggers (NT1 and NT2). Intriguingly, the NT1 could directly hybridize SHP, while the NT2 could act as the target analog to induce another CNDP. The resulting dual-CNDP contributed the striking signal amplification, and only a very weak blank noise existed since the ligation template of target was not involved. In this case, the target could be detected in a wide linear range (5 orders of magnitude), and a low detection limit (78 fM) was obtained, which is superior to most of the existing fluorescent methods. Moreover, the dual-CNDP sensing system provided a high selectivity towards target DNA against mismatched target and was successfully applied to analysis of target gene extracted from cancer cells or in human serum-contained samples, indicating its great potential for practical applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Purification of Single-Stranded cDNA Based on RNA Degradation Treatment and Adsorption Chromatography.

Science.gov (United States)

Trujillo-Esquivel, Elías; Franco, Bernardo; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Mora-Montes, Héctor M

2016-08-02

Analysis of gene expression is a common research tool to study networks controlling gene expression, the role of genes with unknown function, and environmentally induced responses of organisms. Most of the analytical tools used to analyze gene expression rely on accurate cDNA synthesis and quantification to obtain reproducible and quantifiable results. Thus far, most commercial kits for isolation and purification of cDNA target double-stranded molecules, which do not accurately represent the abundance of transcripts. In the present report, we provide a simple and fast method to purify single-stranded cDNA, exhibiting high purity and yield. This method is based on the treatment with RNase H and RNase A after cDNA synthesis, followed by separation in silica spin-columns and ethanol precipitation. In addition, our method avoids the use of DNase I to eliminate genomic DNA from RNA preparations, which improves cDNA yield. As a case report, our method proved to be useful in the purification of single-stranded cDNA from the pathogenic fungus Sporothrix schenckii.

Rolling replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro

International Nuclear Information System (INIS)

Shavitt, O.; Livneh, Z.

1989-01-01

Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers

Synthesis of a wild-type and three mutant Cucurbita maxima trypsin inhibitor-encoding genes by a single-strand approach.

Science.gov (United States)

Botes, D P; Qobose, M D; Corfield, V A

1991-09-15

A single-strand approach to gene assembly, based on a modification of an in vitro complementary oligodeoxyribonucleotide template-directed ligation of the desired sequence to a linearized vector [Chen et al., Nucleic Acids Res. 18 (1990) 871-878], is described. The gene coding for the wild-type Cucurbita maxima trypsin inhibitor of 29 amino acid residues [Bode et al., FEBS Lett. 242 (1989) 285-292], as well as three mutant forms of the gene, in which two of the three disulfide bonds have been replaced singly or as a pair, have been synthesized in a single synthesis run with minimal manual intervention. Subsequent to ligation to pUC9 and in vivo gapped duplex repair by Escherichia coli, their sequences have been verified.

Genetics of x-ray induced double strand break repair in saccharomyces cerevisiae

International Nuclear Information System (INIS)

Budd, M.E.

1982-07-01

The possible fates of x-ray-induced double-strand breaks in Saccharomyces cerevisiae were examined. One possible pathway which breaks can follow, the repair pathway, was studied by assaying strains with mutations in the RAD51, RAD54, and RAD57 loci for double-strand break repair. In order of increasing radiation sensitivity one finds: rad57-1(23 0 )> rad51-1(30 0 )> rad54-3(36 0 ). At 36 0 , rad54-3 cells cannot repair double-strand breaks, while 23 0 , they can. Strains with the rad57-1 mutation can rejoin broken chromosomes at both temperatures. However, the low survival at 36 0 shows that the assay is not distinguishing large DNA fragments which allow cell survival from those which cause cell death. A rad51-1 strain could also rejoin broken chromosomes, and was thus capable of incomplete repair. The data can be explained with the hypothesis that rad54-3 cells are blocked in an early step of repair, while rad51-1 and rad57-1 strains are blocked in a later step of repair. The fate of double-strand breaks when they are left unrepaired was investigated with the rad54-3 mutation. If breaks are prevented from entering the RAD54 repair pathway they become uncommitted lesions. These lesions are repaired slower than the original breaks. One possible fate for an uncommitted lesion is conversion into a fixed lesion, which is likely to be an unrepairable or misrepaired double-strand break. The presence of protein synthesis after irradiation increases the probability that a break will enter the repair pathway. Evidence shows that increased probability of repair results from enhanced synthesis of repair proteins shortly after radiation

Human RECQ5 helicase promotes repair of DNA double-strand breaks by synthesis-dependent strand annealing

Czech Academy of Sciences Publication Activity Database

Paliwal, S.; Kanagaraj, R.; Sturzenegger, A.; Burdová, Kamila; Janščák, Pavel

2014-01-01

Roč. 42, č. 4 (2014), s. 2380-2390 ISSN 0305-1048 R&D Projects: GA ČR GA204/09/0565; GA ČR GAP305/10/0281 Grant - others:Swiss National Science Foundation(CH) 31003A-129747; Swiss National Science Foundation(CH) 31003A_146206 Institutional support: RVO:68378050 Keywords : Human RECQ5 helicase * DNA double-strand breaks * mitotic homologous recombination Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 9.112, year: 2014

Poly(ADP-ribose polymerase (PARP-1 is not involved in DNA double-strand break recovery

Directory of Open Access Journals (Sweden)

Fernet Marie

2003-07-01

Full Text Available Abstract Background The cytotoxicity and the rejoining of DNA double-strand breaks induced by γ-rays, H2O2 and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose polymerase (PARP-1 in DNA double-strand break repair. Results PARP-1-/- were considerably more sensitive than PARP-1+/+ 3T3s to induced cell kill by γ-rays and H2O2. However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1-/- 3T3s by retroviral transfection of the full PARP-1 cDNA did not induce any change in neocarzinostatin response. Moreover the incidence and the rejoining kinetics of neocarzinostatin-induced DNA double-strand breaks were identical in PARP-1+/+ and PARP-1-/- 3T3s. Poly(ADP-ribose synthesis following γ-rays and H2O2 was observed in PARP-1-proficient cells only. In contrast neocarzinostatin, even at supra-lethal concentration, was unable to initiate PARP-1 activation yet it induced H2AX histone phosphorylation in both PARP1+/+ and PARP-1-/- 3T3s as efficiently as γ-rays and H2O2. Conclusions The results show that PARP-1 is not a major determinant of DNA double-strand break recovery with either strand break rejoining or cell survival as an endpoint. Even though both PARP-1 and ATM activation are major determinants of the cell response to γ-rays and H2O2, data suggest that PARP-1-dependent poly(ADP-ribose synthesis and ATM-dependent H2AX phosphorylation, are not inter-related in the repair pathway of neocarzinostatin-induced DNA double-strand breaks.

Highly selective detection of single-nucleotide polymorphisms using a quartz crystal microbalance biosensor based on the toehold-mediated strand displacement reaction.

Science.gov (United States)

Wang, Dingzhong; Tang, Wei; Wu, Xiaojie; Wang, Xinyi; Chen, Gengjia; Chen, Qiang; Li, Na; Liu, Feng

2012-08-21

Toehold-mediated strand displacement reaction (SDR) is first introduced to develop a simple quartz crystal microbalance (QCM) biosensor without an enzyme or label at normal temperature for highly selective and sensitive detection of single-nucleotide polymorphism (SNP) in the p53 tumor suppressor gene. A hairpin capture probe with an external toehold is designed and immobilized on the gold electrode surface of QCM. A successive SDR is initiated by the target sequence hybridization with the toehold domain and ends with the unfolding of the capture probe. Finally, the open-loop capture probe hybridizes with the streptavidin-coupled reporter probe as an efficient mass amplifier to enhance the QCM signal. The proposed biosensor displays remarkable specificity to target the p53 gene fragment against single-base mutant sequences (e.g., the largest discrimination factor is 63 to C-C mismatch) and high sensitivity with the detection limit of 0.3 nM at 20 °C. As the crucial component of the fabricated biosensor for providing the high discrimination capability, the design rationale of the capture probe is further verified by fluorescence sensing and atomic force microscopy imaging. Additionally, a recovery of 84.1% is obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of employing this biosensor in detecting SNPs in biological samples.

Long-stem shaped multifunctional molecular beacon for highly sensitive nucleic acids determination via intramolecular and intermolecular interactions based strand displacement amplification.

Science.gov (United States)

Xu, Jianguo; Zheng, Tingting; Le, Jingqing; Jia, Lee

2017-11-20

Occurrence and application of oligonucleotide probes have promoted great progress in the biochemical analysis field due to their unique biological and chemical properties. In this work, a long-stem shaped multifunctional molecular beacon (LS-MMB) that is responsive to a cancer-related gene, p53, is well-prepared. By designing the probe with long-paired bases at its two ends and short-paired bases between the middle region and the 3' end, the LS-MMB is intelligently endowed with the ability to recognize the target analyte, serve as the polymerization primer/template, and signal the hybridization event synchronously, which is distinctly advantageous over the traditional molecular beacons (MBs). Moreover, it is excitingly found that the LS-MMB can be employed to exert intramolecular and intermolecular interactions for strand displacement amplification (SDA) without the involvement of any assistant probes; this therapy results in a really easy and rapid sensing system that provides an extremely low background noise and high target output signal. In this case, an excellent sensitivity and specificity to detect target gene down to picomolar level and resolution to even one nucleotide variation are achieved, respectively. In addition, the application potential for real genomic DNA analysis is realized. We envision that the probe of LS-MMB can act as a universal platform for biosensing and biomedical research.

Specific functions of the Rep and Rep׳ proteins of porcine circovirus during copy-release and rolling-circle DNA replication.

Science.gov (United States)

Cheung, Andrew K

2015-07-01

The roles of two porcine circovirus replication initiator proteins, Rep and Rep׳, in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replication) and generates the single-stranded circular (ssc) genome from the displaced DNA strand. In the process, a minus-genome primer (MGP) necessary for complementary-strand synthesis, from ssc to ccc, is synthesized. Rep׳ cleaves the growing nascent-strand to regenerate the parent ccc molecule. In the process, a Rep׳-DNA hybrid containing the right palindromic sequence (at the origin of DNA replication) is generated. Analysis of the virus particle showed that it is composed of four components: ssc, MGP, capsid protein and a novel Rep-related protein (designated Protein-3). Copyright © 2015. Published by Elsevier Inc.

Methods for the preparation of large quantities of complex single-stranded oligonucleotide libraries.

Science.gov (United States)

Murgha, Yusuf E; Rouillard, Jean-Marie; Gulari, Erdogan

2014-01-01

Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.

Host-Guest Recognition-Assisted Electrochemical Release: Its Reusable Sensing Application Based on DNA Cross Configuration-Fueled Target Cycling and Strand Displacement Reaction Amplification.

Science.gov (United States)

Chang, Yuanyuan; Zhuo, Ying; Chai, Yaqin; Yuan, Ruo

2017-08-15

In this work, an elegantly designed host-guest recognition-assisted electrochemical release was established and applied in a reusable electrochemical biosensor for the detection of microRNA-182-5p (miRNA-182-5p), a prostate cancer biomarker in prostate cancer, based on the DNA cross configuration-fueled target cycling and strand displacement reaction (SDR) amplification. With such a design, the single target miRNA input could be converted to large numbers of single-stranded DNA (S1-Trp and S2-Trp) output, which could be trapped by cucurbit[8]uril methyl viologen (CB-8-MV 2+ ) based on the host-guest recognition, significantly enhancing the sensitivity for miRNA detection. Moreover, the nucleic acids products obtained from the process of cycling amplification could be utilized sufficiently, avoiding the waste and saving the experiment cost. Impressively, by resetting a settled voltage, the proposed biosensor could release S1-Trp and S2-Trp from the electrode surface, attributing that the guest ion methyl viologen (MV 2+ ) was reduced to MV +· under this settled voltage and formed a more-stable CB-8-MV +· -MV +· complex. Once O 2 was introduced in this system, MV +· could be oxidized to MV 2+ , generating the complex of CB-8-MV 2+ for capturing S1-Trp and S2-Trp again in only 5 min. As a result, the simple and fast regeneration of biosensor for target detection was realized on the base of electrochemical redox-driven assembly and release, overcoming the challenges of time-consuming, burdensome operations and expensive experimental cost in traditional reusable biosensors and updating the construction method for a reusable bisensor. Furthermore, the biosensor could be reused for more than 10 times with a regeneration rate of 93.20%-102.24%. After all, the conception of this work provides a novel thought for the construction of effective reusable biosensor to detect miRNA and other biomarkers and has great potential application in the area requiring the release of

Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication.

Science.gov (United States)

Feng, Wenyi; Collingwood, David; Boeck, Max E; Fox, Lindsay A; Alvino, Gina M; Fangman, Walton L; Raghuraman, Mosur K; Brewer, Bonita J

2006-02-01

During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.

Repair of X-ray-induced single-strand breaks by a cell-free system

International Nuclear Information System (INIS)

Seki, Shuji; Ikeda, Shogo; Tsutui, Ken; Teraoka, Hirobumi

1990-01-01

Repair of X-ray-induced single-strand breaks of DNA was studied in vitro using an exonuclease purified from mouse ascites sarcoma (SR-C3H/He) cells. X-ray-dose-dependent unscheduled DNA synthesis was primed by the exonuclease. Repair of X-ray-induced single-strand breaks in pUC19 plasmid DNA was demonstrated by agarose gel electrophoresis after incubating the damaged DNA with the exonuclease, DNA polymerase (Klenow fragment of DNA polymerase I or DNA polymerase β purified from SR-C3H/He cells), four deoxynucleoside triphosphates, ATP and DNA ligase (T4 DNA ligase or DNA ligase I purified from calf thymus). The present results suggested that the exonuclease is involved in the initiation of repair of X-ray-induced single-strand breaks in removing 3' ends of X-ray-damaged DNA. (author)

Multiple-strand displacement and identification of single nucleotide polymorphisms as markers of genotypic variation of Pasteuria penetrans biotypes infecting root-knot nematodes.

Science.gov (United States)

Nong, Guang; Chow, Virginia; Schmidt, Liesbeth M; Dickson, Don W; Preston, James F

2007-08-01

Pasteuria species are endospore-forming obligate bacterial parasites of soil-inhabiting nematodes and water-inhabiting cladocerans, e.g. water fleas, and are closely related to Bacillus spp. by 16S rRNA gene sequence. As naturally occurring bacteria, biotypes of Pasteuria penetrans are attractive candidates for the biocontrol of various Meloidogyne spp. (root-knot nematodes). Failure to culture these bacteria outside their hosts has prevented isolation of genomic DNA in quantities sufficient for identification of genes associated with host recognition and virulence. We have applied multiple-strand displacement amplification (MDA) to generate DNA for comparative genomics of biotypes exhibiting different host preferences. Using the genome of Bacillus subtilis as a paradigm, MDA allowed quantitative detection and sequencing of 12 marker genes from 2000 cells. Meloidogyne spp. infected with P. penetrans P20 or B4 contained single nucleotide polymorphisms (SNPs) in the spoIIAB gene that did not change the amino acid sequence, or that substituted amino acids with similar chemical properties. Individual nematodes infected with P. penetrans P20 or B4 contained SNPs in the spoIIAB gene sequenced in MDA-generated products. Detection of SNPs in the spoIIAB gene in a nematode indicates infection by more than one genotype, supporting the need to sequence genomes of Pasteuria spp. derived from single spore isolates.

Label-free genotyping of cytochrome P450 2D6*10 using ligation-mediated strand displacement amplification with DNAzyme-based chemiluminescence detection.

Science.gov (United States)

Wang, Hong-Qi; Wu, Zhan; Zhang, Yan; Tang, Li-Juan; Yu, Ru-Qin; Jiang, Jian-Hui

2012-01-13

Genotyping of cytochrome P450 monooxygenase 2D6*10 (CYP2D6*10) plays an important role in pharmacogenomics, especially in clinical drug therapy of Asian populations. This work reported a novel label-free technique for genotyping of CYP2D6*10 based on ligation-mediated strand displacement amplification (SDA) with DNAzyme-based chemiluminescence detection. Discrimination of single-base mismatch is firstly accomplished using DNA ligase to generate a ligation product. The ligated product then initiates a SDA reaction to produce aptamer sequences against hemin, which can be probed by chemiluminescence detection. The proposed strategy is used for the assay of CYP2D6*10 target and the genomic DNA. The results reveal that the proposed technique displays chemiluminescence responses in linear correlation to the concentrations of DNA target within the range from 1 pM to 1 nM. A detection limit of 0.1 pM and a signal-to-background ratio of 57 are achieved. Besides such high sensitivity, the proposed CYP2D6*10 genotyping strategy also offers superb selectivity, great robustness, low cost and simplified operations due to its label-free, homogeneous, and chemiluminescence-based detection format. These advantages suggest this technique may hold considerable potential for clinical CYP2D6*10 genotyping and association studies. Copyright © 2011 Elsevier B.V. All rights reserved.

Helicase and Polymerase Move Together Close to the Fork Junction and Copy DNA in One-Nucleotide Steps

Directory of Open Access Journals (Sweden)

Manjula Pandey

2014-03-01

Full Text Available By simultaneously measuring DNA synthesis and dNTP hydrolysis, we show that T7 DNA polymerase and T7 gp4 helicase move in sync during leading-strand synthesis, taking one-nucleotide steps and hydrolyzing one dNTP per base-pair unwound/copied. The cooperative catalysis enables the helicase and polymerase to move at a uniformly fast rate without guanine:cytosine (GC dependency or idling with futile NTP hydrolysis. We show that the helicase and polymerase are located close to the replication fork junction. This architecture enables the polymerase to use its strand-displacement synthesis to increase the unwinding rate, whereas the helicase aids this process by translocating along single-stranded DNA and trapping the unwound bases. Thus, in contrast to the helicase-only unwinding model, our results suggest a model in which the helicase and polymerase are moving in one-nucleotide steps, DNA synthesis drives fork unwinding, and a role of the helicase is to trap the unwound bases and prevent DNA reannealing.

Inhibition and recovery of DNA synthesis in human cells after exposure to ultraviolet light

International Nuclear Information System (INIS)

Painter, R.B.

1985-01-01

The inhibition of DNA synthesis in normal human cells by UV is a complex function of fluence because it has several causes. At low fluences, inhibition of replicon initiation is most important. This is made clear by the fact that it occurs to a lesser degree in cells from patients with ataxia telangiectasia (AT). Assuming that only leading strand synthesis is blocked by UV-induced lesions, single lesions between replicons in parental strands for leading strand synthesis inhibit DNA synthesis by acting as temporary blocks until they are replicated by extension of the lagging strand of the adjacent replicon. A more severe inhibition occurs when two lesions are induced between adjacent growing replicons, because one in four possible configurations may result in a long-lived unreplicated region (LLUR). In the absence of excision repair, these may eventually be replicated by activation of an otherwise unused origin within the LLUR. The frequency of LLURs increases steeply with fluence. Activation of normally unused origins to replicate LLURs may facilitate recovery from inhibition of DNA synthesis, but repair of lesions is probably more important. In excision-repair-defective cells, an LLUR without an origin to initiate its replication may be a lethal lesion. (orig.)

Mechanical properties of rubberwood oriented strand lumber (OSL: The effect of strand length

Directory of Open Access Journals (Sweden)

Buhnnum Kyokong

2005-09-01

Full Text Available Effect of strand length on mechanical properties (tension, compression and bending of oriented strand lumber (OSL made of rubberwood (Hevea brasiliensis Muell. Arg. was reported. Three strand lengths of 50 mm, 100 mm, and 150 mm with 1 mm thickness and 15 mm width were used. The strands were mixed with 5% pMDI glue (weight basis in a tumble mixer. The OSL specimens were formed by hot pressing process of unidirectionally aligned strands. Average specific gravity and moisture content were 0.76 and 8.34%, respectively. Tension and compression tests were carried out for directions both parallel and perpendicular to grain while bending test was performed only in parallel direction. Ultimate stresses and moduli of elasticity were examined from the stress-strain curves. It was found that for the parallel-to-grain direction, the longer strand OSL gave higher strength. The role of the strand length did not appear for the direction normal to the grain. The relationship between the mechanical properties of OSL and strand length was well described by the modified Hankinson formula.

Base damage within single-strand DNA underlies in vivo hypermutability induced by a ubiquitous environmental agent.

Directory of Open Access Journals (Sweden)

Kin Chan

Full Text Available Chromosomal DNA must be in single-strand form for important transactions such as replication, transcription, and recombination to occur. The single-strand DNA (ssDNA is more prone to damage than double-strand DNA (dsDNA, due to greater exposure of chemically reactive moieties in the nitrogenous bases. Thus, there can be agents that damage regions of ssDNA in vivo while being inert toward dsDNA. To assess the potential hazard posed by such agents, we devised an ssDNA-specific mutagenesis reporter system in budding yeast. The reporter strains bear the cdc13-1 temperature-sensitive mutation, such that shifting to 37°C results in telomere uncapping and ensuing 5' to 3' enzymatic resection. This exposes the reporter region, containing three closely-spaced reporter genes, as a long 3' ssDNA overhang. We validated the ability of the system to detect mutagenic damage within ssDNA by expressing a modified human single-strand specific cytosine deaminase, APOBEC3G. APOBEC3G induced a high density of substitutions at cytosines in the ssDNA overhang strand, resulting in frequent, simultaneous inactivation of two reporter genes. We then examined the mutagenicity of sulfites, a class of reactive sulfur oxides to which humans are exposed frequently via respiration and food intake. Sulfites, at a concentration similar to that found in some foods, induced a high density of mutations, almost always as substitutions at cytosines in the ssDNA overhang strand, resulting in simultaneous inactivation of at least two reporter genes. Furthermore, sulfites formed a long-lived adducted 2'-deoxyuracil intermediate in DNA that was resistant to excision by uracil-DNA N-glycosylase. This intermediate was bypassed by error-prone translesion DNA synthesis, frequently involving Pol ζ, during repair synthesis. Our results suggest that sulfite-induced lesions in DNA can be particularly deleterious, since cells might not possess the means to repair or bypass such lesions

3′-Terminal Sequence in Poliovirus Negative-Strand Templates Is the Primary cis-Acting Element Required for VPgpUpU-Primed Positive-Strand Initiation

OpenAIRE

Sharma, Nidhi; O'Donnell, Brian J.; Flanegan, James B.

2005-01-01

The 5′ cloverleaf in poliovirus RNA has a direct role in regulating the stability, translation, and replication of viral RNA. In this study, we investigated the role of stem a in the 5′ cloverleaf in regulating the stability and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Our results showed that disrupting the duplex structure of stem a destabilized viral RNA and inhibited efficient negative-strand synthesis. Surprisingly, the duplex structure of stem a was no...

A Major Role of DNA Polymerase δ in Replication of Both the Leading and Lagging DNA Strands.

Science.gov (United States)

Johnson, Robert E; Klassen, Roland; Prakash, Louise; Prakash, Satya

2015-07-16

Genetic studies with S. cerevisiae Polδ (pol3-L612M) and Polε (pol2-M644G) mutant alleles, each of which display a higher rate for the generation of a specific mismatch, have led to the conclusion that Polε is the primary leading strand replicase and that Polδ is restricted to replicating the lagging strand template. Contrary to this widely accepted view, here we show that Polδ plays a major role in the replication of both DNA strands, and that the paucity of pol3-L612M-generated errors on the leading strand results from their more proficient removal. Thus, the apparent lack of Polδ contribution to leading strand replication is due to differential mismatch removal rather than differential mismatch generation. Altogether, our genetic studies with Pol3 and Pol2 mutator alleles support the conclusion that Polδ, and not Polε, is the major DNA polymerase for carrying out both leading and lagging DNA synthesis. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Energy Technology Data Exchange (ETDEWEB)

Ngaojampa, C.; Nimmanpipug, P. [Computer Simulation and Modeling Laboratory (CSML), Department of Chemistry and Center for Innovation Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Yu, L.D., E-mail: yuld@fnrf.science.cmu.ac.t [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Anuntalabhochai, S. [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Lee, V.S., E-mail: vannajan@gmail.co [Computer Simulation and Modeling Laboratory (CSML), Department of Chemistry and Center for Innovation Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand)

2011-02-15

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.

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

International Nuclear Information System (INIS)

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

2011-01-01

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.

Repair of abasic sites in DNA

Energy Technology Data Exchange (ETDEWEB)

Dianov, Grigory L.; Sleeth, Kate M.; Dianova, Irina I.; Allinson, Sarah L

2003-10-29

Repair of both normal and reduced AP sites is activated by AP endonuclease, which recognizes and cleaves a phosphodiester bond 5' to the AP site. For a short period of time an incised AP site is occupied by poly(ADP-ribose) polymerase and then DNA polymerase {beta} adds one nucleotide into the repair gap and simultaneously removes the 5'-sugar phosphate. Finally, the DNA ligase III/XRCC1 complex accomplishes repair by sealing disrupted DNA ends. However, long-patch BER pathway, which is involved in the removal of reduced abasic sites, requires further DNA synthesis resulting in strand displacement and the generation of a damage-containing flap that is later removed by the flap endonuclease. Strand-displacement DNA synthesis is accomplished by DNA polymerase {delta}/{epsilon} and DNA ligase I restores DNA integrity. DNA synthesis by DNA polymerase {delta}/{epsilon} is dependent on proliferating cell nuclear antigen, which also stimulates the DNA ligase I and flap endonuclease. These repair events are supported by multiple protein-protein interactions.

Stranded costs and exit fees

International Nuclear Information System (INIS)

2002-01-01

The New Brunswick Market Design Committee has been directed to examine the issue of stranded costs since it is a major component of restructuring within the electricity sector. When regulated monopolies are faced with competition, they could find that some of their embedded costs cannot be recovered. These costs are referred to as stranded costs. Common sources include large capital investments in uneconomic plants or expensive power purchase contracts or fuel supply contracts. In general, stranded costs do not include gains or losses associated with normal business risks experienced by regulated utilities. This report presents recommendations for mitigation of stranded costs, valuation methodologies and cost-recovery mechanisms. It also presents a summary of experience with stranded costs in other jurisdictions such as California, Rhode Island, Pennsylvania and Ontario. Stranded costs are often recovered through an obligatory charge on all customers, particularly in jurisdictions where retail competition exists. In the New Brunswick market, however, the only customers who can create stranded costs are those eligible to choose their own suppliers. It is argued that since most customers will not have a choice of electricity suppliers, they cannot generate stranded costs and therefore, should not have to pay costs stranded by others. A method to quantify stranded costs is presented, along with a review of transmission-related stranded costs in New Brunswick. Expansion of self-generation in New Brunswick could strand transmission assets. Currently, self-generators only contribute a small amount to fixed charges of the transmission system. However, under new recommended tariffs, the amount could increase. It is likely that the net amount of stranded transmission costs will not be large. 2 refs., 1 fig

Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements

Directory of Open Access Journals (Sweden)

Modesto Redrejo-Rodríguez

2017-11-01

Full Text Available Family B DNA polymerases (PolBs play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB, that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance.

Role of deoxyribonucleic acid polymerases and deoxyribonucleic acid ligase in x-ray-induced repair synthesis in toluene-treated Escherichia coli K-12

International Nuclear Information System (INIS)

Billen, D.; Hellermann, G.R.

1976-01-01

Toluene-treated Escherichia coli mutants have been used to study the roles of deoxyribonucleic acid (DNA) polymerases I, II, and III, and of DNA ligase in repair synthesis and strand rejoining following X-irradiation. In cells possessing all three DNA polymerases, both a greater amount of repair synthesis (''exaggerated'' repair synthesis) and failure of ligation are observed when DNA ligase activity is inhibited. In a mutant lacking the polymerizing activity of DNA polymerase I, exaggerated repair synthesis is not observed, and strand rejoining does not occur even if DNA ligase is fully activated. In a mutant possessing the polymerizing activity of DNA polymerase I but lacking its 5' → 3' exonuclease activity, exaggerated repair synthesis is minimal. After irradiation, DNA polymerases II and III are capable of carrying out an adenosine 5'-triphosphate-dependent repair synthesis, but rejoining of strand breaks does not occur and exaggerated synthesis is not seen whether DNA ligase is active or not. These results suggest that DNA polymerase I and DNA ligase act together to limit repair synthesis after X irradiation and that both are necessary in toluene-treated cells for strand rejoining. DNA polymerases II and III apparently cannot complete chain elongation and gap filling, and therefore repair carried out by these enzymes does not respond to ligase action

Spontaneous unscheduled DNA synthesis in human lymphocytes

International Nuclear Information System (INIS)

Forell, B.; Myers, L.S. Jr.; Norman, A.

1979-01-01

The rate of spontaneous unscheduled DNA synthesis in human lymphocytes was estimated from measurements of tritiated thymidine incorporation into double-stranded DNA (ds-DNA) during incubation of cells in vitro. The contribution of scheduled DNA synthesis to the observed incorporation was reduced by inhibiting replication with hydroxyurea and by separating freshly replicated single-stranded DNA (ss-DNA) from repaired ds-DNA by column chromatography. The residual contribution of scheduled DNA synthesis was estimated by observing effects on thymidine incorporation of: (a) increasing the rate of production of apurinic sites, and alternatively, (b) increasing the number of cells in S-phase. Corrections based on estimates of endogenous pool size were also made. The rate of spontaneous unscheduled DNA synthesis is estimated to be 490 +- 120 thymidine molecules incorporated per cell per hour. These results compare favorably with estimates made from rates of depurination and depyrimidination of DNA, measured in molecular systems if we assume thymidine is incorporated by a short patch mechanism which incorporates an average of four bases per lesion

Identification of a Single Strand Origin of Replication in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis.

Directory of Open Access Journals (Sweden)

Laurel D Wright

2015-10-01

Full Text Available We identified a functional single strand origin of replication (sso in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons are DNA elements typically found integrated into a bacterial chromosome where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICEs become activated and excise from the host chromosome and can transfer to neighboring cells via the element-encoded conjugation machinery. Activated ICEBs1 undergoes autonomous rolling circle replication that is needed for the maintenance of the excised element in growing and dividing cells. Rolling circle replication, used by many plasmids and phages, generates single-stranded DNA (ssDNA. In many cases, the presence of an sso enhances the conversion of the ssDNA to double-stranded DNA (dsDNA by enabling priming of synthesis of the second DNA strand. We initially identified sso1 in ICEBs1 based on sequence similarity to the sso of an RCR plasmid. Several functional assays confirmed Sso activity. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1 maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2 stable acquisition of ICEBs1 following transfer to a new host. We identified sequences similar to known plasmid sso's in several other ICEs. Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements.

Arranging eukaryotic nuclear DNA polymerases for replication: Specific interactions with accessory proteins arrange Pols α, δ, and ϵ in the replisome for leading-strand and lagging-strand DNA replication.

Science.gov (United States)

Kunkel, Thomas A; Burgers, Peter M J

2017-08-01

Biochemical and cryo-electron microscopy studies have just been published revealing interactions among proteins of the yeast replisome that are important for highly coordinated synthesis of the two DNA strands of the nuclear genome. These studies reveal key interactions important for arranging DNA polymerases α, δ, and ϵ for leading and lagging strand replication. The CMG (Mcm2-7, Cdc45, GINS) helicase is central to this interaction network. These are but the latest examples of elegant studies performed in the recent past that lead to a much better understanding of how the eukaryotic replication fork achieves efficient DNA replication that is accurate enough to prevent diseases yet allows evolution. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

The prion protein has DNA strand transfer properties similar to retroviral nucleocapsid protein.

Science.gov (United States)

Gabus, C; Auxilien, S; Péchoux, C; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W; Nandi, P; Darlix, J L

2001-04-06

The transmissible spongiform encephalopathies are fatal neurodegenerative diseases that are associated with the accumulation of a protease-resistant form of the cellular prion protein (PrP). Although PrP is highly conserved and widely expressed in vertebrates, its function remains a matter of speculation. Indeed PrP null mice develop normally and are healthy. Recent results show that PrP binds to nucleic acids in vitro and is found associated with retroviral particles. Furthermore, in mice the scrapie infectious process appears to be accelerated by MuLV replication. These observations prompted us to further investigate the interaction between PrP and nucleic acids, and compare it with that of the retroviral nucleocapsid protein (NC). As the major nucleic acid-binding protein of the retroviral particle, NC protein is tightly associated with the genomic RNA in the virion nucleocapsid, where it chaperones proviral DNA synthesis by reverse transcriptase. Our results show that the human prion protein (huPrP) functionally resembles NCp7 of HIV-1. Both proteins form large nucleoprotein complexes upon binding to DNA. They accelerate the hybridization of complementary DNA strands and chaperone viral DNA synthesis during the minus and plus DNA strand transfers necessary to generate the long terminal repeats. The DNA-binding and strand transfer properties of huPrP appear to map to the N-terminal fragment comprising residues 23 to 144, whereas the C-terminal domain is inactive. These findings suggest that PrP could be involved in nucleic acid metabolism in vivo. Copyright 2001 Academic Press.

Displacer Diameter Effect in Displacer Pulse Tube Refrigerator

Science.gov (United States)

Zhu, Shaowei

2017-12-01

Gas driving displacer pulse tube refrigerators are one of the work recovery type of pulse tube refrigerators whose theoretical efficiency is the same as Stirling refrigerators'. Its cooling power is from the displacement of the displacer. Displace diameter, rod diameter and pressure drop of the regenerator influence the displacement, which are investigated by numerical simulation. It is shown that the displacement ratio of the displacer over the piston is almost not affected by the displacer diameter at the same rod diameter ratio, or displacer with different diameters almost has the same performance.

Helical self-organization and hierarchical self-assembly of an oligoheterocyclic pyridine-pyridazine strand into extended supramolecular fibers.

Science.gov (United States)

Cuccia, Louis A; Ruiz, Eliseo; Lehn, Jean-Marie; Homo, Jean-Claude; Schmutz, Marc

2002-08-02

The synthesis and characterization of an alternating pyridine-pyridazine strand comprising thirteen heterocycles are described. Spontaneous folding into a helical secondary structure is based on a general molecular self-organization process enforced by the conformational information encoded within the primary structure of the molecular strand itself. Conformational control based on heterocyclic "helicity codons" illustrates a strategy for designing folding properties into synthetic oligomers (foldamers). Strong intermolecular interactions of the highly ordered lock-washer subunits of compound 3 results in hierarchical supramolecular self-assembly into protofibrils and fibrils. Compound 3 also forms mechanically stable two-dimensional Langmuir-Blodgett and cast thin films.

Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators.

Directory of Open Access Journals (Sweden)

Yumiko Kurokawa

2008-04-01

Full Text Available In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 (the S. pombe Rad52 homolog and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 (the S. pombe Rad51 homolog-mediated homologous recombination (HR and HR repair. Here, we have demonstrated an in vitro reconstitution of the central step of DNA strand exchange during HR. Our system consists entirely of homogeneously purified proteins, including Rhp51, the two mediators, and replication protein A (RPA, which reflects genetic requirements in vivo. Using this system, we present the first robust biochemical evidence that concerted action of the two mediators directs the loading of Rhp51 onto single-stranded DNA (ssDNA precoated with RPA. Dissection of the reaction reveals that Rad22 overcomes the inhibitory effect of RPA on Rhp51-Swi5-Sfr1-mediated strand exchange. In addition, Rad22 negates the requirement for a strict order of protein addition to the in vitro system. However, despite the presence of Rad22, Swi5-Sfr1 is still essential for strand exchange. Importantly, Rhp51, but neither Rad22 nor the Swi5-Sfr1 mediator, is the factor that displaces RPA from ssDNA. Swi5-Sfr1 stabilizes Rhp51-ssDNA filaments in an ATP-dependent manner, and this stabilization is correlated with activation of Rhp51 for the strand exchange reaction. Rad22 alone cannot activate the Rhp51 presynaptic filament. AMP-PNP, a nonhydrolyzable ATP analog, induces a similar stabilization of Rhp51, but this stabilization is independent of Swi5-Sfr1. However, hydrolysis of ATP is required for processive strand transfer, which results in the formation of a long heteroduplex. Our in vitro reconstitution system has revealed that the two mediators have indispensable, but distinct, roles for mediating Rhp51 loading onto RPA-precoated ssDNA.

Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1.

Science.gov (United States)

Gloor, Jason W; Balakrishnan, Lata; Campbell, Judith L; Bambara, Robert A

2012-08-01

In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼ 30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication.

Science.gov (United States)

Herzig, Eytan; Voronin, Nickolay; Kucherenko, Nataly; Hizi, Amnon

2015-08-01

The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription

Assembling of G-strands into novel tetra-molecular parallel G4-DNA nanostructures using avidin-biotin recognition.

Science.gov (United States)

Borovok, Natalia; Iram, Natalie; Zikich, Dragoslav; Ghabboun, Jamal; Livshits, Gideon I; Porath, Danny; Kotlyar, Alexander B

2008-09-01

We describe a method for the preparation of novel long (hundreds of nanometers), uniform, inter-molecular G4-DNA molecules composed of four parallel G-strands. The only long continuous G4-DNA reported so far are intra-molecular structures made of a single G-strand. To enable a tetra-molecular assembly of the G-strands we developed a novel approach based on avidin-biotin biological recognition. The steps of the G4-DNA production include: (i) Enzymatic synthesis of long poly(dG)-poly(dC) molecules with biotinylated poly(dG)-strand; (ii) Formation of a complex between avidin-tetramer and four biotinylated poly(dG)-poly(dC) molecules; (iii) Separation of the poly(dC) strands from the poly(dG)-strands, which are connected to the avidin; (iv) Assembly of the four G-strands attached to the avidin into tetra-molecular G4-DNA. The average contour length of the formed structures, as measured by AFM, is equal to that of the initial poly(dG)-poly(dC) molecules, suggesting a tetra-molecular mechanism of the G-strands assembly. The height of tetra-molecular G4-nanostructures is larger than that of mono-molecular G4-DNA molecules having similar contour length. The CD spectra of the tetra- and mono-molecular G4-DNA are markedly different, suggesting different structural organization of these two types of molecules. The tetra-molecular G4-DNA nanostructures showed clear electrical polarizability. This suggests that they may be useful for molecular electronics.

KARAKTERISTIK ORIENTED STRAND BOARD DARI KAYU AKASIA DAN AFRIKA BERDASARKAN PENYUSUNAN ARAH STRAND

Directory of Open Access Journals (Sweden)

Nurhaida

2008-04-01

Full Text Available The research objectives arc to evaluate physical and mechanical properties of OSB based on strands orientation; and to evaluate physical and mechanical properties of OSB made from akasia wood (Acacia mangium Wild and afrika wood (Maesopsis eminii Engl. Akasia and afrika wood are used for OSB strand material with phenol formaldehyde (PF as adhesives and addition of paraffin. OSB made in this research is consist of three plies whereas are differed into eight (8 strand orientations. In the making process, hot press was carried out at 160OC and pressure 25kg.cm-2 for 15 minutes. Determination of OSB physical and mechanical properties is referred to JIS A 5908-2003. Result showed that strand orientations has no affect to OSB physical properties except for linicr swelling 24h, but it significantly influence all mechanical properties of OSB. Wood species have an effect on mechanical properties of OSB in the dry test, wet MOE lengthwise test and OSB physical properties, particularly to OSB density and water absorbing capability at 2h and 24h. All of OSB physical properties arc meet JIS A 5908-2003 standard, but not all of the mechanical properties such as dry MOE lengthwise, dry MOE and MOR widthwise. The best physical and mechanical properties is presented by OSB made from akasia wood in strand orientation F, G, Band C whereas all parameters meet JIS A 5908-2003 standard. In comparation with strand orientation B that is frequent used in industry, strand orientation F and G arc proficient to raise the modulus elasticity value (MOE and strength (MOR as much as 167.81-231.65% and 89.73-109.87%, respectively; especially in widthwise board application. Furthermore, strand orientation F and G arc more flexible as structural components

Structural features of single-stranded integron cassette attC sites and their role in strand selection.

Directory of Open Access Journals (Sweden)

Marie Bouvier

2009-09-01

Full Text Available We recently showed that cassette integration and deletion in integron platforms were occurring through unconventional site-specific recombination reactions involving only the bottom strand of attC sites. The lack of sequence conservation among attC sites led us to hypothesize that sequence-independent structural recognition determinants must exist within attC sites. The structural data obtained from a synaptic complex of the Vibrio cholerae integrase with the bottom strand of an attC site has shown the importance of extra helical bases (EHB inside the stem-loop structure formed from the bottom strand. Here, we systematically determined the contribution of three structural elements common to all known single-stranded attC site recombination substrates (the EHBs, the unpaired central spacer (UCS, and the variable terminal structure (VTS to strand choice and recombination. Their roles have been evaluated in vivo in the attIxattC reaction context using the suicide conjugation assay we previously developed, but also in an attCxattC reaction using a deletion assay. Conjugation was used to deliver the attC sites in single-stranded form. Our results show that strand choice is primarily directed by the first EHB, but the presence of the two other EHBs also serves to increase this strand selection. We found that the structure of the central spacer is essential to achieve high level recombination of the bottom strand, suggesting a dual role for this structure in active site exclusion and for hindering the reverse reaction after the first strand exchange. Moreover, we have shown that the VTS has apparently no role in strand selectivity.

UV light-induced DNA synthesis arrest in HeLa cells is associated with changes in phosphorylation of human single-stranded DNA-binding protein

International Nuclear Information System (INIS)

Carty, M.P.; Zernik-Kobak, M.; McGrath, S.; Dixon, K.

1994-01-01

We show that DNA replication activity in extracts of human HeLa cells decreases following UV irradiation. Alterations in replication activity in vitro parallel the UV-induced block in cell cycle progression of these cells in culture. UV irradiation also induces specific changes in the pattern of phosphorylation of the 34 kDa subunit of a DNA replication protein, human single-stranded DNA-binding protein (hSSB). The appearance of a hyperphosphorylated form of hSSB correlates with reduced in vitro DNA replication activity in extracts of UV-irradiated cells. Replication activity can be restored to these extracts in vitro by addition of purified hSSB. These results suggest that UV-induced DNA synthesis arrest may be mediated in part through phosphorylation-related alterations in the activity of hSSB, an essential component of the DNA replication apparatus. (Author)

DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease

Energy Technology Data Exchange (ETDEWEB)

Ahn, Byungchan, E-mail: bbccahn@mail.ulsan.ac.kr [Department of Life Sciences, University of Ulsan, Ulsan (Korea, Republic of); Bohr, Vilhelm A. [Laboratory of Molecular Gerontology, Biomedical Research Center, National Institute on Aging, Baltimore, MD (United States)

2011-08-12

Highlights: {yields} In this study, we investigated the effect of a DNA secondary structure on the two WRN activities. {yields} We found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. {yields} These results imply that WRN helicase and exonuclease activities can act independently. -- Abstract: Werner syndrome (WS) is an autosomal recessive premature aging disorder characterized by aging-related phenotypes and genomic instability. WS is caused by mutations in a gene encoding a nuclear protein, Werner syndrome protein (WRN), a member of the RecQ helicase family, that interestingly possesses both helicase and exonuclease activities. Previous studies have shown that the two activities act in concert on a single substrate. We investigated the effect of a DNA secondary structure on the two WRN activities and found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. These results imply that WRN helicase and exonuclease activities can act independently, and we propose that the uncoordinated action may be relevant to the in vivo activity of WRN.

Excess single-stranded DNA inhibits meiotic double-strand break repair.

Directory of Open Access Journals (Sweden)

Rebecca Johnson

2007-11-01

Full Text Available During meiosis, self-inflicted DNA double-strand breaks (DSBs are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE, in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects

Strand transfer and elongation of HIV-1 reverse transcription is facilitated by cell factors in vitro.

Directory of Open Access Journals (Sweden)

David Warrilow

Full Text Available Recent work suggests a role for multiple host factors in facilitating HIV-1 reverse transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 reverse transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor reverse transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of reverse transcription. Instead, the cell factor(s enhanced first strand transfer and synthesis of late reverse transcription suggesting it stabilized the reverse transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous reverse transcription (ERT system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded reverse transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s suggesting a lack of involvement of the capsid in the in vitro assay. In addition, reverse transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s may improve reverse transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the reverse transcription products, but it is unclear if this is related to improved DNA synthesis.

Factors affecting mutational specificity in mammalian cells: [Informal technical progress report], February 1, 1986-January 31, 1987

International Nuclear Information System (INIS)

Strauss, B.S.

1987-01-01

We analyzed the different c-H-ras mutations produced in cells after treatment with chemical carcinogens. The overall goal of this work is an understanding of the changes produced by environmental mutagens and carcinogens to learn how closely the results obtained with bacterial and cellular assay systems apply to the in vivo situation. Our work continues to utilize the technique for the study of DNA synthesis termination in vitro developed in this laboraory and utilized for the past several years. This technology can be characterized as a Sanger dideoxy sequencing technique with mutation induced lesions in the template strand serving as chain terminators instead of the dideoxynucleotides used in the Sanger technique to terminate the growing strand. We have added to this technique a modification for the study of termination on double stranded templates as possibly modeling more closely the natural situation. For this modification, double stranded M13 DNA is split once with a restriction enzyme which makes a unique cut and the linearized DNA is hybridized with single stranded circular DNA to make a uniquely nicked double stranded circle with a single 3'OH to serve as a primer for DNA synthesis which can occur by either strand displacement or nick translation

Radiobiology of DNA strand breakage

International Nuclear Information System (INIS)

Johansen, I.

1975-01-01

The yield of single-strand breaks in lambda DNA within lysogenic host bacteria was measured after exposure to 4-MeV electrons (50 msec) and rapid transfer (45 msec) to alkaline detergent. In nitrogen anoxia the yield was 1.2 x 10 -12 DNA single-strand breaks per rad per dalton, and under full oxygenation the yield increased to 5 x 10 -12 breaks per rad per dalton. A search for the presence of fast repair mechanisms failed to demonstrate the presence of any mechanism for repair of strand breaks operating within a fraction of a second. Strand breaks produced in the presence of oxygen were repaired in 30--40 sec, while breaks produced under anoxia were rejoined even slower. A functional product from the polAl gene was needed for the rejoining of the broken molecules. Intermediate levels of DNA strand breakage seen at low concentrations of oxygen are dependent on the concentration of cellular sulfhydryl compounds, suggesting that in strand breakage oxygen and hydrogen donors compete for reactions with radiation-induced transients in the DNA. Intercomparisons of data on radiation-induced lethality of cells and single-strand breaks in episomal DNA allow the distinction between two classes of radiation-induced radicals, R 1 and R 2 , with different chemical properties; R 1 reacts readily with oxygen and N-oxyls under formation of potentially lethal products. The reactivity of oxygen in this reaction is 30--40 times higher than that of TMPN. R 2 reacts 16 times more readily than R 1 with oxygen under formation of single-strand breaks in the DNA. R 2 does not react with N-oxyls

Theoretical analysis on ac loss properties of two-strand parallel conductors composed of superconducting multifilamentary strands

CERN Document Server

Iwakuma, M; Funaki, K

2002-01-01

The ac loss properties of two-strand parallel conductors composed of superconducting multifilamentary strands were theoretically investigated. The constituent strands generally need to be insulated and transposed for the sake of uniform current distribution and low ac loss. In case the transposition points deviate from the optimum ones, shielding current is induced according to the interlinkage magnetic flux of the twisted loop enclosed by the insulated strands and the contact resistances at the terminals. It produces an additional ac loss. Supposing a simple situation where a two-strand parallel conductor with one-point transposition is exposed to a uniform ac magnetic field, the basic equations for the magnetic field were proposed and the theoretical expressions of the additional ac losses derived. As a result, the following features were shown. The additional ac loss in the non-saturation case, where the induced shielding current is less than the critical current of a strand, is proportional to the square ...

DNA hybridization kinetics: zippering, internal displacement and sequence dependence.

Science.gov (United States)

Ouldridge, Thomas E; Sulc, Petr; Romano, Flavio; Doye, Jonathan P K; Louis, Ard A

2013-10-01

Although the thermodynamics of DNA hybridization is generally well established, the kinetics of this classic transition is less well understood. Providing such understanding has new urgency because DNA nanotechnology often depends critically on binding rates. Here, we explore DNA oligomer hybridization kinetics using a coarse-grained model. Strand association proceeds through a complex set of intermediate states, with successful binding events initiated by a few metastable base-pairing interactions, followed by zippering of the remaining bonds. But despite reasonably strong interstrand interactions, initial contacts frequently dissociate because typical configurations in which they form differ from typical states of similar enthalpy in the double-stranded equilibrium ensemble. Initial contacts must be stabilized by two or three base pairs before full zippering is likely, resulting in negative effective activation enthalpies. Non-Arrhenius behavior arises because the number of base pairs required for nucleation increases with temperature. In addition, we observe two alternative pathways-pseudoknot and inchworm internal displacement-through which misaligned duplexes can rearrange to form duplexes. These pathways accelerate hybridization. Our results explain why experimentally observed association rates of GC-rich oligomers are higher than rates of AT- rich equivalents, and more generally demonstrate how association rates can be modulated by sequence choice.

Contact mechanical characteristics of Nb3Sn strands under transverse electromagnetic loads in the CICC cross-section

International Nuclear Information System (INIS)

Zhu Jiyue; Luo Wei; Zhou Youhe; Zheng Xiaojing

2012-01-01

This paper presents a new code for the two-dimensional discrete element method (DEM) and relevant simulations to quantitatively characterize the contact force behavior of the Nb 3 Sn strands in the ITER CICC cross-section under a transverse electromagnetic load. In order to obtain the essential parameters in the contact force model employed in the DEM, a simulation of the experiments conducted by Nijhuis et al (2004 IEEE Trans. Appl. Supercond. 14 1489–94) is first performed, where the load–displacement curve predicted by the code is in good agreement with the measurements. After that, the contact force chain between strands and its distribution is quantitatively analyzed by the code. It is found that the contact force distribution among strands is heterogeneous and strongly anisotropic. In other words, the force chain distribution, which determines the behavior of the assembly of strands with discrete media, and the distribution of area average magnitude of the contact force are obviously inhomogeneous. To describe this inhomogeneity, here, the probability density function (PDF) is used in the statistical analysis. The numerical results show that the PDFs of the magnitudes of the resultant contact force, normal contact force, and tangential contact force all decay with an exponential law, and that PDFs of the directions of the contact forces are all anisotropic and exhibit about six periodic changes in which the peak values in the direction parallel to the applied electromagnetic load are appreciably larger than the other peaks. (paper)

The multiple personalities of Watson and Crick strands.

Science.gov (United States)

Cartwright, Reed A; Graur, Dan

2011-02-08

In genetics it is customary to refer to double-stranded DNA as containing a "Watson strand" and a "Crick strand." However, there seems to be no consensus in the literature on the exact meaning of these two terms, and the many usages contradict one another as well as the original definition. Here, we review the history of the terminology and suggest retaining a single sense that is currently the most useful and consistent. The Saccharomyces Genome Database defines the Watson strand as the strand which has its 5'-end at the short-arm telomere and the Crick strand as its complement. The Watson strand is always used as the reference strand in their database. Using this as the basis of our standard, we recommend that Watson and Crick strand terminology only be used in the context of genomics. When possible, the centromere or other genomic feature should be used as a reference point, dividing the chromosome into two arms of unequal lengths. Under our proposal, the Watson strand is standardized as the strand whose 5'-end is on the short arm of the chromosome, and the Crick strand as the one whose 5'-end is on the long arm. Furthermore, the Watson strand should be retained as the reference (plus) strand in a genomic database. This usage not only makes the determination of Watson and Crick unambiguous, but also allows unambiguous selection of reference stands for genomics. This article was reviewed by John M. Logsdon, Igor B. Rogozin (nominated by Andrey Rzhetsky), and William Martin.

The multiple personalities of Watson and Crick strands

Directory of Open Access Journals (Sweden)

Graur Dan

2011-02-01

Full Text Available Abstract Background In genetics it is customary to refer to double-stranded DNA as containing a "Watson strand" and a "Crick strand." However, there seems to be no consensus in the literature on the exact meaning of these two terms, and the many usages contradict one another as well as the original definition. Here, we review the history of the terminology and suggest retaining a single sense that is currently the most useful and consistent. Proposal The Saccharomyces Genome Database defines the Watson strand as the strand which has its 5'-end at the short-arm telomere and the Crick strand as its complement. The Watson strand is always used as the reference strand in their database. Using this as the basis of our standard, we recommend that Watson and Crick strand terminology only be used in the context of genomics. When possible, the centromere or other genomic feature should be used as a reference point, dividing the chromosome into two arms of unequal lengths. Under our proposal, the Watson strand is standardized as the strand whose 5'-end is on the short arm of the chromosome, and the Crick strand as the one whose 5'-end is on the long arm. Furthermore, the Watson strand should be retained as the reference (plus strand in a genomic database. This usage not only makes the determination of Watson and Crick unambiguous, but also allows unambiguous selection of reference stands for genomics. Reviewers This article was reviewed by John M. Logsdon, Igor B. Rogozin (nominated by Andrey Rzhetsky, and William Martin.

Asymmetric Modification of Hepatitis B Virus (HBV) Genomes by an Endogenous Cytidine Deaminase inside HBV Cores Informs a Model of Reverse Transcription.

Science.gov (United States)

Nair, Smita; Zlotnick, Adam

2018-05-15

Cytidine deaminases inhibit replication of a broad range of DNA viruses by deaminating cytidines on single-stranded DNA (ssDNA) to generate uracil. While several lines of evidence have revealed hepatitis B virus (HBV) genome editing by deamination, it is still unclear which nucleic acid intermediate of HBV is modified. Hepatitis B virus has a relaxed circular double-stranded DNA (rcDNA) genome that is reverse transcribed within virus cores from a RNA template. The HBV genome also persists as covalently closed circular DNA (cccDNA) in the nucleus of an infected cell. In the present study, we found that in HBV-producing HepAD38 and HepG2.2.15 cell lines, endogenous cytidine deaminases edited 10 to 25% of HBV rcDNA genomes, asymmetrically with almost all mutations on the 5' half of the minus strand. This region corresponds to the last half of the minus strand to be protected by plus-strand synthesis. Within this half of the genome, the number of mutations peaks in the middle. Overexpressed APOBEC3A and APOBEC3G could be packaged in HBV capsids but did not change the amount or distribution of mutations. We found no deamination on pregenomic RNA (pgRNA), indicating that an intact genome is encapsidated and deaminated during or after reverse transcription. The deamination pattern suggests a model of rcDNA synthesis in which pgRNA and then newly synthesized minus-sense single-stranded DNA are protected from deaminase by interaction with the virus capsid; during plus-strand synthesis, when enough dsDNA has been synthesized to displace the remaining minus strand from the capsid surface, the single-stranded DNA becomes deaminase sensitive. IMPORTANCE Host-induced mutation of the HBV genome by APOBEC proteins may be a path to clearing the virus. We examined cytidine-to-thymidine mutations in the genomes of HBV particles grown in the presence or absence of overexpressed APOBEC proteins. We found that genomes were subjected to deamination activity during reverse transcription

Variation in sperm displacement and its association with accessory gland protein loci in Drosophila melanogaster.

Science.gov (United States)

Clark, A G; Aguadé, M; Prout, T; Harshman, L G; Langley, C H

1995-01-01

Genes that influence mating and/or fertilization success may be targets for strong natural selection. If females remate frequently relative to the duration of sperm storage and rate of sperm use, sperm displacement may be an important component of male reproductive success. Although it has long been known that mutant laboratory stocks of Drosophila differ in sperm displacement, the magnitude of the naturally occurring genetic variation in this character has not been systematically quantified. Here we report the results of a screen for variation in sperm displacement among 152 lines of Drosophilia melanogaster that were made homozygous for second and/or third chromosomes recovered from natural populations. Sperm displacement was assayed by scoring the progeny of cn;bw females that had been mated sequentially to cn;bw and tested males in either order. Highly significant differences were seen in both the ability to displace sperm that is resident in the female's reproductive tract and in the ability to resist displacement by subsequent sperm. Most lines exhibited nearly complete displacement, having nearly all progeny sired by the second male, but several lines had as few as half the progeny fathered by the second male. Lines that were identified in the screen for naturally occurring variation in sperm displacement were also characterized for single-strand conformation polymorphisms (SSCP) at seven accessory gland protein (Acp) genes, Glucose dehydrogenase (Gld), and Esterase-6 (Est-6). Acp genes encode proteins that are in some cases known to be transmitted to the female in the seminal fluid and are likely candidates for genes that might mediate the phenomenon of sperm displacement. Significant associations were found between particular Acp alleles at four different loci (Acp26Aa/Ab, Acp29B, Acp36DE and Acp53E) and the ability of males to resist displacement by subsequent sperm. There was no correlation between the ability to displace resident sperm and the ability

Sea Turtle Stranding Network Reports

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — The Sea Turtle Stranding and Salvage Network (STSSN) was formally established in 1980 to collect information on and document the stranding of marine turtles along...

A ratiometric electrochemical biosensor for the exosomal microRNAs detection based on bipedal DNA walkers propelled by locked nucleic acid modified toehold mediate strand displacement reaction.

Science.gov (United States)

Zhang, Jing; Wang, Liang-Liang; Hou, Mei-Feng; Xia, Yao-Kun; He, Wen-Hui; Yan, An; Weng, Yun-Ping; Zeng, Lu-Peng; Chen, Jing-Hua

2018-04-15

Sensitive and selective detection of microRNAs (miRNAs) in cancer cells derived exosomes have attracted rapidly growing interest owing to their potential in diagnostic and prognostic applications. Here, we design a ratiometric electrochemical biosensor based on bipedal DNA walkers for the attomolar detection of exosomal miR-21. In the presence of miR-21, DNA walkers are activated to walk continuously along DNA tracks, resulting in conformational changes as well as considerable increases of the signal ratio produced by target-respond and target-independent reporters. With the signal cascade amplification of DNA walkers, the biosensor exhibits ultrahigh sensitivity with the limit of detection (LOD) down to 67 aM. Furthermore, owing to the background-correcting function of target-independent reporters termed as reference reporters, the biosensor is robust and stable enough to be applied in the detection of exosomal miR-21 extracted from breast cancer cell lines and serums. In addition, because locked nucleic acid (LNA) modified toehold mediate strand displacement reaction (TMSDR) has extraordinary discriminative ability, the biosensor displays excellent selectivity even against the single-base-mismatched target. It is worth mentioning that our sensor is regenerative and stable for at least 5 cycles without diminution in sensitivity. In brief, the high sensitivity, selectivity and reproducibility, together with cheap, make the proposed biosensor a promising approach for exosomal miRNAs detection, in conjunction with early point-of-care testing (POCT) of cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Displaced Sense: Displacement, Religion and Sense-making

OpenAIRE

Naidu, Maheshvari

2016-01-01

Whether formally categorized as refugees or not, displaced migrants experience varying degrees of vulnerability in relation to where they find themselves displaced. The internally displaced furthermore squat invisibly and outside the boundaries of the legal framework and incentive structures accorded to those classified as 'refugee'. They are thus arguably, by and large, left to source sustaining solutions for themselves. This article works through the theoretical prism of sense-making theory...

Estimation of the long-term slip, surface uplift and block rotation along the northern strand of the North Anatolian Fault Zone: Inferences from geomorphology of the Almacık Block

Science.gov (United States)

Yıldırım, Cengiz; Tüysüz, Okan

2017-11-01

The Almacık Block is one of the key morphotectonic units in the eastern Marmara Region associated with the long-term slip partitioning within the North Anatolian Fault Zone (NAFZ). In this study, we provide new geomorphic reconstructions of offset drainage basins, morphometric analysis of topography, and longitudinal profiles of the rivers crossing different flanks of the Almacık Block. Our geomorphic reconstructions of offset drainage basins along the Hendek and Karadere faults imply mean offsets of 2.3 ± 0.4 km and 8.4 ± 0.7 km, respectively, during the Quaternary. Our dataset also imply that slip partitioning occurs in a broader zone than previously proposed, and that the total 10.7 ± 0.6 km offset along the Hendek and Karadere faults of the northern strand must be taken into account for long-term slip partitioning in the Eastern Marmara Region. Together with previously suggested 10 km offset along the southern strand (Yaltırak, 2002), 16 ± 1.0 km offset along the middle strand (Özalp et al., 2013) and the 52 ± 1.0 km offset along the Mudurnu Segment of the northern strand (Akbayram et al., 2016) our newly proposed geomorphic markers raise the cumulative offset in the eastern Marmara region associated with the NAF to 89 ± 1.0 km since the Latest Pliocene - Quaternary. In addition to these lateral displacements, our morphometric analysis and longitudinal profiles of the rivers imply up to 1130 ± 130 m surface uplift of the Almacık Block as a combined result of vertical displacement within the deformation zone of the northern strand of the NAFZ. Finally, by assuming that river basins act as passive deformation markers, our basin azimuth analyses imply 20° ± 2° clockwise rotation of the Almacık Block associated with the NAFZ.

RPA Stabilization of Single-Stranded DNA Is Critical for Break-Induced Replication.

Science.gov (United States)

Ruff, Patrick; Donnianni, Roberto A; Glancy, Eleanor; Oh, Julyun; Symington, Lorraine S

2016-12-20

DNA double-strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair of DSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and by facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular break-induced replication (BIR). The BIR defects of the rfa1 mutants could be partially suppressed by eliminating the Sgs1-Dna2 resection pathway, suggesting that Dna2 nuclease attacks the ssDNA formed during end resection when not fully protected by RPA. Overexpression of Rad51 was also found to suppress the rfa1 BIR defects. We suggest that Rad51 binding to the ssDNA formed by excessive end resection and during D-loop migration can partially compensate for dysfunctional RPA. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

[Expression and purification of a novel thermophilic bacterial single-stranded DNA-binding protein and enhancement the synthesis of DNA and cDNA].

Science.gov (United States)

Jia, Xiao-Wei; Zhang, Guo-Hui; Shi, Hai-Yan

2012-12-01

Express a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription. We express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction. The plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15. kod-ssb may in future be used to enhance DNA and cDNA amplification.

Intracellular generation of single-strand template increases the knock-in efficiency by combining CRISPR/Cas9 with AAV.

Science.gov (United States)

Xiao, Qing; Min, Taishan; Ma, Shuangping; Hu, Lingna; Chen, Hongyan; Lu, Daru

2018-04-18

Targeted integration of transgenes facilitates functional genomic research and holds prospect for gene therapy. The established microhomology-mediated end-joining (MMEJ)-based strategy leads to the precise gene knock-in with easily constructed donor, yet the limited efficiency remains to be further improved. Here, we show that single-strand DNA (ssDNA) donor contributes to efficient increase of knock-in efficiency and establishes a method to achieve the intracellular linearization of long ssDNA donor. We identified that the CRISPR/Cas9 system is responsible for breaking double-strand DNA (dsDNA) of palindromic structure in inverted terminal repeats (ITRs) region of recombinant adeno-associated virus (AAV), leading to the inhibition of viral second-strand DNA synthesis. Combing Cas9 plasmids targeting genome and ITR with AAV donor delivery, the precise knock-in of gene cassette was achieved, with 13-14% of the donor insertion events being mediated by MMEJ in HEK 293T cells. This study describes a novel method to integrate large single-strand transgene cassettes into the genomes, increasing knock-in efficiency by 13.6-19.5-fold relative to conventional AAV-mediated method. It also provides a comprehensive solution to the challenges of complicated production and difficult delivery with large exogenous fragments.

Fair Exchange in Strand Spaces

Directory of Open Access Journals (Sweden)

Joshua D. Guttman

2009-10-01

Full Text Available Many cryptographic protocols are intended to coordinate state changes among principals. Exchange protocols coordinate delivery of new values to the participants, e.g. additions to the set of values they possess. An exchange protocol is fair if it ensures that delivery of new values is balanced: If one participant obtains a new possession via the protocol, then all other participants will, too. Fair exchange requires progress assumptions, unlike some other protocol properties. The strand space model is a framework for design and verification of cryptographic protocols. A strand is a local behavior of a single principal in a single session of a protocol. A bundle is a partially ordered global execution built from protocol strands and adversary activities. The strand space model needs two additions for fair exchange protocols. First, we regard the state as a multiset of facts, and we allow strands to cause changes in this state via multiset rewriting. Second, progress assumptions stipulate that some channels are resilient-and guaranteed to deliver messages-and some principals are assumed not to stop at certain critical steps. This method leads to proofs of correctness that cleanly separate protocol properties, such as authentication and confidentiality, from invariants governing state evolution. G. Wang's recent fair exchange protocol illustrates the approach.

How quantum entanglement in DNA synchronizes double-strand breakage by type II restriction endonucleases.

Science.gov (United States)

Kurian, P; Dunston, G; Lindesay, J

2016-02-21

Macroscopic quantum effects in living systems have been studied widely in pursuit of fundamental explanations for biological energy transport and sensing. While it is known that type II endonucleases, the largest class of restriction enzymes, induce DNA double-strand breaks by attacking phosphodiester bonds, the mechanism by which simultaneous cutting is coordinated between the catalytic centers remains unclear. We propose a quantum mechanical model for collective electronic behavior in the DNA helix, where dipole-dipole oscillations are quantized through boundary conditions imposed by the enzyme. Zero-point modes of coherent oscillations would provide the energy required for double-strand breakage. Such quanta may be preserved in the presence of thermal noise by the enzyme's displacement of water surrounding the DNA recognition sequence. The enzyme thus serves as a decoherence shield. Palindromic mirror symmetry of the enzyme-DNA complex should conserve parity, because symmetric bond-breaking ceases when the symmetry of the complex is violated or when physiological parameters are perturbed from optima. Persistent correlations in DNA across longer spatial separations-a possible signature of quantum entanglement-may be explained by such a mechanism. Copyright © 2015 Elsevier Ltd. All rights reserved.

Strand exchange of telomeric DNA catalyzed by the Werner syndrome protein (WRN) is specifically stimulated by TRF2

Science.gov (United States)

Edwards, Deanna N.; Orren, David K.; Machwe, Amrita

2014-01-01

Werner syndrome (WS), caused by loss of function of the RecQ helicase WRN, is a hereditary disease characterized by premature aging and elevated cancer incidence. WRN has DNA binding, exonuclease, ATPase, helicase and strand annealing activities, suggesting possible roles in recombination-related processes. Evidence indicates that WRN deficiency causes telomeric abnormalities that likely underlie early onset of aging phenotypes in WS. Furthermore, TRF2, a protein essential for telomere protection, interacts with WRN and influences its basic helicase and exonuclease activities. However, these studies provided little insight into WRN's specific function at telomeres. Here, we explored the possibility that WRN and TRF2 cooperate during telomeric recombination processes. Our results indicate that TRF2, through its interactions with both WRN and telomeric DNA, stimulates WRN-mediated strand exchange specifically between telomeric substrates; TRF2's basic domain is particularly important for this stimulation. Although TRF1 binds telomeric DNA with similar affinity, it has minimal effects on WRN-mediated strand exchange of telomeric DNA. Moreover, TRF2 is displaced from telomeric DNA by WRN, independent of its ATPase and helicase activities. Together, these results suggest that TRF2 and WRN act coordinately during telomeric recombination processes, consistent with certain telomeric abnormalities associated with alteration of WRN function. PMID:24880691

Chemo-mechanical pushing of proteins along single-stranded DNA.

Science.gov (United States)

Sokoloski, Joshua E; Kozlov, Alexander G; Galletto, Roberto; Lohman, Timothy M

2016-05-31

Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3'-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5' to 3' ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5' to 3') pushing of the SSB toward the 3' ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3' to 5') direction, are observed with EcRep and EcUvrD (both 3' to 5' ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage, Senescence and Apoptosis in Colorectal Cancer Cells.

Directory of Open Access Journals (Sweden)

Aruna S Jaiswal

Full Text Available Recently approved chemotherapeutic agents to treat colorectal cancer (CRC have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β activity. Temozolomide (TMZ, an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715. In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC50 of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.

The European Nb3Sn advanced strand development programme

International Nuclear Information System (INIS)

Vostner, A.; Salpietro, E.

2005-01-01

Strands relevant for fusion with high critical current densities and moderate hysteresis losses were developed and already produced on industrial scale. Based on these achievements EFDA-CSU Garching has launched a Nb 3 Sn strand development and procurement action inside Europe in order to assess the current status of the Nb 3 Sn strand production capability. All six addressed companies have replied positively to the strand R and D programme which includes the three major Nb 3 Sn production techniques namely the bronze, internal-tin and powder-in-tube (PIT) route. According to the strand requirements for the ITER TF conductor a critical current density of 800 A/mm 2 (at 12 T, 4.2 K and 10 μV/m) and overall strand hysteresis losses below 500 kJ/m 3 have been specified as the minimum guaranteed strand performance. The second major objective of this programme is to motivate the strand manufacturers to develop and design high performance Nb 3 Sn strands optimised for the ITER conductor. For this purpose, a target critical current density of 1100 A/mm 2 has been added to the specification. This paper describes the strategy behind the strand development programme, the actual status of the strand production as well as first preliminary results obtained from the strand suppliers

DNA synthesis in vitro in human fibroblast preparations

Energy Technology Data Exchange (ETDEWEB)

Kaufmann, W.K.

1983-01-01

When confluent cultures of human fibroblasts were ultraviolet irradiated and either permeabilized or lysed, three types of DNA synthesis were subsequently observed during incubation in vitro: (A) a low level of DNA replication, which ceased after 15-30 min incubation at 37/sup 0/C; (B) radiation-dependent reparative gap-filling, which also ceased after 15 min at 37/sup 0/C; and (C) radiation-independent DNA synthesis, which was not semiconservative and proceeded at a linear rate for 1 hr at 37/sup 0/C. Normal and xeroderma pigmentosum fibroblasts displayed different rates of radiation-dependent reparative gap-filling after lysis but similar rates of radiation-independent DNA synthesis. The rates of DNA replication and radiation-independent DNA synthesis were less in the permeable cell system than in the lysed cell system, whereas radiation-dependent reparative gap-filling was the same in both. Preparations of permeable and lysed cells activated radiation-dependent reparative gap-filling at about 15% of the rate estimated for intact cells. No radiation-dependent DNA strand breaks, as assayed by alkaline elution, were observed in the lysed cell preparation. Some radiation-dependent breaks were observed in the permeable cell preparation, but radiation-dependent DNA breakage was less than that seen in intact cells. This inability to incise DNA at damaged sites could account for the low rate of activation of reparative gap-filling in vitro. DNA strand breaks were produced in fibroblast preparations nonspecifically during lysis or permeabilization and incubation in vitro, and this breakage of DNA probably was responsible for the radiation-independent DNA synthesis.

THE INFLUENCE OF INTERNALLY DISPLACED PERSONS ON THE SOCIAL AND ECONOMIC DEVELOPMENT OF REGIONS IN UKRAINE

Directory of Open Access Journals (Sweden)

Inna Arakelova

2017-12-01

Full Text Available In terms of the issues aggravation concerning internally displaced persons and the disproportions in the social and economic development of regions as a result of the antiterrorist operation in Ukraine, an analysis of social and economic transformations in regional development under the migration processes influence and the search for their overcoming mechanisms becomes relevant. The purpose of the article is to analyse the impact of internally displaced persons flows on the social and economic development of regions in order to improve the regional development mechanisms in Ukraine. The research methodology includes a set of scientific research methods that provide a systematic approach to investigating the impact of internally displaced persons on the social and economic development of Ukrainian regions, theoretical approaches to state regulation of internal migration flows in conditions of decentralization of power and reform of public administration in the country. In the course of investigations, the following methods are used: statistical analysis and synthesis while determining the indicators of the IDPs burden on the regional social and economic systems of Ukraine; analysis while studying the main problems of internally displaced persons and their directions; logical synthesis and synthesis while drawing conclusions. The information base for the study consists of the legislative acts of the Verkhovna Rada of Ukraine, the Decree of the Cabinet of Ministers of Ukraine, statistical and reporting information of the Ministry of Social Policy of Ukraine, development of the UN Agency for Refugees, the UN ReliefWeb Specialized Service for the Coordination of Humanitarian Affairs, Inter-Agency Standing Committee, PROMAN, NGO “Labor and Health Social Initiatives”, Ukrainian Helsinki Human Rights Union, research results of domestic and foreign scientists. Results. The article deals with the issues of effective reforming of regional development

Experimental Investigation on Embedding Strength Perpendicular to Grain of Parallel Strand Bamboo

Directory of Open Access Journals (Sweden)

Junwen Zhou

2018-01-01

Full Text Available Parallel strand bamboo (PSB is a latest construction material; to know more about mechanical properties of PSB, 5 groups of specimens with difference only in bolt diameter were designed to study the impact of the fastener diameter on embedding strength perpendicular to grain of PSB. Based on the tested result, the feasibility for PSB of the theoretical equation in the American code and European code on embedding strength predication was assessed. A controlled displacement was used to load till specimen failure, the stress-displacement curve of all specimens was obtained in terms of the tested results, and the yielding tested strength based on 5% bolt diameter offset proposed by the American code was found. The tested results showed that the yielding strength perpendicular to grain of PSB was stable, the variable coefficient was between 5.88% and 13.34%, and the average yielding strength values were 80.84 MPa, 77.40 MPa, 76.52 MPa, 74.20 MPa, and 67.01 MPa, respectively, which decreased with the increase of bolt diameter, and the average yielding strength values are larger than the calculated results using theoretical formula. Therefore, the theoretical equation on embedding yielding strength of wood in the American code and European code applies to PSB.

Displacement Parameter Inversion for a Novel Electromagnetic Underground Displacement Sensor

Directory of Open Access Journals (Sweden)

Nanying Shentu

2014-05-01

Full Text Available Underground displacement monitoring is an effective method to explore deep into rock and soil masses for execution of subsurface displacement measurements. It is not only an important means of geological hazards prediction and forecasting, but also a forefront, hot and sophisticated subject in current geological disaster monitoring. In previous research, the authors had designed a novel electromagnetic underground horizontal displacement sensor (called the H-type sensor by combining basic electromagnetic induction principles with modern sensing techniques and established a mutual voltage measurement theoretical model called the Equation-based Equivalent Loop Approach (EELA. Based on that work, this paper presents an underground displacement inversion approach named “EELA forward modeling-approximate inversion method”. Combining the EELA forward simulation approach with the approximate optimization inversion theory, it can deduce the underground horizontal displacement through parameter inversion of the H-type sensor. Comprehensive and comparative studies have been conducted between the experimentally measured and theoretically inversed values of horizontal displacement under counterpart conditions. The results show when the measured horizontal displacements are in the 0–100 mm range, the horizontal displacement inversion discrepancy is generally tested to be less than 3 mm under varied tilt angles and initial axial distances conditions, which indicates that our proposed parameter inversion method can predict underground horizontal displacement measurements effectively and robustly for the H-type sensor and the technique is applicable for practical geo-engineering applications.

Does trans-lesion synthesis explain the UV-radiation resistance of DNA synthesis in C.elegans embryos?

International Nuclear Information System (INIS)

Hartman, Phil; Reddy, Jennifer; Svendsen, Betty-Ann

1991-01-01

Over 10-fold larger fluences were required to inhibit both DNA synthesis and cell division in wild-type C.elegans embryos as compared with other model systems or C.elegans rad mutants. In addition, unlike in other organisms, the molecular weight of daughter DNA strands was reduced only after large, superlethal fluences. The molecular weight of nascent DNA fragments exceeded the interdimer distance by up to 19-fold, indicating that C.elegans embryos can replicate through non-instructional lesions. This putative trans-lesion synthetic capability may explain the refractory nature of UV-radiation on embryonic DNA synthesis and nuclear division in C.elegans. (author). 42 refs.; 7 figs

Does trans-lesion synthesis explain the UV-radiation resistance of DNA synthesis in C. elegans embryos

Energy Technology Data Exchange (ETDEWEB)

Hartman, Phil; Reddy, Jennifer; Svendsen, Betty-Ann [Texas Christian Univ., Fort Worth, TX (United States). Dept. of Biology

1991-09-01

Over 10-fold larger fluences were required to inhibit both DNA synthesis and cell division in wild-type C.elegans embryos as compared with other model systems or C.elegans rad mutants. In addition, unlike in other organisms, the molecular weight of daughter DNA strands was reduced only after large, superlethal fluences. The molecular weight of nascent DNA fragments exceeded the interdimer distance by up to 19-fold, indicating that C.elegans embryos can replicate through non-instructional lesions. This putative trans-lesion synthetic capability may explain the refractory nature of UV-radiation on embryonic DNA synthesis and nuclear division in C.elegans. (author). 42 refs.; 7 figs.

[Intra-articular injections of hyaluronic acid for anterior disc displacement of temporomandibular joint].

Science.gov (United States)

Long, X

2017-03-09

Anterior disc displacement (ADD) of temporomandibular joint (TMJ) is regarded as one of the major findings in temporomandibular disorders (TMD). It is related to joint noise, pain, mandibular dysfunction, degenerative change and osteoarthritis. In the mean time, the pathological changes were found in synovial membrane and synovial fluid. Hyaluronic acid is a principal component of the synovial fluid which plays an important role in nutrition, lubrication, anti-inflammation and cartilage repair. The synthesis, molecule weight, and concentration of hyaluronic acid are decreased during TMD and cause TMJ degenerative changes. The clinical conditions, pathological changes, the mechanism of action for hyaluronic acid and the treatment of anterior disc displacement of TMJ are discussed in this article.

Displacement ventilation

DEFF Research Database (Denmark)

Kosonen, Risto; Melikov, Arsen Krikor; Mundt, Elisabeth

The aim of this Guidebook is to give the state-of-the art knowledge of the displacement ventilation technology, and to simplify and improve the practical design procedure. The Guidebook discusses methods of total volume ventilation by mixing ventilation and displacement ventilation and it gives...... insights of the performance of the displacement ventilation. It also shows practical case studies in some typical applications and the latest research findings to create good local micro-climatic conditions....

DNA strand breaks, repair, and survival in x-irradiated mammalian cells

International Nuclear Information System (INIS)

Dugle, D.L.; Gillespie, C.J.; Chapman, J.D.

1976-01-01

The yields of unrepairable single- and double-strand breaks in the DNA of x-irradiated Chinese hamster cells were measured by low-speed neutral and alkaline sucrose density gradient sedimentation in order to investigate the relation between these lesions and reproductive death. After maximal single-strand rejoining, at all doses, the number of residual single-strand breaks was twice the number of residual double-strand breaks. Both double-strand and unrepairable single-strand breaks were proportional to the square of absorbed dose, in the range 10-50 krad. No rejoining of double-strand breaks was observed. These observations suggest that, in mammalian cells, most double-strand breaks are not repairable, while all single-strand breaks are repaired except those that are sufficiently close on complementary strands to constitute double-strand breaks. Comparison with cell survival measurements at much lower doses suggests that loss of reproductive capacity corresponds to induction of approximately one double-strand break

Synthesis and optical resolution of a Cu(I) double-stranded helicate with ketimine-bridged tris(bipyridine) ligands.

Science.gov (United States)

Furusho, Yoshio; Goto, Hidetoshi; Itomi, Ken; Katagiri, Hiroshi; Miyagawa, Toyoharu; Yashima, Eiji

2011-09-21

A tetranuclear Cu(I) double-stranded helicate was synthesized from ketimine-bridged tris(bipyridine) ligands and Cu(I) ions, and the racemate was successfully resolved by diastereomeric salt formation using an optically pure phosphate anion followed by anion exchange with NaPF(6) without racemization.

Detecting single-abasic residues within a DNA strand immobilized in a biological nanopore using an integrated CMOS sensor.

Science.gov (United States)

Kim, Jungsuk; Maitra, Raj D; Pedrotti, Ken; Dunbar, William B

2013-02-01

In this paper, we demonstrate the application of a novel current-measuring sensor (CMS) customized for nanopore applications. The low-noise CMS is fabricated in a 0.35μm CMOS process and is implemented in experiments involving DNA captured in an α-hemolysin (α-HL) nanopore. Specifically, the CMS is used to build a current amplitude map as a function of varying positions of a single-abasic residue within a homopolymer cytosine single-stranded DNA (ssDNA) that is captured and held in the pore. Each ssDNA is immobilized using a biotin-streptavidin linkage. Five different DNA templates are measured and compared: one all-cytosine ssDNA, and four with a single-abasic residue substitution that resides in or near the ~1.5nm aperture of the α-HL channel when the strand is immobilized. The CMOS CMS is shown to resolves the ~5Å displacements of the abasic residue within the varying templates. The demonstration represents an advance in application-specific circuitry that is optimized for small-footprint nanopore applications, including genomic sequencing.

Job Displacement and Crime

DEFF Research Database (Denmark)

Bennett, Patrick; Ouazad, Amine

We use a detailed employer-employee data set matched with detailed crime information (timing of crime, fines, convictions, crime type) to estimate the impact of job loss on an individual's probability to commit crime. We focus on job losses due to displacement, i.e. job losses in firms losing...... a substantial share of their workers, for workers with at least three years of tenure. Displaced workers are more likely to commit offenses leading to conviction (probation, prison terms) for property crimes and for alcohol-related traffic violations in the two years following displacement. We find no evidence...... that displaced workers' propensity to commit crime is higher than non-displaced workers before the displacement event; but it is significantly higher afterwards. Displacement impacts crime over and above what is explained by earnings losses and weeks of unemployment following displacement....

Comparison of the Rate and Extent of Deoxyribonucleic Acid Repair and Semi-Conservative Synthesis in Bacteria Exposed to Ultra-Violet Light

Energy Technology Data Exchange (ETDEWEB)

Billen, D. [Radiation Biology Laboratory and Departments of Microbiology and Radiology, College of Medicine, University of Florida, Gainesville, FL (United States)

1968-08-15

Many bacterial strains possess the ability to repair genetic damage resulting from ultra-violet light (u.v. ) exposure. Of major importance is the occurrence of a 'repair' type of deoxyribonucleic acid (DNA) replication during 'dark repair', which presumably results in the replacement of the damaged portion of the genome. With deuterium, {sup 15}N and {sup 13}C as a density label, and buoyant density centrifugation in CsCl as a means of separating pre and post-irradiation synthesized DNA strands, the rate and extent of DNA repair synthesis in exponential - phase Escherichia coli strain B/r were determined. After u.v. exposure, {sup 3}H-thymine incorporation into the 'heavy' parental DNA strands was used to measure repair synthesis, while {sup 3}H-thymine incorporation into 'light' and newly synthesized DNA strands measured semi-conservative replication. The rate of bases incorporated by repair synthesis in the initial 15 minures of post-irradiation incubation at 37 Degree-Sign C appears to be saturated at a dose of approximately 100 ergs/mm{sup 2}. At higher doses (up to 600 ergs/mm{sup 2}) the increase observed was not proportional to dose. During this initial 15 minutes, less than 1% of the chromosomal DNA was replaced. The amount of DNA synthesized by semi-conservative replication during the initial 15 minutes was reduced with increasing u.v. dose. After exposure to 600 ergs/mm{sup 2}, repair and semiconservative DNA synthesis were nearly equivalent in the irradiated cells after 15 minutes of incubation. Repair synthesis was observed to be terminated by 45 minutes in bacteria exposed to 160 or 500 ergs/mm{sup 2} (64% and 10% survivors, respectively). The amount of genome replaced by repair synthesis at several doses was determined. Starvation for a required amino acid (resulting in an inhibition of protein and ribonucleic acid synthesis) did not prevent the repair synthesis nor grossly alter its extent. The restoration of the semi-conservative mo d e of DNA

Southeast US Historical Marine Mammal Stranding Database

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — Data on marine mammal strandings are collected by the Southeast Marine Mammal Stranding Network. Basic data on the location, species identification, animal...

The stranding anomaly as population indicator

DEFF Research Database (Denmark)

Peltier, Helene; Baagøe, Hans J.; Camphuysen, Kees C. J.

2013-01-01

Ecological indicators for monitoring strategies are expected to combine three major characteristics: ecological significance, statistical credibility, and cost-effectiveness. Strategies based on stranding networks rank highly in cost-effectiveness, but their ecological significance and statistica...... surveys, mostly SCANS surveys (1994 and 2005). This new indicator could be applied to cetacean populations across the world and more widely to marine megafauna....... credibility are disputed. Our present goal is to improve the value of stranding data as population indicator as part of monitoring strategies by constructing the spatial and temporal null hypothesis for strandings. The null hypothesis is defined as: small cetacean distribution and mortality are uniform...

Formation of double-strand breaks in DNA of γ-irradiated bacteria depending on the function of fast repair processes of DNA single-strand breaks

International Nuclear Information System (INIS)

Petrov, S.I.; Gaziev, A.I.

1980-01-01

The formation of double-strand breaks in DNA of γ-irradiated ( 60 Co)Ex coli bacteria depending on the function of fast repair processes of DNA single-strand breaks, is investigated. The profiles of sedimentation of DNA Ex coli cells, irradiated at 0-2 deg C in the salt medium and in EDTA-borate buffer, are presented. It is shown that when irradiating cells in EDTA-borate buffer, the output of single- and double strand breaks in DNA is much higher than in the case of their irradiation in the minimum salt medium. The dependence of output of single-strand and double-strand breaks depending on the radiatier doze of E coli cells in the salt medium and EDTA-borate buffer, is studied. The supposition is made on the presence of a regulative interaction between the accumulation of DNA single-breaks and their repair with the formation of double-strand breaks. The functionating of fast and superfast repair processes considerably affects the formation of double-strand breaks in DNA of a bacterium cell. A considerable amount of double-breaks registered immediately after irradiation forms due to a close position of single-strand breaks on the opposite DNA strands

Displacement Ventilation

DEFF Research Database (Denmark)

Nielsen, Peter Vilhelm

Displacement ventilation is an interesting new type of air distribution principle which should be considered in connection with design of comfort ventilation in both smal1 and large spaces. Research activities on displacement ventilation are large all over the world and new knowledge of design...... methods appears continuously. This book gives an easy introduction to the basis of displacement ventilation and the chapters are written in the order which is used in a design procedure. The main text is extended by five appendices which show some of the new research activities taking place at Aalborg...

TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA.

Science.gov (United States)

Flynn, Rachel Litman; Centore, Richard C; O'Sullivan, Roderick J; Rai, Rekha; Tse, Alice; Songyang, Zhou; Chang, Sandy; Karlseder, Jan; Zou, Lee

2011-03-24

Maintenance of telomeres requires both DNA replication and telomere 'capping' by shelterin. These two processes use two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomeres 1 (POT1). Although RPA and POT1 each have a critical role at telomeres, how they function in concert is not clear. POT1 ablation leads to activation of the ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase at telomeres, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. Unexpectedly, we found that purified POT1 and its functional partner TPP1 are unable to prevent RPA binding to telomeric ssDNA efficiently. In cell extracts, we identified a novel activity that specifically displaces RPA, but not POT1, from telomeric ssDNA. Using purified protein, here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) recapitulates the RPA displacing activity. The RPA displacing activity is inhibited by the telomeric repeat-containing RNA (TERRA) in early S phase, but is then unleashed in late S phase when TERRA levels decline at telomeres. Interestingly, TERRA also promotes POT1 binding to telomeric ssDNA by removing hnRNPA1, suggesting that the re-accumulation of TERRA after S phase helps to complete the RPA-to-POT1 switch on telomeric ssDNA. Together, our data suggest that hnRNPA1, TERRA and POT1 act in concert to displace RPA from telomeric ssDNA after DNA replication, and promote telomere capping to preserve genomic integrity.

Alaska Beluga Strandings

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — NMFS is mandated by the Marine Mammal Protection Act (MMPA) to collect reports of marine mammal stranding events. This particular catalogue item is specific to the...

Low-residue euthanasia of stranded mysticetes.

Science.gov (United States)

Harms, Craig A; McLellan, William A; Moore, Michael J; Barco, Susan G; Clarke, Elsburgh O; Thayer, Victoria G; Rowles, Teresa K

2014-01-01

Euthanasia of stranded large whales poses logistic, safety, pharmaceutical, delivery, public relations, and disposal challenges. Reasonable arguments may be made for allowing a stranded whale to expire naturally. However, slow cardiovascular collapse from gravitational effects outside of neutral buoyancy, often combined with severely debilitating conditions, motivate humane efforts to end the animal's suffering. The size of the animal and prevailing environmental conditions often pose safety concerns for stranding personnel, which take priority over other considerations. When considering chemical euthanasia, the size of the animal also necessitates large quantities of euthanasia agents. Drug residues are a concern for relay toxicity to scavengers, particularly for pentobarbital-containing euthanasia solutions. Pentobarbital is also an environmental concern because of its stability and long persistence in aquatic environments. We describe a euthanasia technique for stranded mysticetes using readily available, relatively inexpensive, preanesthetic and anesthetic drugs (midazolam, acepromazine, xylazine) followed by saturated KCl delivered via custom-made needles and a low-cost, basic, pressurized canister. This method provides effective euthanasia while moderating personnel exposure to hazardous situations and minimizing drug residues of concern for relay toxicity.

Stranded Specimen Data

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — The Marine Mammal and Turtle Division collects data about individual cetaceans and sea turtles that come ashore, or strand on the beach. The date and location of...

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

Science.gov (United States)

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.

Current redistribution in cables made of insulated, soldered, or oxidized strands

International Nuclear Information System (INIS)

Turck, B.

1979-07-01

Current redistributions are compared in cables made of insulated strands, soldered, or oxidized strands and insulated strands with periodic joints. After discussing the different current redistributions in the cases of a rapidly changing current and a dc current, several particular situations are investigated: what happens if a strand is broken, or if a local normal zone appears that does not affect all the strands equally, the detection of this normal zone, and the influence of short circuits between strands

The contribution of mitochondrial thymidylate synthesis in preventing the nuclear genome stress

OpenAIRE

Lee, Ming-Hsiang; Wang, Liya; Chang, Zee-Fen

2014-01-01

In quiescent fibroblasts, the expression levels of cytosolic enzymes for thymidine triphosphate (dTTP) synthesis are down-regulated, causing a marked reduction in the dTTP pool. In this study, we provide evidence that mitochondrial thymidylate synthesis via thymidine kinase 2 (TK2) is a limiting factor for the repair of ultraviolet (UV) damage in the nuclear compartment in quiescent fibroblasts. We found that TK2 deficiency causes secondary DNA double-strand breaks formation in the nuclear ge...

Strand SPA & Konverentsikeskus

Index Scriptorium Estoniae

2008-01-01

Strand SPA & Konverentsikeskus on Pärnu suurim äri- ja konverentsiklientidele suunatud hotell, mis klientide seas on hinnatud just selle kompleksuse tõttu, kuna kõik, mida külaline vajab ja soovib, on olemas ühe katuse all

Development of strand burner for solid propellant burning rate studies

International Nuclear Information System (INIS)

Aziz, A; Mamat, R; Ali, W K Wan

2013-01-01

It is well-known that a strand burner is an apparatus that provides burning rate measurements of a solid propellant at an elevated pressure in order to obtain the burning characteristics of a propellant. This paper describes the facilities developed by author that was used in his studies. The burning rate characteristics of solid propellant have be evaluated over five different chamber pressures ranging from 1 atm to 31 atm using a strand burner. The strand burner has a mounting stand that allows the propellant strand to be mounted vertically. The strand was ignited electrically using hot wire, and the burning time was recorded by electronic timer. Wire technique was used to measure the burning rate. Preliminary results from these techniques are presented. This study shows that the strand burner can be used on propellant strands to obtain accurate low pressure burning rate data

Chlamydomonas chloroplasts can use short dispersed repeats and multiple pathways to repair a double-strand break in the genome.

Science.gov (United States)

Odom, Obed W; Baek, Kwang-Hyun; Dani, Radhika N; Herrin, David L

2008-03-01

Certain group I introns insert into intronless DNA via an endonuclease that creates a double-strand break (DSB). There are two models for intron homing in phage: synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR). The Cr.psbA4 intron homes efficiently from a plasmid into the chloroplast psbA gene in Chlamydomonas, but little is known about the mechanism. Analysis of co-transformants selected using a spectinomycin-resistant 16S gene (16S(spec)) provided evidence for both pathways. We also examined the consequences of the donor DNA having only one-sided or no homology with the psbA gene. When there was no homology with the donor DNA, deletions of up to 5 kb involving direct repeats that flank the psbA gene were obtained. Remarkably, repeats as short as 15 bp were used for this repair, which is consistent with the single-strand annealing (SSA) pathway. When the donor had one-sided homology, the DSB in most co-transformants was repaired using two DNAs, the donor and the 16S(spec) plasmid, which, coincidentally, contained a region that is repeated upstream of psbA. DSB repair using two separate DNAs provides further evidence for the SDSA pathway. These data show that the chloroplast can repair a DSB using short dispersed repeats located proximally, distally, or even on separate molecules relative to the DSB. They also provide a rationale for the extensive repertoire of repeated sequences in this genome.

Enhancement of RNA synthesis by promoter duplication in tombusviruses

International Nuclear Information System (INIS)

Panavas, T.; Panaviene, Z.; Pogany, J.; Nagy, P.D.

2003-01-01

Replication of tombusviruses, small plus-strand RNA viruses of plants, is regulated by cis-acting elements present in the viral RNA. The role of cis-acting elements can be studied in vitro by using a partially purified RNA-dependent RNA polymerase (RdRp) preparation obtained from tombusvirus-infected plants , Virology 276, 279- 288). Here, we demonstrate that the minus-strand RNA of tombusviruses contains, in addition to the 3'-terminal minimal plus-strand initiation promoter, a second cis-acting element, termed the promoter proximal enhancer (PPE). The PPE element enhanced RNA synthesis by almost threefold from the adjacent minimal promoter in the in vitro assay. The sequence of the PPE element is 70% similar to the minimal promoter, suggesting that sequence duplication of the minimal promoter may have been the mechanism leading to the generation of the PPE. Consistent with this proposal, replacement of the PPE element with the minimal promoter, which resulted in a perfectly duplicated promoter region, preserved its enhancer-like function. In contrast, mutagenesis of the PPE element or its replacement with an artificial G/C-rich sequence abolished its stimulative effect on initiation of RNA synthesis in vitro. In vivo experiments are also consistent with the role of the PPE element in enhancement of tombusvirus replication. Sequence comparison of several tombusviruses and related carmoviruses further supports the finding that duplication of minimal promoter sequences may have been an important mechanism during the evolution of cis-acting elements in tombusviruses and related RNA viruses

Displacement characteristics of a piezoactuator-based prototype microactuator with a hydraulic displacement amplification system

Energy Technology Data Exchange (ETDEWEB)

Muralidhara [NMAMIT, Nitte (India); Rao, Rathnamala [NITK, Surathkal (India)

2015-11-15

In this study, a new piezoactuator-based prototype microactuator is proposed with a hydraulic displacement amplification system. A piezoactuator is used to deflect a diaphragm which displaces a certain volume of hydraulic fluid into a smaller-diameter piston chamber, thereby amplifying the displacement at the other end of the piston. An electro-mechanical model is implemented to estimate the displacement of a multilayer piezoelectric actuator for the applied input voltage considering the hysteresis behavior. The displacement characteristics of the proposed microactuator are studied for triangular actuation voltage signal. Results of the experiments and simulation of the displacement behavior of the stacked piezoactuator and the amplified displacement of the prototype actuator were compared. Experimental results suggest that the mathematical model developed for the new piezoactuator-based prototype actuator is capable of estimating its displacement behavior accurately, within an error of 1.2%.

Displacement characteristics of a piezoactuator-based prototype microactuator with a hydraulic displacement amplification system

International Nuclear Information System (INIS)

Muralidhara; Rao, Rathnamala

2015-01-01

In this study, a new piezoactuator-based prototype microactuator is proposed with a hydraulic displacement amplification system. A piezoactuator is used to deflect a diaphragm which displaces a certain volume of hydraulic fluid into a smaller-diameter piston chamber, thereby amplifying the displacement at the other end of the piston. An electro-mechanical model is implemented to estimate the displacement of a multilayer piezoelectric actuator for the applied input voltage considering the hysteresis behavior. The displacement characteristics of the proposed microactuator are studied for triangular actuation voltage signal. Results of the experiments and simulation of the displacement behavior of the stacked piezoactuator and the amplified displacement of the prototype actuator were compared. Experimental results suggest that the mathematical model developed for the new piezoactuator-based prototype actuator is capable of estimating its displacement behavior accurately, within an error of 1.2%.

Two modes of interaction of the single-stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase-thioredoxin complex

KAUST Repository

Ghosh, Sharmistha; Hamdan, Samir; Richardson, Charles C.

2010-01-01

The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD.gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Two modes of interaction of the single-stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase-thioredoxin complex

KAUST Repository

Ghosh, Sharmistha

2010-04-06

The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD.gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Measuring vulnerability to disaster displacement

Science.gov (United States)

Brink, Susan A.; Khazai, Bijan; Power, Christopher; Wenzel, Friedemann

2015-04-01

Large scale disasters can cause devastating impacts in terms of population displacement. Between 2008 and 2013, on average 27 million people were displaced annually by disasters (Yonetani 2014). After large events such as hurricane Katrina or the Port-au-Prince earthquake, images of inadequate public shelter and concerns about large scale and often inequitable migration have been broadcast around the world. Population displacement can often be one of the most devastating and visible impacts of a natural disaster. Despite the importance of population displacement in disaster events, measures to understand the socio-economic vulnerability of a community often use broad metrics to estimate the total socio-economic risk of an event rather than focusing on the specific impacts that a community faces in a disaster. Population displacement is complex and multi-causal with the physical impact of a disaster interacting with vulnerability arising from the response, environmental issues (e.g., weather), cultural concerns (e.g., expectations of adequate shelter), and many individual factors (e.g., mobility, risk perception). In addition to the complexity of the causes, population displacement is difficult to measure because of the wide variety of different terms and definitions and its multi-dimensional nature. When we speak of severe population displacement, we may refer to a large number of displaced people, an extended length of displacement or associated difficulties such as poor shelter quality, risk of violence and crime in shelter communities, discrimination in aid, a lack of access to employment or other difficulties that can be associated with large scale population displacement. We have completed a thorough review of the literature on disaster population displacement. Research has been conducted on historic events to understand the types of negative impacts associated with population displacement and also the vulnerability of different groups to these impacts. We

Double-Stranded Peptide Nucleic Acids

DEFF Research Database (Denmark)

2001-01-01

A novel class of compounds, known as peptide nucleic acids, form double-stranded structures with one another and with ssDNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker.......A novel class of compounds, known as peptide nucleic acids, form double-stranded structures with one another and with ssDNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

The (not so) immortal strand hypothesis.

Science.gov (United States)

Tomasetti, Cristian; Bozic, Ivana

2015-03-01

Non-random segregation of DNA strands during stem cell replication has been proposed as a mechanism to minimize accumulated genetic errors in stem cells of rapidly dividing tissues. According to this hypothesis, an "immortal" DNA strand is passed to the stem cell daughter and not the more differentiated cell, keeping the stem cell lineage replication error-free. After it was introduced, experimental evidence both in favor and against the hypothesis has been presented. Using a novel methodology that utilizes cancer sequencing data we are able to estimate the rate of accumulation of mutations in healthy stem cells of the colon, blood and head and neck tissues. We find that in these tissues mutations in stem cells accumulate at rates strikingly similar to those expected without the protection from the immortal strand mechanism. Utilizing an approach that is fundamentally different from previous efforts to confirm or refute the immortal strand hypothesis, we provide evidence against non-random segregation of DNA during stem cell replication. Our results strongly suggest that parental DNA is passed randomly to stem cell daughters and provides new insight into the mechanism of DNA replication in stem cells. Copyright © 2015. Published by Elsevier B.V.

DNA strand breakage by 125I-decay in oligoDNA

International Nuclear Information System (INIS)

Lobachevsky, P.; Martin, R.F.

1996-01-01

Full text: A double-stranded oligodeoxynucleotide containing 125 I-dC in a defined location, with 5'- or 3'- 32 P-end-labelling of either strand, was used to investigate DNA strand breakage resulting from 125 I decay. Samples of the 32 P-end-labelled and 125 I-dC containing oligoDNA were incubated in 20 mM phosphate buffer (PB), or PB + 2 M dimethylsulphoxide (DMSO) at 4 deg during 18-20 days. The 32 P-end-labelled DNA fragments produced by 125 I decays were separated on denaturing polyacrylamide gels, and the 3P activity in each fragment was determined by scintillation counting after elution from the gel. The fragment size distribution was then converted to a distribution of single stranded break probabilities at each nucleotide position. The results indicate that each 125 I decay event produces at least one break in the 125 I-dC containing strand, and causes breakage of the opposite strand in 75-80% of events. Thus, the double stranded break is produced by 125 I decay with probability ∼0.8. Most of single stranded breaks (around 90%) occurred within 5-6 nucleotides of the 125 I-dC, however DNA breaks were detected up to 18-20 nucleotides from the decay site. The average numbers of single stranded breaks per decay are 3.7 (PB) and 3.3 (PB+DMSO) in 125 I-dC containing strand, and 1.5 (PB) and 1.3 (PB+DMSO) in the opposite strand. Deconvolution of strand break probabilities as a function of separation from the 125 I, in terms of both distance (to target deoxyribosyl carbon atoms, in B-DNA) and nucleotide number, show that the latter is an important parameter for the shorter-range damage. This could indicate a role for attenuation/dissipation of damage through the stacked bases. In summary, the results represent a much more extensive set of data than available from earlier experiments on DNA breakage from l25 I-decay, and may provide new mechanistic insights

Resonant neutron-induced atomic displacements

Energy Technology Data Exchange (ETDEWEB)

Elmaghraby, Elsayed K., E-mail: e.m.k.elmaghraby@gmail.com

2017-05-01

Highlights: • Neutron induced atomic displacements was investigated based on scattering of energy of neutron. • Model for cascade function (multiplication of displacements with increasing energy transfer) was proposed and justified. • Parameterizations for the dpa induced in all elements were performed. • Table containing all necessary parameters to calculate the displacement density induced by neutron is given. • Contribution of non resonance displacement and resonant-neutron induced displacements are distinguished. - Abstract: A model for displacement cascade function was modified to account for the continuous variation of displacement density in the material in response to neutron exposure. The model is based on the Gaussian distribution of displacement energies of atoms in a material. Analytical treatment for moderated epithermal neutron field was given in which the displacement density was divided into two terms, discrete-resonance term and continuum term. Calculation are done for all isotopes using ENDF/B VII.1 data files and temperature dependent cross section library. Weighted elemental values were reported a fitting was performed to obtain energy-dependent formula of displacement density and reduce the number of parameters. Results relevant the present specification of the cascade function are tabulated for each element to enable calculation of displacement density at any value of displacement energy in the between 5 eV and 55 eV.

Enzymatic Production of Monoclonal Stoichiometric Single-Stranded DNA Oligonucleotides

Science.gov (United States)

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

Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli

International Nuclear Information System (INIS)

Moreau, P.L.

1988-01-01

Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes. (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected. These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein. However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair. Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA

Southeast Region Level A Marine Mammal Stranding Data

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — Data on marine mammal strandings are collected by the Southeast Marine Mammal Stranding Network. Basic data on the location, species identification, animal...

Tissue strands as "bioink" for scale-up organ printing.

Science.gov (United States)

Yu, Yin; Ozbolat, Ibrahim T

2014-01-01

Organ printing, takes tissue spheroids as building blocks together with additive manufacturing technique to engineer tissue or organ replacement parts. Although a wide array of cell aggregation techniques has been investigated, and gained noticeable success, the application of tissue spheroids for scale-up tissue fabrication is still worth investigation. In this paper, we introduce a new micro-fabrication technique to create tissue strands at the scale of 500-700μm as a "bioink" for future robotic tissue printing. Printable alginate micro-conduits are used as semi-permeable capsules for tissue strand fabrication. Mouse insulinoma beta TC3 cell tissue strands were formed upon 4 days post fabrication with reasonable mechanical strength, high cell viability close to 90%, and tissue specific markers expression. Fusion was readily observed between strands when placing them together as early as 24h. Also, tissue strands were deposited with human umbilical vein smooth muscle cells (HUVSMCs) vascular conduits together to fabricated miniature pancreatic tissue analog. Our study provided a novel technique using tissue strands as "bioink" for scale-up bioprinting of tissues or organs.

A methodology to identify stranded generation facilities and estimate stranded costs for Louisiana's electric utility industry

Science.gov (United States)

Cope, Robert Frank, III

1998-12-01

The electric utility industry in the United States is currently experiencing a new and different type of growing pain. It is the pain of having to restructure itself into a competitive business. Many industry experts are trying to explain how the nation as a whole, as well as individual states, will implement restructuring and handle its numerous "transition problems." One significant transition problem for federal and state regulators rests with determining a utility's stranded costs. Stranded generation facilities are assets which would be uneconomic in a competitive environment or costs for assets whose regulated book value is greater than market value. At issue is the methodology which will be used to estimate stranded costs. The two primary methods are known as "Top-Down" and "Bottom-Up." The "Top-Down" approach simply determines the present value of the losses in revenue as the market price for electricity changes over a period of time into the future. The problem with this approach is that it does not take into account technical issues associated with the generation and wheeling of electricity. The "Bottom-Up" approach computes the present value of specific strandable generation facilities and compares the resulting valuations with their historical costs. It is regarded as a detailed and difficult, but more precise, approach to identifying stranded assets and their associated costs. This dissertation develops a "Bottom-Up" quantitative, optimization-based approach to electric power wheeling within the state of Louisiana. It optimally evaluates all production capabilities and coordinates the movement of bulk power through transmission interconnections of competing companies in and around the state. Sensitivity analysis to this approach is performed by varying seasonal consumer demand, electric power imports, and transmission inter-connection cost parameters. Generation facility economic dispatch and transmission interconnection bulk power transfers, specific

Job Displacement and Crime

DEFF Research Database (Denmark)

Bennett, Patrick; Ouazad, Amine

theory of crime. Marital dissolution is more likely post-displacement, and we find small intra-family externalities of adult displacement on younger family members’ crime. The impact of displacement on crime is stronger in municipalities with higher capital and labor income inequalities....

Effect of Twist Pitch in the Strands on the Saturation and Losses in the Nb3Sn Strands for the ITER TF CICC

International Nuclear Information System (INIS)

Martovetsky, N.N.

2007-01-01

ITER TF coils will see a significant longitudinal magnetic field in the event of the plasma disruption. This abrupt change of magnetic fields results in the appearance of an additional electrical field in the strands. The mechanism of this electrical field is the induced currents that expel the flux from the strands. This effect was known since the late 1970's [1-3] and most of the details necessary for the analyses given in this report are presented in [4]. Let's assume for simplicity a zero transport current in the strand. When a longitudinal pulsed field is applied, the outer filaments will carry an induced current repelling the change of flux. The current density of this current is 'critical' in the simplification of Bean's critical state model, where superconducting transition is represented as j=j c at any non-zero electrical field and zero where the electrical field has not penetrated. In reality, since the current density is roughly logarithmic with the electrical field, E=E c *exp[(j-j c )/jo], Bean's model is just a simplification, and current density is slightly nonuniform in the outer filament and more so for the interior strands. The inner portion of the filaments will carry a current of the opposite sign. Even in the Bean's model it is not uniform, but the assumption that it is uniform and less than critical simplifies mathematics significantly and does not deviate far from the real current density distribution. In certain circumstances, the average electrical field in the strands will be high enough to exceed the take-off electrical field averaged across the cross section. In this case, the multifilamentary strand will become unstable and will experience transition to the normal state. With zero transport current, it will eventually recover, of course. This phenomenon is analogous to the flux jump. If the strand carries a transport current, the situation becomes more complicated. If it goes unstable and the transport current is higher than the

Studies on the interaction of the food colorant tartrazine with double stranded deoxyribonucleic acid.

Science.gov (United States)

Basu, Anirban; Suresh Kumar, Gopinatha

2016-05-01

Interaction of the food additive tartrazine with double-stranded DNA was studied by spectroscopic and calorimetric techniques. Absorbance studies revealed that tartrazine exhibited hypochromism in the presence of DNA without any bathochromic effects. Minor groove displacement assay of DAPI and Hoechst 33258 suggested that tartrazine binds in the minor groove of DNA. The complexation was predominantly entropy driven with a smaller but favorable enthalpic contribution to the standard molar Gibbs energy. The equilibrium constant was evaluated to be (3.68 ± .08) × 10(4) M(-1) at 298.15 K. The negative standard molar heat capacity value along with an enthalpy-entropy compensation phenomenon proposed the involvement of dominant hydrophobic forces in the binding process. Tartrazine enhanced the thermal stability of DNA by 7.53 K under saturation conditions.

Inhibition of HIV-1 reverse transcriptase-catalyzed synthesis by intercalated DNA Benzo[a]Pyrene 7,8-Dihydrodiol-9,10-Epoxide adducts.

Directory of Open Access Journals (Sweden)

Parvathi Chary

Full Text Available To aid in the characterization of the relationship of structure and function for human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT, this investigation utilized DNAs containing benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE-modified primers and templates as a probe of the architecture of this complex. BPDE lesions that differed in their stereochemistry around the C10 position were covalently linked to N (6-adenine and positioned in either the primer or template strand of a duplex template-primer. HIV-1 RT exhibited a stereoisomer-specific and strand-specific difference in replication when the BPDE-lesion was placed in the template versus the primer strand. When the C10 R-BPDE adduct was positioned in the primer strand in duplex DNA, 5 nucleotides from the 3΄ end of the primer terminus, HIV-1 RT could not fully replicate the template, producing truncated products; this block to further synthesis did not affect rates of dissociation or DNA binding affinity. Additionally, when the adducts were in the same relative position, but located in the template strand, similar truncated products were observed with both the C10 R and C10 S BPDE adducts. These data suggest that the presence of covalently-linked intercalative DNA adducts distant from the active site can lead to termination of DNA synthesis catalyzed by HIV-1 RT.

Fragmentation in DNA double-strand breaks

International Nuclear Information System (INIS)

Wei Zhiyong; Suzhou Univ., Suzhou; Zhang Lihui; Li Ming; Fan Wo; Xu Yujie

2005-01-01

DNA double strand breaks are important lesions induced by irradiations. Random breakage model or quantification supported by this concept is suitable to analyze DNA double strand break data induced by low LET radiation, but deviation from random breakage model is more evident in high LET radiation data analysis. In this work we develop a new method, statistical fragmentation model, to analyze the fragmentation process of DNA double strand breaks. After charged particles enter the biological cell, they produce ionizations along their tracks, and transfer their energies to the cells and break the cellular DNA strands into fragments. The probable distribution of the fragments is obtained under the condition in which the entropy is maximum. Under the approximation E≅E 0 + E 1 l + E 2 l 2 , the distribution functions are obtained as exp(αl + βl 2 ). There are two components, the one proportional to exp(βl 2 ), mainly contributes to the low mass fragment yields, the other component, proportional to exp(αl), decreases slowly as the mass of the fragments increases. Numerical solution of the constraint equations provides parameters α and β. Experimental data, especially when the energy deposition is higher, support the statistical fragmentation model. (authors)

Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA.

Science.gov (United States)

Darlix, J L; Vincent, A; Gabus, C; de Rocquigny, H; Roques, B

1993-08-01

Two DNA strand transfer reactions take place during reverse transcription of the retroviral genome. The first transfer, that of the minus-strand strong stop DNA from the 5' end of the viral RNA to the 3' end, has been studied in vitro with two RNAs mimicking the 5' and 3' regions of the HIV1 genome and with nucleocapsid protein, NCp7, and reverse transcriptase. The results show that NCp7 strongly activates the 5' to 3' DNA strand transfer during reverse transcription while a basic peptide resembling NCp7 is inactive. Activation of the first transfer by several NCp7 derived peptides and the influence of the terminal redundancies (R) present at the 5' and 3' ends of HIV1 RNA were also examined. The first transfer is optimal in the presence of intact NCp7 and necessitates R on both the 5' and 3' RNAs. Sequencing of full length viral DNA products reveals approximately 40% misincorporations at the first nucleotide beyond the transfer point. If such base misincorporations occur during proviral DNA synthesis with possible homologous recombinations it may well contribute to the high level of genetic variability of HIV.

Manufacture of Strand Board Bonded with Disposal Expanded Polystyrene as Binder

OpenAIRE

Hermawan, Andi; Ohuchi, Takeshi; Fujimoto, Noboru; 大内, 毅; 藤本, 登留

2010-01-01

The objective of this study was to evaluate the physical and mechanical properties of strand board bonded with disposal expanded polystyrene (EPS) as binder. The strand board was manufactured using strand made from Douglas-fir beams selected from construction scrap wood. The strands were oriented, and two types of three-layer (face-core-face) strand board were manufactured: one in which the board bounded with only disposal EPS (P board), and the other in which the board bonded with disposal...

Four-Strand Core Suture Improves Flexor Tendon Repair Compared to Two-Strand Technique in a Rabbit Model

Directory of Open Access Journals (Sweden)

Alice Wichelhaus

2016-01-01

Full Text Available Introduction. This study was designed to investigate the influence of the amount of suture material on the formation of peritendinous adhesions of intrasynovial flexor tendon repairs. Materials and Methods. In 14 rabbits, the flexor tendons of the third and the fourth digit of the right hind leg were cut and repaired using a 2- or 4-strand core suture technique. The repaired tendons were harvested after three and eight weeks. The range of motion of the affected toes was measured and the tendons were processed histologically. The distance between the transected tendon ends, the changes in the peritendinous space, and cellular and extracellular inflammatory reaction were quantified by different staining. Results. A 4-strand core suture resulted in significantly less gap formation. The 2-strand core suture showed a tendency to less adhesion formation. Doubling of the intratendinous suture material was accompanied by an initial increase in leukocyte infiltration and showed a greater amount of formation of myofibroblasts. From the third to the eighth week after flexor tendon repair, both the cellular and the extracellular inflammation decreased significantly. Conclusion. A 4-strand core suture repair leads to a significantly better tendon healing process with less diastasis between the sutured tendon ends despite initially pronounced inflammatory response.

Electro-mechanical behaviors of composite superconducting strand with filament breakage

International Nuclear Information System (INIS)

Wang, Xu; Gao, Yuanwen; Zhou, Youhe

2016-01-01

Highlights: • The electromechanical behaviors of the superconducting (SC) strand are investigated. • A 3D FEM model for bending behaviors and electric properties of strand is developed. • The influence of breakage of filaments on the critical current of SC strand is calculated. • The impact of current transfer length on the electric properties of SC strand is discussed. - Abstract: The bending behaviors of superconducting strand with typical multi-filament twist configuration are investigated based on a three-dimensional finite element method (FEM) model, named as the Multi-filament twist model, of the strand. In this 3D FEM model, the impacts of initial thermal residual stress, filament-breakage and its evaluation are taken into accounts. The mechanical responses of the strand under bending load are studied with the factors taken into consideration one by one. The distribution of the damage of the filaments and its evolution and the movement of the neutral axis caused by it are studied and displayed in detail. Besides, taking the advantages of the Multi-filament twist model, the normalized critical current of the strand under bending load is also calculated based on the invariant temperature and field strain functions. In addition, the non-negligible influences of the pitch length of the filaments on both the mechanical behaviors and the normalized critical current are discussed. The stress-strain characteristics of the strand under tensile load and the normalized critical current of it under axial and bending loads resulting from the Multi-filament twist model show good agreement with the experimental data.

Electro-mechanical behaviors of composite superconducting strand with filament breakage

Energy Technology Data Exchange (ETDEWEB)

Wang, Xu [Key Laboratory of Mechanics on Environment and Disaster in Western China, The Ministry of Education of China, Lanzhou, Gansu 730000 (China); Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou, Gansu 730000 (China); Gao, Yuanwen, E-mail: ywgao@lzu.edu.cn [Key Laboratory of Mechanics on Environment and Disaster in Western China, The Ministry of Education of China, Lanzhou, Gansu 730000 (China); Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou, Gansu 730000 (China); Zhou, Youhe [Key Laboratory of Mechanics on Environment and Disaster in Western China, The Ministry of Education of China, Lanzhou, Gansu 730000 (China); Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou, Gansu 730000 (China)

2016-10-15

Highlights: • The electromechanical behaviors of the superconducting (SC) strand are investigated. • A 3D FEM model for bending behaviors and electric properties of strand is developed. • The influence of breakage of filaments on the critical current of SC strand is calculated. • The impact of current transfer length on the electric properties of SC strand is discussed. - Abstract: The bending behaviors of superconducting strand with typical multi-filament twist configuration are investigated based on a three-dimensional finite element method (FEM) model, named as the Multi-filament twist model, of the strand. In this 3D FEM model, the impacts of initial thermal residual stress, filament-breakage and its evaluation are taken into accounts. The mechanical responses of the strand under bending load are studied with the factors taken into consideration one by one. The distribution of the damage of the filaments and its evolution and the movement of the neutral axis caused by it are studied and displayed in detail. Besides, taking the advantages of the Multi-filament twist model, the normalized critical current of the strand under bending load is also calculated based on the invariant temperature and field strain functions. In addition, the non-negligible influences of the pitch length of the filaments on both the mechanical behaviors and the normalized critical current are discussed. The stress-strain characteristics of the strand under tensile load and the normalized critical current of it under axial and bending loads resulting from the Multi-filament twist model show good agreement with the experimental data.

Double-Strand DNA Break Repair in Mycobacteria.

Science.gov (United States)

Glickman, Michael S

2014-10-01

Discontinuity of both strands of the chromosome is a lethal event in all living organisms because it compromises chromosome replication. As such, a diversity of DNA repair systems has evolved to repair double-strand DNA breaks (DSBs). In part, this diversity of DSB repair systems has evolved to repair breaks that arise in diverse physiologic circumstances or sequence contexts, including cellular states of nonreplication or breaks that arise between repeats. Mycobacteria elaborate a set of three genetically distinct DNA repair pathways: homologous recombination, nonhomologous end joining, and single-strand annealing. As such, mycobacterial DSB repair diverges substantially from the standard model of prokaryotic DSB repair and represents an attractive new model system. In addition, the presence in mycobacteria of a DSB repair system that can repair DSBs in nonreplicating cells (nonhomologous end joining) or when DSBs arise between repeats (single-strand annealing) has clear potential relevance to Mycobacterium tuberculosis pathogenesis, although the exact role of these systems in M. tuberculosis pathogenesis is still being elucidated. In this article we will review the genetics of mycobacterial DSB repair systems, focusing on recent insights.

The (not so) Immortal Strand Hypothesis

OpenAIRE

Tomasetti, Cristian; Bozic, Ivana

2015-01-01

Background: Non-random segregation of DNA strands during stem cell replication has been proposed as a mechanism to minimize accumulated genetic errors in stem cells of rapidly dividing tissues. According to this hypothesis, an “immortal” DNA strand is passed to the stem cell daughter and not the more differentiated cell, keeping the stem cell lineage replication error-free. After it was introduced, experimental evidence both in favor and against the hypothesis has been presented. Principal...

A new cable-in-conduit conductor magnet with insulated strands

International Nuclear Information System (INIS)

Yamaguchi, Satarou; Yamamoto, Junya; Motojima, Osamu.

1995-09-01

Many studies have used cable-in-conduit conductor (CICC) coils in trying to develop an AC superconducting magnet because of its enormous potential if AC losses were low and insulation voltage was high. The strands in the most recent CICC magnets are coated with chromium or another metal with high electrical resistance to order to induce current re-distribution among the strands and to avoid a quench caused by a current imbalance. Current re-distribution is highly complex and very difficult to analyze because the conditions of the strand surfaces and the contact areas vary greatly with the operation of the conductor. If, however, the cable currents were well-balanced, insulating the strands would be the best way to reduce AC losses. We propose a new CICC magnet structure featuring a current lead that balances the strand currents via its resistance. Having calculated current balances, we find that strand currents are well within the present parameters for nuclear fusion experiments and superconducting magnet energy storages. (author)

The risk of stranded assets for utilities in Canada

International Nuclear Information System (INIS)

Schroeder, W.

1998-01-01

The problems of dealing with stranded assets in Canada and the United States were discussed. Compared to the United States, the risk associated with stranded assets for utilities in Canada was considered to be relatively low because of the following factors: (1) low variable cost, (2) isolation, (3) lack of transmission interconnection capacity, (4) lack of tight synchronization in North America, (5) the likelihood of an increase in natural gas prices, (6) the absence of jurisdictional disputes such as FERC versus the states, (7) social considerations, (8) the learning curve, (9) politics, (10) weak balance sheets, (11) relatively low electricity prices, (12) the weak Canadian dollar, and (13) the possibility of refinancing at lower interest rates. Ontario Hydro, New Brunswick and Nova Scotia Power are the three Canadian utilities that may have stranded costs. For Ontario Hydro and New Brunswick Power the stranded costs would be related to nuclear generator problems, whereas for Nova Scotia Power, the stranded costs would be related to the thermal generating base, the threat from Sable Island Gas and the changing tax structure of the utility. Some other reasons why stranded assets could be created in Canada would include low variable costs and high fixed costs, over capacity of at least 30 per cent in generation, limited domestic energy growth, competitive threat from gas, reliability and safety of nuclear plants, and technology change. Five factors in terms of which stranded assets can be expressed are: (1) variable cost definition, (2) total cost definition, (3) operating profit definition, (4) wide geographic definition, and (5) free market definition. In calculating stranded assets, the number of years over which the assets are recovered and the discount rate are considered to be key factors. 26 tabs

Internal Displacement: Livelihood saving responses

OpenAIRE

Deborah Hines

2001-01-01

Deborah Hines explores how to assist the internally displaced and those prone to displacement. She considers the major causes of internal displacement, making the case for a more comprehensive set of policy and operational actions in response to situations of internal displacement. Development (2001) 44, 34–39. doi:10.1057/palgrave.development.1110289

A mass stranding of the squid martialia hyadesi Rochebrune and ...

African Journals Online (AJOL)

1997-02-11

Feb 11, 1997 ... All animals were immature, with females in lower stages of maturity than males. No predatory marine mammals were seen in the area during or after the stranding event. An interpretation of the stranding is presented with reference to historical reports of squid strandings worldwide. Evidence suggests some ...

Radiation induced strand breaks and time scale for repair of broken strands in superinfecting phage lambda DNA in Escherichia coli lysogenic for lambda

International Nuclear Information System (INIS)

Johansen, I.; Boye, E.; Brustad, T.

1975-01-01

The production of the first radiation induced break in covalent lambda DNA molecules in pol + and pol A 1 lysogenic host cells was measured after exposure to electrons from a linear accelerator and transfer to alkaline detergent within 100 ms from the onset of irradiation. The results revealed the presence of an oxygen effect in DNA strand breakage. In both pol + and pol A 1 host cells the rate of production in nitrogen was 1.2x10 -12 DNA single strand breaks per rad per dalton as compared to 5x10 -12 in oxygen. The yields of strand breaks in lambda DNA in pol + host cells under oxygenated or anoxic conditions are independent of whether the cells are irradiated in buffer at room temperature, in buffer at ice temperature, or in growth medium at 37 0 C. These results indicate that enzymic repair of DNA strand breaks before analysis is insignificant in these experiments. The presence of an oxygen effect in DNA strand breakage under these conditions suggest that an actual difference exists between initial number of breaks produced in nitrogen and in oxygen. The kinetics of rejoining of broken molecules under optimal growth conditions was measured by incubating the irradiated host cells prior to lysis. In pol + host cells 50% of the lambda DNA molecules broken in presence of oxygen are rejoined within 10 to 20 seconds of incubation. A significantly lower recovery is seen in pol + host cells after irradiation in nitrogen. The rejoining of broken lambda DNA strands in pol A 1 host cells is impaired after irradiation in presence of oxygen as well as under anoxia. These results show that DNA polymerase I is needed for the rapid rejoining of radiation induced strand breaks in the DNA, and that oxygen promoted strand breaks are more easily rejoined than are those produced in nitrogen. (author)

Viral RNA polymerase scanning and the gymnastics of Sendai virus RNA synthesis

International Nuclear Information System (INIS)

Kolakofsky, Daniel; Le Mercier, Philippe; Iseni, Frederic; Garcin, Dominique

2004-01-01

mRNA synthesis from nonsegmented negative-strand RNA virus (NNV) genomes is unique in that the genome RNA is embedded in an N protein assembly (the nucleocapsid) and the viral RNA polymerase does not dissociate from the template after release of each mRNA, but rather scans the genome RNA for the next gene-start site. A revised model for NNV RNA synthesis is presented, in which RNA polymerase scanning plays a prominent role. Polymerase scanning of the template is known to occur as the viral transcriptase negotiates gene junctions without falling off the template

The replisome uses mRNA as a primer after colliding with RNA polymerase.

Science.gov (United States)

Pomerantz, Richard T; O'Donnell, Mike

2008-12-11

Replication forks are impeded by DNA damage and protein-nucleic acid complexes such as transcribing RNA polymerase. For example, head-on collision of the replisome with RNA polymerase results in replication fork arrest. However, co-directional collision of the replisome with RNA polymerase has little or no effect on fork progression. Here we examine co-directional collisions between a replisome and RNA polymerase in vitro. We show that the Escherichia coli replisome uses the RNA transcript as a primer to continue leading-strand synthesis after the collision with RNA polymerase that is displaced from the DNA. This action results in a discontinuity in the leading strand, yet the replisome remains intact and bound to DNA during the entire process. These findings underscore the notable plasticity by which the replisome operates to circumvent obstacles in its path and may explain why the leading strand is synthesized discontinuously in vivo.

Induction of DNA strand breaks in 14C-labelled cells

International Nuclear Information System (INIS)

Sundell-Bergman, S.; Johanson, K.J.

1979-01-01

Chinese hamster cells grown in vitro were labelled with 14 C-thymidine for 18 hours and after 3 hours in non-radioactive medium they were stored at 0 0 C for various periods ( 1 to 12 hours). During this treatment a number of DNA strand breaks were induced by 14 C decay which were not repaired at 0 0 C. The number of DNA strand breaks was determined using the DNA unwinding technique. At 0.5-1 dpm per cell a detectable number of DNA strand breaks were found. Treatment for six hours (1 dpm per cell) reduced the percentage of double-stranded DNA from 80 to 70%, corresponding to about 750 DNA strand breaks per cell. The rejoining of DNA strand breaks was studied after treatment for 12 hours at 0 0 C followed by incubation of the cells for various periods at 37 0 C. Most of the DNA strand breaks induced by 14 C decay at 0 0 C were repaired after incubation at 37 0 C for 15 minutes. Assuming an absorbed dose of 1.8 mGy per 14 C decay to the cell nucleus an RBE value close to 1 was found for internal irradiation from 14 C decay as compared with 60 Co-gamma irradiation. (author)

New views on strand asymmetry in insect mitochondrial genomes.

Directory of Open Access Journals (Sweden)

Shu-Jun Wei

Full Text Available Strand asymmetry in nucleotide composition is a remarkable feature of animal mitochondrial genomes. Understanding the mutation processes that shape strand asymmetry is essential for comprehensive knowledge of genome evolution, demographical population history and accurate phylogenetic inference. Previous studies found that the relative contributions of different substitution types to strand asymmetry are associated with replication alone or both replication and transcription. However, the relative contributions of replication and transcription to strand asymmetry remain unclear. Here we conducted a broad survey of strand asymmetry across 120 insect mitochondrial genomes, with special reference to the correlation between the signs of skew values and replication orientation/gene direction. The results show that the sign of GC skew on entire mitochondrial genomes is reversed in all species of three distantly related families of insects, Philopteridae (Phthiraptera, Aleyrodidae (Hemiptera and Braconidae (Hymenoptera; the replication-related elements in the A+T-rich regions of these species are inverted, confirming that reversal of strand asymmetry (GC skew was caused by inversion of replication origin; and finally, the sign of GC skew value is associated with replication orientation but not with gene direction, while that of AT skew value varies with gene direction, replication and codon positions used in analyses. These findings show that deaminations during replication and other mutations contribute more than selection on amino acid sequences to strand compositions of G and C, and that the replication process has a stronger affect on A and T content than does transcription. Our results may contribute to genome-wide studies of replication and transcription mechanisms.

Getting Frustrated: Modelling Emotion Contagion in Stranded Passengers

NARCIS (Netherlands)

van der Wal, C. Natalie; Couwenberg, Maik; Bosse, T.

2017-01-01

Train passengers can get stranded due to a variety of events, such as a delay, technical malfunctioning or a natural disaster. Stranded passengers can get frustrated, which could escalate in misbehaviours. Examples are verbal and physical violence or dangerous behaviours such as opening emergency

Simple Laboratory methods to measure cell proliferation using DNA synthesis property

Directory of Open Access Journals (Sweden)

Madhavan H N

2007-01-01

Full Text Available This is a mini-review on the techniques to measure proliferation of cells by estimation of DNA synthesis. This is not an exhaustive review of literature, but a bird’s eye view of a few selected articles which may provide the technical details to the readers.The nucleus of a cell occupies about 10-30% of the cells space, depends on the type of genetic material (DNA -DeoxyriboNucleic Acid. DNA is a long, double-stranded, helical molecule which carries the genetic information. Duplication of the DNA takes place by the phenomena of replication. One copy of double-stranded DNA molecule forms two double-stranded DNA molecules. DNA replication is the fundamental process used in all living organisms as it is the basis for biological inheritance. This process is known also as Mitosis in somatic cells. In Mitosis, the duplication process results in two genetically identical "daughter" cells from a single "parent" cell. The resulting double-stranded DNA molecules are identical; proof reading and error-checking mechanisms exist to ensure near perfect pair. Mitosis is divided into six phases: prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis.

Inter-strand resistance measurements in the termination of the ITER SULTAN samples

International Nuclear Information System (INIS)

Cau, F; Bruzzone, P

2009-01-01

In cabled conductors a perfect uniformity of the current among the strands is hardly reached, due to the non-homogeneity of the contact resistance distribution between the strands and the copper of the electrical terminations. In the case of large current unbalance, the overloaded strands hit the critical surface at high field early, developing a current sharing voltage, which drives the redistribution of the current, mainly in the electrical terminations where the inter-strand resistance is lower than in the high field conductor. If the inter-strand resistance in the termination is low, the voltage levels are sufficiently low to allow an effective redistribution of the current to the less loaded strands. The inter-strand resistance of three different termination layouts of ITER short length samples is measured to make a database available which can be used to qualify the layout of the joints and their capability of redistributing the current among the strands.

RRP Nb3Sn strand studies for LARP

International Nuclear Information System (INIS)

Barzi, Emanuela; Bossert, Rodger; Caspi, Shlomo; Dietderich, Daniel R.; Ferracin, Paolo; Ghosh, Arup; Turrioni, Daniele

2006-01-01

The Nb 3 Sn strand chosen for the next step in the magnet R and D of the U.S. LHC Accelerator Research Program is the 54/61 sub-element Restacked Rod Process by Oxford Instruments, Superconducting Technology. To ensure that the 0.7 mm RRP strands to be used in the upcoming LARP magnets are suitable, extensive studies were performed. Measurements included the critical current, e , using the voltage-current (V-I) method, the stability current, I S , as the minimal quench current obtained with the voltage-field (V-H) method, and RRR. Magnetization was measured at low and high fields to determine the effective filament size and to detect flux jumps. Effects of heat treatment temperature and durations on I e and I S were also studied. Using strand billet qualification and tests of strands extracted from cables, the short sample limits of magnet performance were obtained. The details and the results of this investigation are herein described

RRP Nb3Sn Strand Studies for LARP

International Nuclear Information System (INIS)

Barzi, Emanuela; Bossert, Rodger; Caspi, Shlomo; Dietderich, Daniel R.; Ferracin, Paolo; Ghosh, Arup; Turrioni, Daniele

2007-01-01

The Nb 3 Sn strand chosen for the next step in the magnet R and D of the U.S. LHC Accelerator Research Program is the 54/61 sub-element Restacked Rod Process by Oxford Instruments, Superconducting Technology. To ensure that the 0.7 mm RRP strands to be used in the upcoming LARP magnets are suitable, extensive studies were performed. Measurements included the critical current, I c , using the voltage-current (V-I) method, the stability current, I S , as the minimal quench current obtained with the voltage-field (V-H) method, and RRR. Magnetization was measured at low and high fields to determine the effective filament size and to detect flux jumps. Effects of heat treatment temperature and durations on I c and I S were also studied. Using strand billet qualification and tests of strands extracted from cables, the short sample limits of magnet performance were obtained. The details and the results of this investigation are herein described

Flexural behavior of bonded post-tensioned concrete beams under strand corrosion

International Nuclear Information System (INIS)

Zhang, Xuhui; Wang, Lei; Zhang, Jianren; Ma, Yafei; Liu, Yongming

2017-01-01

Highlights: • Flexural behavior of bonded PT beams with strand corrosion is experimental tested. • Cracking, stiffness, ultimate strength, failure & ductility of beams are clarified. • A coefficient is proposed to measure incompatible strain between strand & concrete. - Abstract: An experimental test is performed to investigate the flexural behavior of bonded post-tensioned concrete beams under strand corrosion. Eight beams are designed and subjected to accelerated method to different corrosion levels. The initial stiffness of beams is observed by cyclic loading-unloading test during the corrosion procedure. Corrosion effects on concrete cracking, post-cracking stiffness, ultimate strength, failure mode and ductility are then clarified by the flexural test. And, a coefficient is introduced to quantify the incompatible strain between corroded strand and concrete. Results show that the prestress force loss of strand has almost the linear relation with corrosion loss. Strand corrosion affects slightly the initial stiffness of beam before flexural cracking, but degrades significantly the post-cracking stiffness of beam as the corrosion loss exceeds 27.0%. Slight corrosion of strand has little effects on beams flexural behavior. The severe corrosion, however, decreases the number of crack, changes the failure mode form the concrete crushing to strand rupture, degrades the ductility and the ultimate strength of beams, and leads to the incompatible strain between strand and concrete. In the present test, the incompatible strain decreases about 20% of the flexural strength as the corrosion loss exceeds 27.0%.

Flexural behavior of bonded post-tensioned concrete beams under strand corrosion

Energy Technology Data Exchange (ETDEWEB)

Zhang, Xuhui [College of Civil Engineering and Mechanics, Xiangtan University, 411105 Xiangtan (China); School of Civil Engineering and Architecture, Changsha University of Science & Technology, 410114 Changsha (China); Industry Key Laboratory of Traffic Infrastructure Security Risk Management (CSUST), 410114 Changsha (China); Wang, Lei, E-mail: leiwlei@hotmail.com [School of Civil Engineering and Architecture, Changsha University of Science & Technology, 410114 Changsha (China); Industry Key Laboratory of Traffic Infrastructure Security Risk Management (CSUST), 410114 Changsha (China); Zhang, Jianren; Ma, Yafei [School of Civil Engineering and Architecture, Changsha University of Science & Technology, 410114 Changsha (China); Industry Key Laboratory of Traffic Infrastructure Security Risk Management (CSUST), 410114 Changsha (China); Liu, Yongming [School for Engineering of Matter, Transport and Energy, Arizona State University, 85281 Tempe, AZ (United States)

2017-03-15

Highlights: • Flexural behavior of bonded PT beams with strand corrosion is experimental tested. • Cracking, stiffness, ultimate strength, failure & ductility of beams are clarified. • A coefficient is proposed to measure incompatible strain between strand & concrete. - Abstract: An experimental test is performed to investigate the flexural behavior of bonded post-tensioned concrete beams under strand corrosion. Eight beams are designed and subjected to accelerated method to different corrosion levels. The initial stiffness of beams is observed by cyclic loading-unloading test during the corrosion procedure. Corrosion effects on concrete cracking, post-cracking stiffness, ultimate strength, failure mode and ductility are then clarified by the flexural test. And, a coefficient is introduced to quantify the incompatible strain between corroded strand and concrete. Results show that the prestress force loss of strand has almost the linear relation with corrosion loss. Strand corrosion affects slightly the initial stiffness of beam before flexural cracking, but degrades significantly the post-cracking stiffness of beam as the corrosion loss exceeds 27.0%. Slight corrosion of strand has little effects on beams flexural behavior. The severe corrosion, however, decreases the number of crack, changes the failure mode form the concrete crushing to strand rupture, degrades the ductility and the ultimate strength of beams, and leads to the incompatible strain between strand and concrete. In the present test, the incompatible strain decreases about 20% of the flexural strength as the corrosion loss exceeds 27.0%.

LHC superconducting strand

CERN Multimedia

Patrice Loiez

1999-01-01

This cross-section through a strand of superconducting matieral as used in the LHC shows the 8000 Niobium-Titanium filaments embedded like a honeycomb in copper. When cooled to 1.9 degrees above absolute zero in the LHC accelerator, these filaments will have zero resistance and so will carry a high electric current with no energy loss.

A Stress Measurement Method for Steel Strands Based on LC Oscillation

Directory of Open Access Journals (Sweden)

Dongjun Chen

2018-01-01

Full Text Available The prestress loss is one of the main factors affecting the safety of prestressed concrete structure. While the detecting signals like sound and light are difficult to spread in steel strands, there is no effective method for prestress detection of the bonded prestressed steel strands in existing structures yet. In this paper, taking into consideration that the electromagnetic oscillation characteristic can make the signal propagate effectively on the bonded prestressed steel strands, a nondestructive prestress detection method based on the electromagnetic effect to detect oscillation frequency is proposed. In a detection circuit, the steel strands are simulated as an inductance component, in which an induced electromagnetic signal passes through the steel strands to form resonance. And then, a frequency meter is used to detect the oscillation frequency of the resonant circuit. The oscillation frequency is supposed to have relationship with the prestress loading on the steel strands. A section of steel strands with a length of 1.2 m is adopted to test the correlation of stress and oscillation frequency. Both the theoretical and experimental results show that the resonant frequency of the circuit decreases with the increase of the stress of the strand and is linear in a certain range.

A model capturing novel strand symmetries in bacterial DNA

International Nuclear Information System (INIS)

Sobottka, Marcelo; Hart, Andrew G.

2011-01-01

Highlights: → We propose a simple stochastic model to construct primitive DNA sequences. → The model provide an explanation for Chargaff's second parity rule in primitive DNA sequences. → The model is also used to predict a novel type of strand symmetry in primitive DNA sequences. → We extend the results for bacterial DNA sequences and compare distributional properties intrinsic to the model to statistical estimates from 1049 bacterial genomes. → We find out statistical evidences that the novel type of strand symmetry holds for bacterial DNA sequences. -- Abstract: Chargaff's second parity rule for short oligonucleotides states that the frequency of any short nucleotide sequence on a strand is approximately equal to the frequency of its reverse complement on the same strand. Recent studies have shown that, with the exception of organellar DNA, this parity rule generally holds for double-stranded DNA genomes and fails to hold for single-stranded genomes. While Chargaff's first parity rule is fully explained by the Watson-Crick pairing in the DNA double helix, a definitive explanation for the second parity rule has not yet been determined. In this work, we propose a model based on a hidden Markov process for approximating the distributional structure of primitive DNA sequences. Then, we use the model to provide another possible theoretical explanation for Chargaff's second parity rule, and to predict novel distributional aspects of bacterial DNA sequences.

Stranding Events of Kogia Whales along the Brazilian Coast.

Directory of Open Access Journals (Sweden)

Jailson F Moura

Full Text Available The genus Kogia, which comprises only two extant species, Kogia sima and Kogia breviceps, represents one of the least known groups of cetaceans in the global ocean. In some coastal regions, however, stranding events of these species have been relatively common over the last decades. Stranding provides the opportunity to investigate the biology of these cetaceans and to explore the epidemiological aspects associated with the mortality of the organisms found on the beach. A number of disturbances (including pelagic fisheries, chemical pollution, boat strikes, and noise pollution have been confirmed to pose a particular threat to the Kogia species. However, no study has yet investigated potential relationships between environmental conditions and stranding events. Here we analyse how a collection of environmental, physical, and biological variables, such as wind, sea surface temperature (SST, water depth, and chlorophyll-a, correlate to Kogia stranding events along the Brazilian coast. The results of our statistical analyses suggest that K. sima is more likely found in warm tropical waters, which provide an explanation for the high frequency of stranding in northeastern Brazilian coast. In contrast, K. breviceps appears to have a preference for temperate and productive waters. Wind speed results to be also an important factor for predicting Kogia strandings in Brazilian coast. Additionally, literature information in combination with our own data and analyses of stomach contents confirms that oceanic cephalopods constitute the primary nutritional source of both Kogia species. By using the available information as a qualitative proxy for habitat preference and feeding ecology, our study provides a novel and comprehensive assessment of Kogia stranding data in relation to environmental conditions along the Brazilian coast.

75 FR 36678 - Prestressed Concrete Steel Wire Strand From China; Determinations

Science.gov (United States)

2010-06-28

... prestressed concrete steel wire strand (PC strand), provided for in subheading 7312.10.30 of the Harmonized... Publication 4162 (June 2010), entitled Prestressed Concrete Steel Wire Strand from China: Investigation Nos... INTERNATIONAL TRADE COMMISSION [Investigation Nos. 701-TA-464 and 731-TA-1160 (Final)] Prestressed...

Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand*

Science.gov (United States)

Teasley, Daniel C.; Parajuli, Shankar; Nguyen, Mai; Moore, Hayley R.; Alspach, Elise; Lock, Ying Jie; Honaker, Yuchi; Saharia, Abhishek; Piwnica-Worms, Helen; Stewart, Sheila A.

2015-01-01

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer. PMID:25922071

Characteristics of Cu stabilized Nb3Al strands with low Cu ratio

Energy Technology Data Exchange (ETDEWEB)

Kikuchi, A.; Yamada, R.; Barzi, E.; Kobayashi, M.; Lamm, M.; Nakagawa, K.; Sasaki, K.; Takeuchi, T.; Turrioni, D.; Zlobin, A.V.; /NIMC, Tsukuba /Fermilab /Hitachi, Tsuchiura Works /KEK, Tsukuba

2008-12-01

Characteristics of recently developed F4-Nb{sub 3}Al strand with low Cu ratio are described. The overall J{sub c} of the Nb{sub 3}Al strand could be easily increased by decreasing of the Cu ratio. Although the quench of a pulse-like voltage generation is usually observed in superconducting unstable conductor, the F4 strand with a low Cu ratio of 0.61 exhibited an ordinary critical transition of gradual voltage generation. The F4 strand does not have magnetic instabilities at 4.2 K because of the tantalum interfilament matrix. The overall J{sub c} of the F4 strand achieved was 80-85% of the RRP strand. In the large mechanical stress above 100 MPa, the overall J{sub c} of the F4 strand might be comparable to that of high J{sub c} RRP-Nb{sub 3}Sn strands. The Rutherford cable with a high packing factor of 86.5% has been fabricated using F4 strands. The small racetrack magnet, SR07, was also fabricated by a 14 m F4 cable. The quench current, I{sub q}, of SR07 were obtained 22.4 kA at 4.5 K and 25.2 kA at 2.2 K. The tantalum matrix Nb{sub 3}Al strands are promising for the application of super-cooled high-field magnets as well as 4.2 K operation magnets.

Characterization of a parallel-stranded DNA hairpin

International Nuclear Information System (INIS)

Germann, M.W.; Vogel, H.J.; Pon, R.T.; van de Sande, J.H.

1989-01-01

Recently, the authors have shown that synthetic DNA containing homooligomeric A-T base pairs can form a parallel-stranded intramolecular hairpin structure. In the present study, they have employed NMR and optical spectroscopy to investigate the structure of the parallel-stranded (PS) DNA hairpin 3'-d(T) 8 C 4 (A) 8 -3' and the related antiparallel (APS) hair 5'-d(T) 8 C 4 (A) 8 -3'. The parallel orientation of the strands in the PS oligonucleotide is achieved by introducing a 5'-5' phosphodiester linkage in the hairpin loop. Ultraviolet spectroscopic and fluorescence data of drug binding are consistent with the formation of PS and APS structures, respectively, in these two hairpins. Vacuum circular dichroism measurements in combination with theoretical CD calculations indicate that the PS structure forms a right-handed helix. 31 P NMR measurements indicate that the conformation of the phosphodiester backbone of the PS structure is not drastically different from that of the APS control. The presence of slowly exchanging imino protons at 14 ppm and the observation of nuclear Overhauser enhancement between imino protons and the AH-2 protons demonstrate that similar base pairing and base stacking between T and A residues occur in both hairpins. On the basis of NOESY measurements, they find that the orientation of the bases is in the anti region and that the sugar puckering is in the 2'-endo range. The results indicate a B-like conformation for each of the strands in the stem part of the PS hairpin and reverse Watson-Crick base pairing between the T and A residues. These data are consistent with a previously calculated structure for parallel-stranded DNA

Torsional regulation of hRPA-induced unwinding of double-stranded DNA

NARCIS (Netherlands)

De Vlaminck, I.; Vidic, I.; Van Loenhout, M.T.J.; Kanaar, R.; Lebbink, J.H.G.; Dekker, C.

2010-01-01

All cellular single-stranded (ss) DNA is rapidly bound and stabilized by single stranded DNA-binding proteins (SSBs). Replication protein A, the main eukaryotic SSB, is able to unwind double-stranded (ds) DNA by binding and stabilizing transiently forming bubbles of ssDNA. Here, we study the

Role of stranded gas in increasing global gas supplies

Science.gov (United States)

Attanasi, E.D.; Freeman, P.A.

2013-01-01

This report synthesizes the findings of three regional studies in order to evaluate, at the global scale, the contribution that stranded gas resources can make to global natural gas supplies. Stranded gas, as defined for this study, is natural gas in discovered conventional gas and oil fields that is currently not commercially producible for either physical or economic reasons. The regional studies evaluated the cost of bringing the large volumes of undeveloped gas in stranded gas fields to selected markets. In particular, stranded gas fields of selected Atlantic Basin countries, north Africa, Russia, and central Asia are screened to determine whether the volumes are sufficient to meet Europe’s increasing demand for gas imports. Stranded gas fields in Russia, central Asia, Southeast Asia, and Australia are also screened to estimate development, production, and transport costs and corresponding gas volumes that could be supplied to Asian markets in China, India, Japan, and South Korea. The data and cost analysis presented here suggest that for the European market and the markets examined in Asia, the development of stranded gas provides a way to meet projected gas import demands for the 2020-to-2040 period. Although this is a reconnaissance-type appraisal, it is based on volumes of gas that are associated with individual identified fields. Individual field data were carefully examined. Some fields were not evaluated because current technology was insufficient or it appeared the gas was likely to be held off the export market. Most of the evaluated stranded gas can be produced and delivered to markets at costs comparable to historical prices. Moreover, the associated volumes of gas are sufficient to provide an interim supply while additional technologies are developed to unlock gas diffused in shale and hydrates or while countries transition to making a greater use of renewable energy sources.

Improved multiple displacement amplification (iMDA) and ultraclean reagents.

Science.gov (United States)

Motley, S Timothy; Picuri, John M; Crowder, Chris D; Minich, Jeremiah J; Hofstadler, Steven A; Eshoo, Mark W

2014-06-06

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance

Corrosion characteristics of unprotected post-tensioning strands under stress.

Science.gov (United States)

2014-05-01

An investigation was conducted to determine the effect of stress condition : and environmental exposure on corrosion of post-tensioned strands during ungrouted periods. : Exposures for periods of up to 4 weeks of stressed, as-received strand placed i...

Translation elongation factor 1A facilitates the assembly of the tombusvirus replicase and stimulates minus-strand synthesis.

OpenAIRE

Zhenghe Li; Judit Pogany; Steven Tupman; Anthony M Esposito; Terri Goss Kinzy; Peter D Nagy

2010-01-01

Replication of plus-strand RNA viruses depends on host factors that are recruited into viral replicase complexes. Previous studies showed that eukaryotic translation elongation factor (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. Using a random library of eEF1A mutants, we identified one mutant that decreased and three mutants that increased Tomato bushy stunt virus (TBSV) replication in a yeast model host. Additional in vitro assays with w...

Synthesis of ammonia using sodium melt.

Science.gov (United States)

Kawamura, Fumio; Taniguchi, Takashi

2017-09-14

Research into inexpensive ammonia synthesis has increased recently because ammonia can be used as a hydrogen carrier or as a next generation fuel which does not emit CO 2 . Furthermore, improving the efficiency of ammonia synthesis is necessary, because current synthesis methods emit significant amounts of CO 2 . To achieve these goals, catalysts that can effectively reduce the synthesis temperature and pressure, relative to those required in the Haber-Bosch process, are required. Although several catalysts and novel ammonia synthesis methods have been developed previously, expensive materials or low conversion efficiency have prevented the displacement of the Haber-Bosch process. Herein, we present novel ammonia synthesis route using a Na-melt as a catalyst. Using this route, ammonia can be synthesized using a simple process in which H 2 -N 2 mixed gas passes through the Na-melt at 500-590 °C under atmospheric pressure. Nitrogen molecules dissociated by reaction with sodium then react with hydrogen, resulting in the formation of ammonia. Because of the high catalytic efficiency and low-cost of this molten-Na catalyst, it provides new opportunities for the inexpensive synthesis of ammonia and the utilization of ammonia as an energy carrier and next generation fuel.

Stranded cost recovery in electricity market reforms in the US

International Nuclear Information System (INIS)

Woo, C.K.; Lloyd, D.; Karimov, R.; Tishler, A.

2003-01-01

An important element of an electricity market reform is stranded cost recovery. This paper explains the cause of stranded costs, describes four recovery mechanisms, evaluates these mechanisms using the criteria of recovery certainty, economic efficiency and equity, reviews the financial performance of 12 utilities in the US in connection to stranded cost recovery, and shows why the mechanism used in California has contributed to the reform failure in that state. (Author)

Bioprinting Using Mechanically Robust Core-Shell Cell-Laden Hydrogel Strands.

Science.gov (United States)

Mistry, Pritesh; Aied, Ahmed; Alexander, Morgan; Shakesheff, Kevin; Bennett, Andrew; Yang, Jing

2017-06-01

The strand material in extrusion-based bioprinting determines the microenvironments of the embedded cells and the initial mechanical properties of the constructs. One unmet challenge is the combination of optimal biological and mechanical properties in bioprinted constructs. Here, a novel bioprinting method that utilizes core-shell cell-laden strands with a mechanically robust shell and an extracellular matrix-like core has been developed. Cells encapsulated in the strands demonstrate high cell viability and tissue-like functions during cultivation. This process of bioprinting using core-shell strands with optimal biochemical and biomechanical properties represents a new strategy for fabricating functional human tissues and organs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioelectrochemical sensing of promethazine with bamboo-type multiwalled carbon nanotubes dispersed in calf-thymus double stranded DNA.

Science.gov (United States)

Primo, Emiliano N; Oviedo, M Belén; Sánchez, Cristián G; Rubianes, María D; Rivas, Gustavo A

2014-10-01

We report the quantification of promethazine (PMZ) using glassy carbon electrodes (GCE) modified with bamboo-like multi-walled carbon nanotubes (bCNT) dispersed in double stranded calf-thymus DNA (dsDNA) (GCE/bCNT-dsDNA). Cyclic voltammetry measurements demonstrated that PMZ presents a thin film-confined redox behavior at GCE/bCNT-dsDNA, opposite to the irreversibly-adsorbed behavior obtained at GCE modified with bCNT dispersed in ethanol (GCE/bCNT). Differential pulse voltammetry-adsorptive stripping with medium exchange experiments performed with GCE/bCNT-dsDNA and GCE modified with bCNTs dispersed in single-stranded calf-thymus DNA (ssDNA) confirmed that the interaction between PMZ and bCNT-dsDNA is mainly hydrophobic. These differences are due to the intercalation of PMZ within the dsDNA that supports the bCNTs, as evidenced from the bathochromic displacement of UV-Vis absorption spectra of PMZ and quantum dynamics calculations at DFTB level. The efficient accumulation of PMZ at GCE/bCNT-dsDNA made possible its sensitive quantification at nanomolar levels (sensitivity: (3.50±0.05)×10(8) μA·cm(-2)·M(-1) and detection limit: 23 nM). The biosensor was successfully used for the determination of PMZ in a pharmaceutical product with excellent correlation. Copyright © 2014 Elsevier B.V. All rights reserved.

Mechanical behaviors of multi-filament twist superconducting strand under tensile and cyclic loading

Science.gov (United States)

Wang, Xu; Li, Yingxu; Gao, Yuanwen

2016-01-01

The superconducting strand, serving as the basic unit cell of the cable-in-conduit-conductors (CICCs), is a typical multi-filament twist composite which is always subjected to a cyclic loading under the operating condition. Meanwhile, the superconducting material Nb3Sn in the strand is sensitive to strain frequently relating to the performance degradation of the superconductivity. Therefore, a comprehensive study on the mechanical behavior of the strand helps understanding the superconducting performance of the strained Nb3Sn strands. To address this issue, taking the LMI (internal tin) strand as an example, a three-dimensional structural finite element model, named as the Multi-filament twist model, of the strand with the real configuration of the LMI strand is built to study the influences of the plasticity of the component materials, the twist of the filament bundle, the initial thermal residual stress and the breakage and its evolution of the filaments on the mechanical behaviors of the strand. The effective properties of superconducting filament bundle with random filament breakage and its evolution versus strain are obtained based on the damage theory of fiber-reinforced composite materials proposed by Curtin and Zhou. From the calculation results of this model, we find that the occurrence of the hysteresis loop in the cyclic loading curve is determined by the reverse yielding of the elastic-plastic materials in the strand. Both the initial thermal residual stress in the strand and the pitch length of the filaments have significant impacts on the axial and hysteretic behaviors of the strand. The damage of the filaments also affects the axial mechanical behavior of the strand remarkably at large axial strain. The critical current of the strand is calculated by the scaling law with the results of the Multi-filament twist model. The predicted results of the Multi-filament twist model show an acceptable agreement with the experiment.

Molecular investigation of evaporation of biodroplets containing single-strand DNA on graphene surface.

Science.gov (United States)

Akbari, Fahimeh; Foroutan, Masumeh

2018-02-14

In this study, the water droplet behaviour of four different types of single-strand DNA with homogeneous base sequence on a graphene substrate during evaporation of the droplet was investigated using molecular dynamics (MD) simulation. The simulation results indicated that the evaporation depended on the DNA sequence. The observed changes can be divided into four parts: (i) vaporization mode, (ii) evaporation flux, (iii) mechanism of single-strand placement on the surface, and (iv) consideration of remaining single strands after evaporation. Our simulation observations indicated different evaporation modes for thymine biodroplets as compared to those for other biodroplets. The evaporation of the thymine biodroplets occurred with an increase in the contact angle, while that of the other biodroplets occur in a constant contact angle mode. Moreover, thymine biodroplets generate the lowest contact line compared to other single strands, and it is always placed far away from the centre of the droplets during evaporation. Investigating variations in the evaporation flux shows that thymine has the highest evaporation flux and guanine has the lowest. Moreover, during initial evaporation, the flux of evaporation increases at the triple point of the biodroplets containing thymine single strands, while it decreases in the other biodroplets. The following observation was obtained from the study of the placement of single strands on the substrate: guanine and thymine interacted slower than other single strands during evaporation with graphene, adenine single strand had a higher folding during evaporation, and guanine single strand showed the lowest end-to-end distance. The investigation of single-strand DNA after evaporation shows that adenine produces the most stable structure at the end of evaporation. In addition, cytosine is the most stretched single-strand DNA due to its lack of internal π-π stacking and hydrogen bonding. Therefore, cytosine single strand is more

The use of a displacement device negatively affects the performance of dogs (Canis familiaris) in visible object displacement tasks.

Science.gov (United States)

Müller, Corsin A; Riemer, Stefanie; Range, Friederike; Huber, Ludwig

2014-08-01

Visible and invisible displacement tasks have been used widely for comparative studies of animals' understanding of object permanence, with evidence accumulating that some species can solve invisible displacement tasks and, thus, reach Piagetian stage 6 of object permanence. In contrast, dogs appear to rely on associative cues, such as the location of the displacement device, during invisible displacement tasks. It remains unclear, however, whether dogs, and other species that failed in invisible displacement tasks, do so because of their inability to form a mental representation of the target object, or simply because of the involvement of a more salient but potentially misleading associative cue, the displacement device. Here we show that the use of a displacement device impairs the performance of dogs also in visible displacement tasks: their search accuracy was significantly lower when a visible displacement was performed with a displacement device, and only two of initially 42 dogs passed the sham-baiting control conditions. The negative influence of the displacement device in visible displacement tasks may be explained by strong associative cues overriding explicit information about the target object's location, reminiscent of an overshadowing effect, and/or object individuation errors as the target object is placed within the displacement device and moves along a spatiotemporally identical trajectory. Our data suggest that a comprehensive appraisal of a species' performance in object permanence tasks should include visible displacement tasks with the same displacement device used in invisible displacements, which typically has not been done in the past.

Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand.

Science.gov (United States)

Teasley, Daniel C; Parajuli, Shankar; Nguyen, Mai; Moore, Hayley R; Alspach, Elise; Lock, Ying Jie; Honaker, Yuchi; Saharia, Abhishek; Piwnica-Worms, Helen; Stewart, Sheila A

2015-06-12

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Displacement data assimilation

Energy Technology Data Exchange (ETDEWEB)

Rosenthal, W. Steven [Pacific Northwest Laboratory, Richland, WA 99354 (United States); Venkataramani, Shankar [Department of Mathematics and Program in Applied Mathematics, University of Arizona, Tucson, AZ 85721 (United States); Mariano, Arthur J. [Rosenstiel School of Marine & Atmospheric Science, University of Miami, Miami, FL 33149 (United States); Restrepo, Juan M., E-mail: restrepo@math.oregonstate.edu [Department of Mathematics, Oregon State University, Corvallis, OR 97331 (United States)

2017-02-01

We show that modifying a Bayesian data assimilation scheme by incorporating kinematically-consistent displacement corrections produces a scheme that is demonstrably better at estimating partially observed state vectors in a setting where feature information is important. While the displacement transformation is generic, here we implement it within an ensemble Kalman Filter framework and demonstrate its effectiveness in tracking stochastically perturbed vortices.

Aromatic fluorine compounds. VI. Displacement of aryl fluorine in diazonium salts

Science.gov (United States)

Finger, G.C.; Oesterling, R.E.

1956-01-01

Several chlorofluorobenzenes have been isolated from the Schiemann synthesis of fluorobenzenes. These have been shown to be the products of two side reactions occurring during thermal decomposition of the dry benzenediazonium fluoborate salt containing coprecipitated sodium chloride, an unavoidable contaminant in large preparations involving the use of hydrochloric acid and sodium fluoborate. The major side reaction and its chloro product were unexpected; a unique displacement of fluorine ortho to the diazonium group was observed. Replacement of the diazo group with chlorine was the predicted side reaction which proved to be minor. Conditions causing the side reactions and the isolation and identification of the products are described.

Cetacean strandings along the coast of Izmir Bay, Turkey

NARCIS (Netherlands)

Guclusoy, H.; Veryeri, N.; Cirik, S.

2004-01-01

The present paper provides information on the stranding of cetaceans in Izmir Bay, Aegean Sea, between 1992 and 2004. The data were collected opportunistically during sightings and stranding data collection for Monk Seals. A total of 12 cetaceans, namely Bottle-nosed Dolphin, Tursiops truncatus

DNA turnover and strand breaks in Escherichia coli

International Nuclear Information System (INIS)

Hanawalt, P.; Grivell, A.; Nakayama, H.

1975-01-01

The extent of DNA turnover has been measured in a dnaB mutant of Escherichia coli, temperature sensitive for semiconservative DNA replication. At the nonpermissive temperature about 0.02 percent of the deoxynucleotides in DNA are exchanged per generation period. This turnover rate is markedly depressed in the presence of rifampicin. During thymine starvation strand breaks accumulate in the DNA of E. coli strains that are susceptible to thymineless death. Rifampicin suppresses the appearance of these breaks, consistent with our hypothesis that transcription may be accompanied by repairable single-strand breaks in DNA. DNA turnover is enhanced severalfold in strands containing 5-bromodeoxyuridine in place of thymidine, possibly because the analog (or the deoxyuridine, following debromination) is sometimes recognized and excised

Residual Resistivity Ratio (RRR) Measurements of LHC Superconducting NbTi Cable Strands

CERN Document Server

Charifoulline, Z

2006-01-01

The Rutherford-type superconducting NbTi cables of the LHC accelerator are currently manufactured by six industrial companies. As a part of the acceptance tests, the Residual Resistivity Ratio (RRR) of superconducting strands is systematically measured on virgin strands to qualify the strands before cabling and on extracted strands to qualify the cables and to check the final heat treatment (controlled oxidation to control interstrand resistance). More than 12000 samples of virgin and extracted strands have been measured during last five years. Results show good correlation with the measurements done by the companies and reflect well the technological process of cable production (strand annealing, cabling, cable heat treatment). This paper presents a description of the RRR-test station and the measurement procedure, the summary of the results over all suppliers and finally the correlation between RRR-values of the cables and the magnets.

Displacement cascades in diatomic materials

International Nuclear Information System (INIS)

Parkin, D.M.; Coulter, C.A.

1981-01-01

A new function, the specified-projectile displacement function p/sub ijk/ (E), is introduced to describe displacement cascades in polyatomic materials. This function describes the specific collision events that produce displacements and hence adds new information not previously available. Calculations of p/sub ijk/ (E) for MgO, Al 2 O 3 and TaO are presented and discussed. Results show that the parameters that have the largest effect on displacement collision events are the PKA energy and the mass ratio of the atom types in the material. It is further shown that the microscopic nature of the displacement events changes over the entire recoil energy range relevant to fusion neutron spectra and that these changes are different in materials whose mass ratio is near one than in those where it is far from one

Tensile and dimensional properties of wood strands made from plantation southern pine lumber

Science.gov (United States)

Qinglin Wu; Zhiyong Cai; Jong N. Lee

2005-01-01

Working stresses and performance of strand composite lumber largely depend upon the properties of each individual strand. Southern pine strands from plantation lumber grown in southern Louisiana were investigated in this study in order to understand strand behaviors. The effects of hot-pressing and resin application on tensile modulus, strength, and dimensional...

Improving strand quality of upland oaks for use in oriented strand board

Science.gov (United States)

David B. DeValliance; Jody D. Gray; Shawn T. Grushecky

2013-01-01

Past research estimates that more than 1 million tons of oak logging residues go unused in West Virginia each year. Much research has been done investigating potential products and markets for this underutilized resource. West Virginia is home to an oriented strand board (OSB) producer that consumes large volumes of small diameter, low quality round wood. However, the...

TALEN-Induced Double-Strand Break Repair of CTG Trinucleotide Repeats

Directory of Open Access Journals (Sweden)

Valentine Mosbach

2018-02-01

Full Text Available Trinucleotide repeat expansions involving CTG/CAG triplets are responsible for several neurodegenerative disorders, including myotonic dystrophy and Huntington’s disease. Because expansions trigger the disease, contracting repeat length could be a possible approach to gene therapy for these disorders. Here, we show that a TALEN-induced double-strand break was very efficient at contracting expanded CTG repeats in yeast. We show that RAD51, POL32, and DNL4 are dispensable for double-strand break repair within CTG repeats, the only required genes being RAD50, SAE2, and RAD52. Resection was totally abolished in the absence of RAD50 on both sides of the break, whereas it was reduced in a sae2Δ mutant on the side of the break containing the longest repeat tract, suggesting that secondary structures at double-strand break ends must be removed by the Mre11-Rad50 complex and Sae2. Following the TALEN double-strand break, single-strand annealing occurred between both sides of the repeat tract, leading to repeat contraction.

Influence of vinyl chloride monomer and vinyl chloride monomer derivatives on hepatic DNA synthesis

International Nuclear Information System (INIS)

Brenner, E.A.

1982-01-01

Vinyl chloride monomer (VCM) is used extensively in the chemical industry, mainly in the production of polyvinyl chloride. It has recently been found to cause hepatic angiosarcoma. As VCM has also been shown to be mutagenic after metabolic activation the effect of VCM on DNA synthesis was investigated. [ 3 H]Thymidine incorporation into DNA was used to measure the rate of DNA synthesis in regenerating rat liver. A possible direct toxic effect of VCM or its metabolites on liver cell metabolism was examined by two unrelated techniques, viz. the measurement of adenine nucleotide concentrations in regenerating livers and the influence on transmembrane potentials in hepatocytes. The distribution of radioactivity in subcellular fractions following [ 14 C]VCM administration suggested microsomal conversion of VCM to an active form which was selectively retained in the nuclear fraction. Measurement of the activities of thymidine kinase and DNA polymerase in regenerating liver indicated that the induction of these enzymes which normally occurs after partial hepatectomy was not prevented by VCM treatment. Three techniques were used to test the hypothesis that the retardation in DNA synthesis was due to DNA damage: the prophage lambda induction test for DNA damage, autoradiographic detection of unscheduled thymidine incorporation into DNA, and detection of DNA strand breaks in alkaline sucrose gradients. All three provided evidence of DNA damage and led to the development of a novel technique to confirm these findings. This involved centrifugation in neutral sucrose gradients on intact double-stranded DNA contained in hepatocyte nucleoids and showed conclusively that VCM administration causes DNA strand breaks. Subsequent repair of DNA was also assessed by this technique. The site of the VCM/metabolite: DNA reaction was characterized by DNA thermal denaturation and renaturation studies

Development of a 10 m quasi-isotropic strand assembled from 2G wires

Science.gov (United States)

Kan, Changtao; Wang, Yinshun; Hou, Yanbing; Li, Yan; Zhang, Han; Fu, Yu; Jiang, Zhe

2018-03-01

Quasi-isotropic strands made of second generation (2G) high temperature superconducting (HTS) wires are attractive to applications of high-field magnets at low temperatures and power transmission cables at liquid nitrogen temperature in virtue of their high current carrying capability and well mechanical property. In this contribution, a 10 m length quasi-isotropic strand is manufactured and successfully tested in liquid nitrogen to verify the feasibility of an industrial scale production of the strand by the existing cabling technologies. The strand with copper sheath consists of 72 symmetrically assembled 2G wires. The uniformity of critical properties of long quasi-isotropic strands, including critical current and n-value, is very important for their using. Critical currents as well as n-values of the strand are measured every 1 m respectively and compared with the simulation results. Critical current and n-value of the strand are calculated basing on the self-consistent model solved by the finite element method (FEM). Effects of self-field on the critical current and n-value distributions in wires of the strand are analyzed in detail. The simulation results show good agreement with the experimental data and the 10 m quasi-isotropic strand has good critical properties uniformity.

Corrosion characteristics of post-tensioning strands in ungrouted ducts : summary.

Science.gov (United States)

2011-01-01

To prevent corrosion of post-tensioning strands, FDOT construction specifications currently require post-tensioning ducts to be grouted within seven calendar days of strand installation. This period challenges construction schedules on large projects...

Magnetization and loss measurements on Nb3Sn and NbTi strands for ITER and LHC

International Nuclear Information System (INIS)

Foitl, M.

2001-05-01

Recent developments in high energy physics have led to a demand for high magnetic fields which cannot be generated permanently by conventional magnets wound from Cu cables. The acceleration of protons in a ring accelerator up to particle energies of several TeVs or the magnetic confinement of fusion plasmas of sufficient pressure to generate ∼1 GW of fusion power, or even more, are only two examples. To reach beam to beam collision energies of several TeV for hadrons or heavy ions, accelerator magnets have to provide very high magnetic fields which can only be produced by superconducting magnets. In the case of the Large Hadron Collider (LHC), which is planned to be commissioned in the year 2005, the circumference of the beam line is given by the dimensions of the 27 Km Large Electron Positron (LEP) Collider tunnel. Consequently each superconducting arc dipole has to provide a field of 8.36 T to bend 7 TeV protons around the ring. Apart from the total magnitude of the bending field, which necessitates to operate the superconducting NbTi magnets at 1.9 K, very strict demands have to be drawn to the quality of the field components of the guiding field. Actually, any kind of random field error would dilute the 7-on-7 TeV counter-rotating proton beams and degrade their nominal luminosity of 1034 cm -1 s -2 . The main contributions to random field errors are random displacements of the coil positions with respect to nominal design, persistent current magnetization stemming from current imbalances in the superconducting cables and variations of the strand magnetization which come from irregularities in the strand manufacturing process. Since there will be more than one cable manufacturer, there will certainly be a difference between average magnetization values for each of them. Interfilament proximity coupling could make the magnetization higher at low fields, which could also have an influence on the performance of certain magnets in the machine. In the first part of

Role of alfalfa mosaic virus coat protein in regulation of the balance between viral plus and minus strand RNA synthesis

NARCIS (Netherlands)

van der Kuyl, A. C.; Neeleman, L.; Bol, J. F.

1991-01-01

Replication of wild type RNA 3 of alfalfa mosaic virus (AIMV) and mutants with frameshifts in the P3 or coat protein (CP) genes was studied in protoplasts from tobacco plants transformed with DNA copies of AIMV RNAs 1 and 2. Accumulation of viral plus and minus strand RNAs was monitored with

Stem cell identity and template DNA strand segregation.

Science.gov (United States)

Tajbakhsh, Shahragim

2008-12-01

The quest for stem cell properties to distinguish their identity from that of committed daughters has led to a re-investigation of the notion that DNA strands are not equivalent, and 'immortal' DNA strands are retained in stem cells whereas newly replicated DNA strands segregate to the differentiating daughter cell during mitosis. Whether this process occurs only in stem cells, and also in all tissues, remains unclear. That individual chromosomes can be also partitioned non-randomly raises the question if this phenomenon is related to the immortal DNA hypothesis, and it underscores the need for high-resolution techniques to observe these events empirically. Although initially postulated as a mechanism to avoid DNA replication errors, alternative views including epigenetic regulation and sister chromatid silencing may provide insights into this process.

Mechanical behavior of Nb{sub 3}Sn strands under transverse electromagnetic loads

Energy Technology Data Exchange (ETDEWEB)

Ru, Yanyun; Yong, Huadong, E-mail: yonghd@lzu.edu.cn; Zhou, Youhe

2016-11-15

Highlights: • The contact forces between the strands under transverse electromagnetic load are considered. • The indentation depth for two-dimensional contact problem is calculated. • The strand damage for different layers is presented. • We discuss the effect of bending wavelength on the Nb{sub 3}Sn strand. - Abstract: The performance of Nb{sub 3}Sn cable-in-conduit-conductor (CICC) shows a significant degradation with increasing electromagnetic load in the International Thermonuclear Experimental Reactor (ITER). As the strand is under the compression caused by adjacent strands, the strand damage may occur for high contact force. In this paper, we present a 3D helix model based on the TEMLOP and FEMCAM to simulate the contact force among the strands under transverse load. The maximum linear strain and indentation depth induced by contact stress are calculated with two-dimensional contact model of cylinder. The numerical results are compared with the experimental values. Finally, the indentation depth is discussed for different layers and cross angles based on two models. With the increasing of layer number, the degradation of performance will occur. The short bending wavelength leads to small indentation depth.

Performance optimization of internal tin process multifilamentary Nb{sub 3}Sn strands

Energy Technology Data Exchange (ETDEWEB)

Zhang, Ke, E-mail: zhangke@c-wst.com [State Key Laboratory of Solidification Processing, Northwestern Polytechnical University, Xi’an, Shaanxi 710072 (China); Western Superconducting Technologies Co. Ltd, Xi’an, Shaanxi 710018 (China); State Engineering Lab. of Superconducting Material Preparation, Xi’an, Shaanxi 710018 (China); Zhang, Pingxiang [State Key Laboratory of Solidification Processing, Northwestern Polytechnical University, Xi’an, Shaanxi 710072 (China); Western Superconducting Technologies Co. Ltd, Xi’an, Shaanxi 710018 (China); State Engineering Lab. of Superconducting Material Preparation, Xi’an, Shaanxi 710018 (China); Northwest Institute for Non-ferrous Metal Research, Xi’an, Shaanxi 710016 (China); Shi, Yigong; Liu, Jianwei; Gao, Huixian; Li, Jianfeng; Liu, Xianghong; Feng, Yong [Western Superconducting Technologies Co. Ltd, Xi’an, Shaanxi 710018 (China); State Engineering Lab. of Superconducting Material Preparation, Xi’an, Shaanxi 710018 (China)

2016-11-01

Highlights: • J{sub c} increases by adding Sn cores in the interspaces, hysteresis loss increases at the same time. • Enlarger the number of filaments with the same Sn proportion will increase J{sub c} and hysteresis loss. • Cu split adopted in each sub-element leads to a low hysteresis loss. - Abstract: In this study, internal tin process Nb{sub 3}Sn strands for the International Thermo-nuclear Experimental Reactor are developed in Western Superconducting Technologies. Short cable-in-conduit conductors manufactured with these strands have been qualified. Mass production is accomplished, and all the performances of the strands meet the requirement of ITER project. Besides, more researches on the effect of strand design on critical current density and hysteresis loss for Nb{sub 3}Sn strands are carried out. It is found that critical current density can be enhanced by reducing filament diameter and increasing Sn content, and hysteresis loss of the strands can be significantly reduced through Cu split adopted in sub-element.

Ataxia-telangiectasia cells are not uniformly deficient in poly(ADP-ribose) synthesis following X-irradiation

International Nuclear Information System (INIS)

Zwelling, L.A.; Kerrigan, D.; Mattern, M.R.

1983-01-01

The synthesis of poly(adenosine diphosphoribose [poly(ADP-R)] follows the DNA strand breakage produced by a number of physical and chemical agents, including X-radiation, and may be important for repair of several types of DNA damage. The reduction or abolition of its synthesis following X-irradiation might explain the enhanced sensitivity of ataxia-telangiectasia (A-T) cells to X-ray. We have examined 8 lines of human fibroblasts (including 4 A-T lines) for stimulation of the synthesis of poly(ADP-R) by X-irradiation. Similar amounts of X-ray-stimulated synthesis of poly(ADP-R) were detected in 4 lines of A-T fibroblasts, and in fibrolasts from a xeroderma pigmentosum (XP) patient, a Fanconi's anemia (FA) patient and 2 normal patients. 6 lines of human lymphoblastoid lines were also examined for X-ray-stimulated poly(ADP-R) synthesis. 4 A-T lines displayed an unusually high synthesis of poly(ADP-R) in unirradiated cells compared with 2 normal lines. (orig./AJ)

Ataxia-telangiectasia cells are not uniformly deficient in poly(ADP-ribose) synthesis following X-irradiation

Energy Technology Data Exchange (ETDEWEB)

Zwelling, L.A.; Kerrigan, D. (National Cancer Inst., Bethesda, MD (USA). Lab. of Molecular Pharmacology); Mattern, M.R. (National Cancer Inst., Bethesda, MD (USA). Lab. of Molecular Carcinogenesis)

1983-04-01

The synthesis of poly(adenosine diphosphoribose (poly(ADP-R)) follows the DNA strand breakage produced by a number of physical and chemical agents, including X-radiation, and may be important for repair of several types of DNA damage. The reduction or abolition of its synthesis following X-irradiation might explain the enhanced sensitivity of ataxia-telangiectasia (A-T) cells to X-ray. We have examined 8 lines of human fibroblasts (including 4 A-T lines) for stimulation of the synthesis of poly(ADP-R) by X-irradiation. Similar amounts of X-ray-stimulated synthesis of poly(ADP-R) were detected in 4 lines of A-T fibroblasts, and in fibrolasts from a xeroderma pigmentosum (XP) patient, a Fanconi's anemia (FA) patient and 2 normal patients. 6 lines of human lymphoblastoid lines were also examined for X-ray-stimulated poly(ADP-R) synthesis. 4 A-T lines displayed an unusually high synthesis of poly(ADP-R) in unirradiated cells compared with 2 normal lines.

Metallographic investigation of fracture behavior in ITER-style Nb$_{3}$Sn superconducting strands

CERN Document Server

Jewell, M C; Larbalestier, D C; Nijhuis, A

2009-01-01

In this work we specify the extent to which fracture in two ITER-style Nb$_{3}$Sn composite strands occurs in a collective or individual manner, under mechanical tension and bending from the TARSIS apparatus at the University of Twente. A bronze-route strand from European Advanced Superconductors (EAS), which has very uniform, well-spaced filaments, has a widely distributed (200 μm) fracture field and exhibits a composite of individual and collective cracks. An internal tin strand from Oxford Instruments – Superconducting Technology (OST) demonstrates much more localized, collective fracture behavior. The filaments in this strand are about four times larger (in area) than the filaments in the EAS strand, and also agglomerate significantly during heat treatment upon conversion of the Nb to Nb$_{3}$Sn. These results demonstrate that the architecture of the strand can play a significant role in determining the mechanical toughness of the composite, and that strand design should incorporate mechanical consider...

MRI of displaced meniscal fragments

International Nuclear Information System (INIS)

Dunoski, Brian; Zbojniewicz, Andrew M.; Laor, Tal

2012-01-01

A torn meniscus frequently requires surgical fixation or debridement as definitive treatment. Meniscal tears with associated fragment displacement, such as bucket handle and flap tears, can be difficult to recognize and accurately describe on MRI, and displaced fragments can be challenging to identify at surgery. A displaced meniscal fragment can be obscured by synovium or be in a location not usually evaluated at arthroscopy. We present a pictorial essay of meniscal tears with displaced fragments in patients referred to a pediatric hospital in order to increase recognition and accurate interpretation by the radiologist, who in turn can help assist the surgeon in planning appropriate therapy. (orig.)

MRI of displaced meniscal fragments

Energy Technology Data Exchange (ETDEWEB)

Dunoski, Brian [University of Cincinnati College of Medicine, Department of Radiology, Cincinnati Children' s Hospital Medical Center, Cincinnati, OH (United States); Children' s Hospital of Michigan, Department of Radiology, Detroit, MI (United States); Zbojniewicz, Andrew M.; Laor, Tal [University of Cincinnati College of Medicine, Department of Radiology, Cincinnati Children' s Hospital Medical Center, Cincinnati, OH (United States)

2012-01-15

A torn meniscus frequently requires surgical fixation or debridement as definitive treatment. Meniscal tears with associated fragment displacement, such as bucket handle and flap tears, can be difficult to recognize and accurately describe on MRI, and displaced fragments can be challenging to identify at surgery. A displaced meniscal fragment can be obscured by synovium or be in a location not usually evaluated at arthroscopy. We present a pictorial essay of meniscal tears with displaced fragments in patients referred to a pediatric hospital in order to increase recognition and accurate interpretation by the radiologist, who in turn can help assist the surgeon in planning appropriate therapy. (orig.)

Importance of protamine phosphorylation to histone displacement in spermatids: can the disruption of this process be used for male contraception

Energy Technology Data Exchange (ETDEWEB)

Balhorn, R.; Hud, N.V.; Corzett, M.; Mazrimas, J.

1995-06-01

Protamine is a small protein that packages DNA in the sperm of most vertebrates. Shortly after its synthesis, the serine and threonine residues in each protamine are phosphorylated and the modified proteins are deposited onto DNA, displacing the histones and other chromatin proteins. We have hypothesized that the phosphorylation of protamine 1 induces protamine dimerization and these dimers are required for efficient histone displacement. Histone displacement by protamines in late-step spermatids appears to be essential for the production of fertile sperm in man and other mammals, and the disruption of this process could provide a new approach for male contraception. As a first step towards testing this theory, we have initiated a set of in vitro experiments to determine whether of not protamine phosphorylation is essential for histone displacement. Thee results of these experiments, although incomplete, confirm that unphosphorylated protamine cannot effectively displace histone from DNA. Polyarginine molecules twice the size of a protamine molecule and salmine dimer were found to be more effective. These results are consistent with the theory that the disruption of protamine phosphorylation may prove to be a useful new approach for male contraception if it can be shown to facilitate or induce protamine dimerization.

Microwave wood strand drying: energy consumption, VOC emission and drying quality

Energy Technology Data Exchange (ETDEWEB)

Wang, S.; Du, G.; Zhang, Y. [Tennessee Univ., Knoxville, TN (United States). Dept. of Forestry, Wildlife and Fisheries

2005-07-01

The objective of this research was to develop microwave drying technology for wood strand drying for oriented strand board (OSB) manufacturing. The advantages of microwave drying included a reduction in the drying time of wood strands and a reduction in the release of volatile organic compounds (VOC) through a decrease in the thermal degradation of the wood material. Temperature and moisture content changes under different microwave drying conditions were investigated. The effects of microwave drying on VOC emissions were evaluated and analyzed using gas chromatography and mass spectrometry. Microwave power input and the mass of drying materials in the microwave oven were found to have a dominant effect on drying quality. Results indicated that an increase in microwave power input and a decrease in sample weights resulted in high drying temperatures, short drying times and a high drying rate. The effect of microwave drying on the strand surfaces was also investigated. Different strand geometries and initial moisture content resulted in varying warm-up curves, but did not influence final moisture content. VOC emissions were quantified by comparing alpha-pinene concentrations. The microwave drying resulted in lower VOC emissions compared with conventional drying methods. It was concluded that the microwave drying technique provided faster strand drying and reduced energy consumption by up to 50 per cent. In addition, the surface wettability of wood strands dried with microwaves was better than with an industrial rotary drum drier. 12 refs., 3 tabs., 5 figs.

Toxin MqsR Cleaves Single-Stranded mRNA with Various 5 Ends

Science.gov (United States)

2016-08-24

either protein ORIGINAL RESEARCH Toxin MqsR cleaves single- stranded mRNA with various 5’ ends Nityananda Chowdhury1,*, Brian W. Kwan1,*, Louise C...in which a single 5′- GCU site was predicted to be single- stranded (ssRNA), double- stranded (dsRNA), in the loop of a stem - loop (slRNA), or in a...single- stranded 5′- GCU sites since cleavage was approximately 20- fold higher than cleavage seen with the 5′- GCU site in the stem - loop and

Effects of threshold displacement energy on defect production by displacement cascades in α, β and γ-LiAlO2

International Nuclear Information System (INIS)

Tsuchihira, H.; Oda, T.; Tanaka, S.

2013-01-01

Threshold displacement energy evaluation and a series of displacement cascade simulations in α, β, and γ-LiAlO 2 were performed using molecular dynamics. Threshold displacement energy evaluations indicated that higher absolute ionic charge values and larger densities both increase threshold displacement energy. The displacement cascade simulations suggest that the influence of different crystal structures on the number of interstitial atoms generated in a displacement cascade is explainable almost entirely by the difference of the threshold displacement energy

Effect of displaced versus non-displaced pelvic fractures on long-term racing performance in 31 Thoroughbred racehorses.

Science.gov (United States)

Hennessy, S E; Muurlink, M A; Anderson, G A; Puksmann, T N; Whitton, R C

2013-06-01

To evaluate the long-term racing prognosis for Thoroughbred racehorses with displaced versus non-displaced fractures of the pelvis identified by scintigraphy. Retrospective case analysis. Medical records of 31 Thoroughbred racehorses presenting to the University of Melbourne Equine Centre with fractures of the pelvis that were identified by scintigraphy were reviewed. Pelvic fracture site was determined and defined as displaced or non-displaced based on ultrasound and/or radiographic findings. Race records were analysed for each horse, with a minimum of 24 months' follow-up, and correlated with fracture type to determine long-term prognosis for racing. Results are expressed as median and range. Fractures at a single site were more common (n = 22) than fractures involving two sites (n = 9) and the ilial wing was the most commonly affected (n = 12). Thoroughbred racehorses with displaced pelvic fractures at any site (n = 12) raced fewer times within 24 months of diagnosis than horses with non-displaced fractures (n = 19) (median 0.5, range 0-13 vs 7, 0-24; P = 0.037), but there was no clear statistical difference in race earnings between the two groups (median A$0, range A$0-$123,250 vs A$14,440, A$0-$325,500, respectively; P = 0.080). Four horses with displaced fractures (33%) were euthanased on humane grounds because of persistent severe pain. When these horses were excluded from the analysis, there were no differences in performance variables between horses with a displaced or non-displaced pelvic fracture. Thoroughbred racehorses with a displaced or non-displaced pelvic fracture that survive the initial post-injury period have a good prognosis for racing. © 2013 The Authors. Australian Veterinary Journal © 2013 Australian Veterinary Association.

World-Wide Benchmarking of ITER Nb$_{3}$Sn Strand Test Facilities

CERN Document Server

Jewell, MC; Takahashi, Yoshikazu; Shikov, Alexander; Devred, Arnaud; Vostner, Alexander; Liu, Fang; Wu, Yu; Jewell, Matthew C; Boutboul, Thierry; Bessette, Denis; Park, Soo-Hyeon; Isono, Takaaki; Vorobieva, Alexandra; Martovetsky, Nicolai; Seo, Kazutaka

2010-01-01

The world-wide procurement of Nb$_{3}$Sn and NbTi for the ITER superconducting magnet systems will involve eight to ten strand suppliers from six Domestic Agencies (DAs) on three continents. To ensure accurate and consistent measurement of the physical and superconducting properties of the composite strand, a strand test facility benchmarking effort was initiated in August 2008. The objectives of this effort are to assess and improve the superconducting strand test and sample preparation technologies at each DA and supplier, in preparation for the more than ten thousand samples that will be tested during ITER procurement. The present benchmarking includes tests for critical current (I-c), n-index, hysteresis loss (Q(hys)), residual resistivity ratio (RRR), strand diameter, Cu fraction, twist pitch, twist direction, and metal plating thickness (Cr or Ni). Nineteen participants from six parties (China, EU, Japan, South Korea, Russia, and the United States) have participated in the benchmarking. This round, cond...

Self-Field Effects in Magneto-Thermal Instabilities for Nb-Sn Strands

CERN Document Server

Bordini, B; Fehér, S; Rossi, L; Zlobin, A V

2008-01-01

Recent advancements in the critical current density (Jc) of Nb$_{3}$Sn conductors, coupled with a large effective filament size, have drawn attention to the problem of magnetothermal instabilities. At low magnetic fields, the quench current of such high Jc Nb$_{3}$Sn strands is significantly lower than their critical current because of the above-mentioned instabilities. An adiabatic model to calculate the minimum current at which a strand can quench due to magneto-thermal instabilities is developed. The model is based on an 'integral' approach already used elsewhere [1]. The main difference with respect to the previous model is the addition of the self-field effect that allows to describe premature quenches of non-magnetized Nb$_{3}$Sn strands and to better calculate the quench current of strongly magnetized strands. The model is in good agreement with experimental results at 4.2 K obtained at Fermilab using virgin Modified Jelly Roll (MJR) strands with a low Residual Resistivity Ratio (RRR) of the stabilizin...

Fundamentals of displacement production in irradiated metals

International Nuclear Information System (INIS)

Doran, D.G.

1975-09-01

Radioinduced displacement damage in metals is described. Discussions are included on the displacement event itself, calculation of displacement rates in general, the manner in which different types of radiation interact with metals to produce displacements, the similarities and differences in the types of damage produced, the current status of computer simulations of displacement cascades, experimental evidence regarding cascades, and aspects of correlating damage produced by different types of radiation

Consistent errors in first strand cDNA due to random hexamer mispriming.

Directory of Open Access Journals (Sweden)

Thomas P van Gurp

Full Text Available Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly.

The role of cytosine methylation on charge transport through a DNA strand

Energy Technology Data Exchange (ETDEWEB)

Qi, Jianqing, E-mail: jqqi@uw.edu; Anantram, M. P., E-mail: anantmp@uw.edu [Department of Electrical Engineering, University of Washington, Seattle, Washington 98195-2500 (United States); Govind, Niranjan, E-mail: niri.govind@pnnl.gov [William R. Wiley Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352 (United States)

2015-09-07

Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.

Measurement of interfacial displacement of a liquid film in microchannels using laser focus displacement meter

International Nuclear Information System (INIS)

Hazuku, Tatsuya; Fukamachi, Norihiro; Takamasa, Tomoji; Hibiki, Takashi

2004-01-01

This paper presents a new method for measuring the interfacial displacement of a liquid film in microchannels using a laser focus displacement meter (LFD). The purpose of the study is to clarify the effectiveness of the new method for obtaining detailed information concerning interfacial displacement, especially in the case of a thin liquid film, in micro- and mini-channels. To prevent the tube wall signal from disturbing that of the gas-liquid interface, a fluorocarbon tube with water box was used; the refraction index of this device is same as that for water. With this method, accurate instantaneous measurements of interfacial displacement of the liquid film were achieved. The error caused by refraction of the laser beam passing through the acrylic water box and fluorocarbon tube was estimated analytically and experimentally. The formulated analytical equation can estimate the real interface displacement using measured displacement in a fluorocarbon tube of 25 μm to 2.0 mm I.D. A preliminary test using fluorocarbon tubes of 1 and 2 mm I.D. showed that the corrected interface displacement calculated by the equation agreed with real displacement within a 1% margin of error. It was also confirmed that the LFD in the system could measure a liquid film of 0.25 μm at the thinnest. We made simultaneous measurements of the interface in fluorocarbon tubes of 0.5 and 1 mm I.D. using the LFD and a high-speed video camera with a microscope. These showed that the LFD could measure the interface of a liquid film with high spatial and temporal resolution during annular, slug, and piston flow regimes. The data also clarified the existence of a thin liquid film less than 1 μm in thickness in slug and annular flow regions. (author)

Ketahanan Papan Unting (Oriented Strand Board) terhadap Serangan Rayap Tanah dan Rayap Kayu Kering

OpenAIRE

Gea, Bud diman

2011-01-01

Oriented strand board is panel of wood raw material from which strand composed of cross and upright structure. In order to oriented strand board can be used for interior and exterior needs, than necessary to be applied various thenology of quality improvement like durabelities and endurance of oriented strand board. For it does found endurance oriented strand board from attack of ground termite and wood dry termite, necessary doing grave yard trial and wood dry termite trial. This research ta...

Higher 5-hydroxymethylcytosine identifies immortal DNA strand chromosomes in asymmetrically self-renewing distributed stem cells.

Science.gov (United States)

Huh, Yang Hoon; Cohen, Justin; Sherley, James L

2013-10-15

Immortal strands are the targeted chromosomal DNA strands of nonrandom sister chromatid segregation, a mitotic chromosome segregation pattern unique to asymmetrically self-renewing distributed stem cells (DSCs). By nonrandom segregation, immortal DNA strands become the oldest DNA strands in asymmetrically self-renewing DSCs. Nonrandom segregation of immortal DNA strands may limit DSC mutagenesis, preserve DSC fate, and contribute to DSC aging. The mechanisms responsible for specification and maintenance of immortal DNA strands are unknown. To discover clues to these mechanisms, we investigated the 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) content on chromosomes in mouse hair follicle DSCs during nonrandom segregation. Although 5-methylcytosine content did not differ significantly, the relative content of 5hmC was significantly higher in chromosomes containing immortal DNA strands than in opposed mitotic chromosomes containing younger mortal DNA strands. The difference in relative 5hmC content was caused by the loss of 5hmC from mortal chromosomes. These findings implicate higher 5hmC as a specific molecular determinant of immortal DNA strand chromosomes. Because 5hmC is an intermediate during DNA demethylation, we propose a ten-eleven translocase enzyme mechanism for both the specification and maintenance of nonrandomly segregated immortal DNA strands. The proposed mechanism reveals a means by which DSCs "know" the generational age of immortal DNA strands. The mechanism is supported by molecular expression data and accounts for the selection of newly replicated DNA strands when nonrandom segregation is initiated. These mechanistic insights also provide a possible basis for another characteristic property of immortal DNA strands, their guanine ribonucleotide dependency.

Synthesis of Indium Nanowires by Galvanic Displacement and Their Optical Properties

Directory of Open Access Journals (Sweden)

Hope Greg

2008-01-01

Full Text Available Abstract Single crystalline indium nanowires were prepared on Zn substrate which had been treated in concentrated sulphuric acid by galvanic displacement in the 0.002 mol L−1In2(SO43-0.002 mol L−1SeO2-0.02 mol L−1SDS-0.01 mol L−1citric acid aqueous solution. The typical diameter of indium nanowires is 30 nm and most of the nanowires are over 30 μm in length. XRD, HRTEM, SAED and structural simulation clearly demonstrate that indium nanowires are single-crystalline with the tetragonal structure, the growth direction of the nanowires is along [100] facet. The UV-Vis absorption spectra showed that indium nanowires display typical transverse resonance of SPR properties. The surfactant (SDS and the pretreatment of Zn substrate play an important role in the growth process. The mechanism of indium nanowires growth is the synergic effect of treated Zn substrate (hard template and SDS (soft template.

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange*

Science.gov (United States)

Borgogno, María V.; Monti, Mariela R.; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E.; Pezza, Roberto J.

2016-01-01

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. PMID:26709229

Estimation of Prestress Force Distribution in the Multi-Strand System of Prestressed Concrete Structures

Directory of Open Access Journals (Sweden)

Keunhee Cho

2015-06-01

Full Text Available Prestressed concrete (PSC is one of the most reliable, durable and widely used construction materials, which overcomes the weakness of concrete in tension by the introduction of a prestress force. Smart strands enabling measurement of the prestress force have recently been developed to maintain PSC structures throughout their lifetime. However, the smart strand cannot give a representative indication of the whole prestress force when used in multi-strand systems since each strand sustains a different prestress force. In this paper, the actual distribution of the prestress force in a multi-strand system is examined using elastomagnetic (EM sensors to develop a method for tracking representative indicators of the prestress force using smart strands.

Estimation of Prestress Force Distribution in the Multi-Strand System of Prestressed Concrete Structures.

Science.gov (United States)

Cho, Keunhee; Park, Sung Yong; Cho, Jeong-Rae; Kim, Sung Tae; Park, Young-Hwan

2015-06-15

Prestressed concrete (PSC) is one of the most reliable, durable and widely used construction materials, which overcomes the weakness of concrete in tension by the introduction of a prestress force. Smart strands enabling measurement of the prestress force have recently been developed to maintain PSC structures throughout their lifetime. However, the smart strand cannot give a representative indication of the whole prestress force when used in multi-strand systems since each strand sustains a different prestress force. In this paper, the actual distribution of the prestress force in a multi-strand system is examined using elastomagnetic (EM) sensors to develop a method for tracking representative indicators of the prestress force using smart strands.

Replicating animal mitochondrial DNA

Directory of Open Access Journals (Sweden)

Emily A. McKinney

2013-01-01

Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

Mechanism and manipulation of DNA:RNA hybrid G-quadruplex formation in transcription of G-rich DNA.

Science.gov (United States)

Zhang, Jia-yu; Zheng, Ke-wei; Xiao, Shan; Hao, Yu-hua; Tan, Zheng

2014-01-29

We recently reported that a DNA:RNA hybrid G-quadruplex (HQ) forms during transcription of DNA that bears two or more tandem guanine tracts (G-tract) on the nontemplate strand. Putative HQ-forming sequences are enriched in the nearby 1000 nt region right downstream of transcription start sites in the nontemplate strand of warm-blooded animals, and HQ regulates transcription under both in vitro and in vivo conditions. Therefore, knowledge of the mechanism of HQ formation is important for understanding the biological function of HQ as well as for manipulating gene expression by targeting HQ. In this work, we studied the mechanism of HQ formation using an in vitro T7 transcription model. We show that RNA synthesis initially produces an R-loop, a DNA:RNA heteroduplex formed by a nascent RNA transcript and the template DNA strand. In the following round of transcription, the RNA in the R-loop is displaced, releasing the RNA in single-stranded form (ssRNA). Then the G-tracts in the RNA can jointly form HQ with those in the nontemplate DNA strand. We demonstrate that the structural cascade R-loop → ssRNA → HQ offers opportunities to intercept HQ formation, which may provide a potential method to manipulate gene expression.

Synthesizing Configurable Biochemical Implementation of Linear Systems from Their Transfer Function Specifications.

Directory of Open Access Journals (Sweden)

Tai-Yin Chiu

Full Text Available The ability to engineer synthetic systems in the biochemical context is constantly being improved and has a profound societal impact. Linear system design is one of the most pervasive methods applied in control tasks, and its biochemical realization has been proposed by Oishi and Klavins and advanced further in recent years. However, several technical issues remain unsolved. Specifically, the design process is not fully automated from specification at the transfer function level, systems once designed often lack dynamic adaptivity to environmental changes, matching rate constants of reactions is not always possible, and implementation may be approximative and greatly deviate from the specifications. Building upon the work of Oishi and Klavins, this paper overcomes these issues by introducing a design flow that transforms a transfer-function specification of a linear system into a set of chemical reactions, whose input-output response precisely conforms to the specification. This system is implementable using the DNA strand displacement technique. The underlying configurability is embedded into primitive components and template modules, and thus the entire system is adaptive. Simulation of DNA strand displacement implementation confirmed the feasibility and superiority of the proposed synthesis flow.

Synthesis of Carbon–Metal Multi-Strand Nanocomposites by Discharges in Heptane Between Two Metallic Electrodes

KAUST Repository

Hamdan, Ahmad; Kabbara, H.; Courty, M.-A.; Cha, Min; Martinez, J.-M.; Belmonte, T.

2017-01-01

We studied composite wires assembled from electric field-driven nanoparticles in a dielectric liquid (heptane) to elucidate the exact processes and controlling factors involved in the synthesis of the multi-phase nanocomposites. Filamentary wires

DNA strand breakage repair in ataxia telangiectasia fibroblast-like cells

Energy Technology Data Exchange (ETDEWEB)

Vincent, Jr, R A; Sheridan, III, R B; Huang, P C [Johns Hopkins Univ., Baltimore, Md. (USA). Dept. of Environmental and Biophysical Sciences

1975-12-01

Human diploid fibroblast-like cells derived from four patients with the genetic disease ataxia telangiectasia and from two non-mutant donors were examined for the repair of x-ray induced strand breaks in DNA. The ataxia telangiectasia cultures showed no significant differences from the non-mutant cultures in the kinetics and extent of strand repair. This suggests that the increased spontaneous and x-ray induced chromatid aberrations observed in ataxia telangiectasia cells are not caused by a defect in the repair of single strand breaks as might be suspected from a general model of aberration production.

Survey of stranded gas and delivered costs to Europe of selected gas resources

Science.gov (United States)

Attanasi, E.D.; Freeman, P.A.

2011-01-01

Two important trends affecting the expected growth of global gas markets are (1) the shift by many industrialized countries from coal-fired electricity generation to the use of natural gas to generate electricity and (2) the industrialization of the heavily populated Asian countries of India and China. This paper surveys discovered gas in stranded conventional gas accumulations and presents estimates of the cost of developing and producing stranded gas in selected countries. Stranded gas is natural gas in discovered or identified fields that is not currently commercially producible for either physical or economic reasons. Published reserves of gas at the global level do not distinguish between volumes of gas in producing fields and volumes in nonproducing fields. Data on stranded gas reported here-that is the volumes, geographical distribution, and size distributions of stranded gas fields at the country and regional level-are based on the examination of individual-field data and represent a significant improvement in information available to industry and government decision makers. Globally, stranded gas is pervasive, but large volumes in large accumulations are concentrated in only a few areas. The cost component of the paper focuses on stranded conventional gas accumulations in Africa and South America that have the potential to augment supplies to Europe. The methods described for the computation of extraction and transport costs are innovative in that they use information on the sizes and geographical distribution of the identified stranded gas fields. The costs are based on industry data specific to the country and geologic basin where the stranded gas is located. Gas supplies to Europe can be increased significantly at competitive costs by the development of stranded gas. Net extraction costs of producing the identified gas depend critically on the natural-gas-liquids (NGLs) content, the prevailing prices of liquids, the size of the gas accumulation, and the

Knotting probabilities after a local strand passage in unknotted self-avoiding polygons

International Nuclear Information System (INIS)

Szafron, M L; Soteros, C E

2011-01-01

We investigate, both theoretically and numerically, the knotting probabilities after a local strand passage is performed in an unknotted self-avoiding polygon (SAP) on the simple cubic lattice. In the polygons studied, it is assumed that two polygon segments have already been brought close together for the purpose of performing a strand passage. This restricts the polygons considered to those that contain a specific pattern called Θ at a fixed location; an unknotted polygon containing Θ is called a Θ-SAP. It is proved that the number of n-edge Θ-SAPs grows exponentially (with n) at the same rate as the total number of n-edge unknotted SAPs (those with no prespecified strand passage structure). Furthermore, it is proved that the same holds for subsets of n-edge Θ-SAPs that yield a specific after-strand-passage knot-type. Thus, the probability of a given after-strand-passage knot-type does not grow (or decay) exponentially with n. Instead, it is conjectured that these after-strand-passage knot probabilities approach, as n goes to infinity, knot-type dependent amplitude ratios lying strictly between 0 and 1. This conjecture is supported by numerical evidence from Monte Carlo data generated using a composite (aka multiple) Markov chain Monte Carlo BFACF algorithm developed to study Θ-SAPs. A new maximum likelihood method is used to estimate the critical exponents relevant to this conjecture. We also obtain strong numerical evidence that the after-strand-passage knotting probability depends on the local structure around the strand-passage site. If the local structure and the crossing sign at the strand-passage site are considered, then we observe that the more 'compact' the local structure, the less likely the after-strand-passage polygon is to be knotted. This trend for compactness versus knotting probability is consistent with results obtained for other strand-passage models; however, we are the first to note the influence of the crossing-sign information. We

Displacement functions for diatomic materials

International Nuclear Information System (INIS)

Panrkin, D.M.; Coulter, C.A.

1979-01-01

An extension of the methods of Lindhard et at. was used to calculate the total displacement function n/sub ij/(E) for a number of diatomic materials, where n/sub ij/(E) is defined to be the average number of atoms of type j which are displaced from their sites in a displacement cascade initiated by a PKA of type i and energy E. From the n/sub ij/(E) one can calculate the fraction n/sub ij/(E) of the displacements produced by a type i PKA with energy E which are of type j. Values of the n/sub ij/ for MgO, CaO, Al 2 O 3 , and TaO are presented. It is shown that for diatomic materials with mass ratios reasonably near one (e.g., MgO, Al 2 O 3 ) and equal displacement thresholds for the two species the n/sub ij/ become independent of the PKA type i at energies only a few times threshold. However, for larger mass ratios the n/sub ij/ do not become independent of i until much larger, energies are reached - e.g. > 10 5 eV for TaO. In addition, it is found that the n/sub ij/ depend sensitively on the displacement thresholds, with very dramatic charges occuring when the two thresholds become significantly different from one another

Oriented-strand-board- the wave of the future- for the building trade

Science.gov (United States)

Linda Ashton

1984-01-01

Move over, plywood. Oriented-strand board is here. It's less expensive. It's as durable. It has as many uses. And it is the wave of the future. "Oriented-strand board is a direct substitute for plywood" said Jerry Buckner, plant manager for the Martco oriented-strand board plant in Lemoyen. OSB, as it is commonly called, is a structural panel made...

75 FR 62820 - Screening Framework Guidance for Providers of Synthetic Double-Stranded DNA

Science.gov (United States)

2010-10-13

... Providers of Synthetic Double- Stranded DNA AGENCY: Department of Health and Human Services, Office of the.... Government has developed Guidance that provides a framework for screening synthetic double-stranded DNA (dsDNA). This document, the Screening Framework Guidance for Providers of Synthetic Double-Stranded DNA...

Stress-rupture lifetimes of organic fiber-epoxy strands and pressure vessels

International Nuclear Information System (INIS)

Hahn, H.T.; Chiu, I.L.; Gates, T.L.

1979-01-01

Long-term behavior of filament-wound pressure vessels were tested, Kevlar 49 epoxy strands were studied in stress-rupture for more than a year. Because the strands are the smallest structural unit in filament winding, their behavior directly controls the performance of vessels. Five different stress levels were studied: 86, 80, 74, 68, and 50% of the mean ultimate tensile strength (UTS). At each stress level, approximately one-hundred strands were hung in a room maintained at 22 to 24 0 C and below 20% relative humidity. Failure times were automatically recorded by a data acquisition system. Lifetimes were analyzed statistically using a two-parameter Weibull distribution. The maximum-likelihood method was used to estimate the parameters. The shape parameter, which is a measure of scatter and failure-rate change, increased with decreasing stress level. Less scatter and increasing failure rates were observed at lower stresses. There was no sign of an endurance limit down to 68% UTS. At 50% UTS no failure had yet occurred after 9000 h. The strand data were compared with data on lifetimes of pressure vessels wound with the same fiber and epoxy. The strands had slightly longer characteristic lifetimes, except at 86% UTS, and slightly less scatter, except at 68% UTS. The results of this study indicate that strands can provide valuable information about the long-term performance of filament-wound pressure vessels

Etiopathogenesis of abomasal displacement in cattle

Directory of Open Access Journals (Sweden)

Šamanc Horea

2003-01-01

Full Text Available Abomasal displacement presents topographic gastropathy, where this organ has changed its position, and there is simultaneous dilatation which can vary in intensity. The incidence of this disorder in herds of high-yield dairy cows varies to a great degree (1 to 18 %. Abomasal displacement was established in herds of East-Frisian cows in 1 to 3% animals, and in Holstein cow herds in 5 to 18 % animals. The most frequent abomasal displacement is to the left (88%. There is significant seasonal variation in the incidence of abomasal displacement. About two-thirds of cases of abomasal displacement are diagnosed from October until April. The disorder appears more frequently in cows with repeated lactations. It has been established that it appears after the first calving in 27.8% cases, after the second to fifth calving in 66.7% cases, and after the sixth and seventh calving in 5.5% of the cows. The response of endocrine pancreas B-cells for insulin secretion to hyperglycaemia caused by applying an excess-glucose test is reduced in cows with left abomasal displacement, and there is constant hyperglycaemia in cows with right abomasal displacement. The excess-glucose test indicates a disrupted function of the endocrine pancreas in diseased animals. It has been determined through examinations of Aml genotypes in Holstein cow herds in connection with the appearance of abomasal displacement, that the occurrence of this disorder cannot be attributed to a genetic predisposition.

Conflict, displacement and health in the Middle East.

Science.gov (United States)

Mowafi, Hani

2011-01-01

Displacement is a hallmark of modern humanitarian emergencies. Displacement itself is a traumatic event that can result in illness or death. Survivors face challenges including lack of adequate shelter, decreased access to health services, food insecurity, loss of livelihoods, social marginalisation as well as economic and sexual exploitation. Displacement takes many forms in the Middle East and the Arab World. Historical conflicts have resulted in long-term displacement of Palestinians. Internal conflicts have driven millions of Somalis and Sudanese from their homes. Iraqis have been displaced throughout the region by invasion and civil strife. In addition, large numbers of migrants transit Middle Eastern countries or live there illegally and suffer similar conditions as forcibly displaced people. Displacement in the Middle East is an urban phenomenon. Many displaced people live hidden among host country populations in poor urban neighbourhoods - often without legal status. This represents a challenge for groups attempting to access displaced populations. Furthermore, health information systems in host countries often do not collect data on displaced people, making it difficult to gather data needed to target interventions towards these vulnerable populations. The following is a discussion of the health impacts of conflict and displacement in the Middle East. A review was conducted of published literature on migration and displacement in the region. Different cases are discussed with an emphasis on the recent, large-scale and urban displacement of Iraqis to illustrate aspects of displacement in this region.

Gender-based violence in conflict and displacement: qualitative findings from displaced women in Colombia.

Science.gov (United States)

Wirtz, Andrea L; Pham, Kiemanh; Glass, Nancy; Loochkartt, Saskia; Kidane, Teemar; Cuspoca, Decssy; Rubenstein, Leonard S; Singh, Sonal; Vu, Alexander

2014-01-01

Gender-based violence (GBV) is prevalent among, though not specific to, conflict affected populations and related to multifarious levels of vulnerability of conflict and displacement. Colombia has been marked with decades of conflict, with an estimated 5.2 million internally displaced persons (IDPs) and ongoing violence. We conducted qualitative research to understand the contexts of conflict, displacement and dynamics with GBV. This as part of a multi-phase, mixed method study, in collaboration with UNHCR, to develop a screening tool to confidentially identify cases of GBV for referral among IDP women who were survivors of GBV. Qualitative research was used to identify the range of GBV, perpetrators, contexts in conflict and displacement, barriers to reporting and service uptake, as well as to understand experiences of service providers. Thirty-five female IDPs, aged 18 years and older, who self-identified as survivors of GBV were enrolled for in-depth interviews in San Jose de Guaviare and Quibdo, Colombia in June 2012. Thirty-one service providers participated in six focus group discussions and four interviews across these sites. Survivors described a range of GBV across conflict and displacement settings. Armed actors in conflict settings perpetrated threats of violence and harm to family members, child recruitment, and, to a lesser degree, rape and forced abortion. Opportunistic violence, including abduction, rape, and few accounts of trafficking were more commonly reported to occur in the displacement setting, often perpetrated by unknown individuals. Intrafamilial violence, intimate partner violence, including physical and sexual violence and reproductive control were salient across settings and may be exacerbated by conflict and displacement. Barriers to reporting and services seeking were reported by survivors and providers alike. Findings highlight the need for early identification of GBV cases, with emphasis on confidential approaches and active

Transformation of Saccharomyces cerevisiae with UV-irradiated single-stranded plasmid.

Science.gov (United States)

Zgaga, Z

1991-08-01

UV-irradiated single-stranded replicative plasmids were used to transform different yeast strains. The low doses of UV used in this study (10-75 J/m2) caused a significant decrease in the transforming efficiency of plasmid DNA in the Rad+ strain, while they had no effect on transformation with double-stranded plasmids of comparable size. Neither the rev3 mutation, nor the rad18 or rad52 mutations influenced the efficiency of transformation with irradiated single-stranded plasmid. However, it was found to be decreased in the double rev3 rad52 mutant. Extracellular irradiation of plasmid that contains both URA3 and LEU2 genes (psLU) gave rise to up to 5% Leu- transformants among selected Ura+ ones in the repair-proficient strain. Induction of Leu- transformants was dose-dependent and only partially depressed in the rev3 mutant. These results suggest that both mutagenic and recombinational repair processes operate on UV-damaged single-stranded DNA in yeast.

Metallographic autopsies of full-scale ITER prototype cable-in-conduit conductors after full cyclic testing in SULTAN: II. Significant reduction of strand movement and strand damage in short twist pitch CICCs

International Nuclear Information System (INIS)

Sanabria, Carlos; Lee, Peter J; Starch, William; Larbalestier, David C; Devred, Arnaud

2015-01-01

Prototype cable-in-conduit-conductors (CICCs) destined for use in the toroidal field and central solenoid coils of the ITER experimental fusion reactor underwent severe cyclic loading in the SULTAN facility. Their autopsies revealed significant and permanent transverse strand migration due to the large Lorentz forces of the SULTAN test. The movement resulted in a 3%–7% void fraction increase on the low pressure (LP) side of the longer twist pitch CICCs. However, short twist pitch conductors exhibited less than 1% void fraction increase in the LP side, as well as a complete absence of the Nb 3 Sn filament fractures observed in the longer twist pitch conductors. We report here a detailed strand-to-cable analysis of short and longer ‘baseline’ twist pitch CICCs. It was found that the use of internal tin (IT) strands in the longer ‘baseline’ twist pitch CICCs can be beneficial possibly because of their superior stiffness—which better resist strand movement—while the use of bronze process strands showed more movement and poorer cyclic test performance. This was not the case for the short twist pitch CICC. Such conductor design seems to work well with both strand types. But it was found that despite the absence of filament fractures, the short twist pitch CICC made from the IT strands studied here developed severe strand distortion during cabling which resulted in diffusion barrier breaks and Sn contamination of the Cu stabilizer during the heat treatment. Conversely, the short twist pitch CICC made from bronze process strands preserved diffusion barrier integrity. (paper)

Workshop on effects of chromium coating on Nb3Sn superconductor strand: Proceedings

International Nuclear Information System (INIS)

1994-01-01

This report discusses the following topics: Chromium coating on superconductor strand -- an overview; technology of chromium plating; comparison of wires plated by different platers; search for chromium in copper; strand manufactures' presentations; chromium plating at the Lawrence Livermore National Laboratory; a first look at a chromium plating process development project tailored for T.P.X. and I.T.E.R. strand; and influence of chromium diffusion and related phenomena on the reference ratios of bare and chromium plated Nb 3 Sn strand

Effect of structure variation of the aptamer-DNA duplex probe on the performance of displacement-based electrochemical aptamer sensors.

Science.gov (United States)

Pang, Jie; Zhang, Ziping; Jin, Haizhu

2016-03-15

Electrochemical aptamer-based (E-AB) sensors employing electrode-immobilized, redox-tagged aptamer probes have emerged as a promising platform for the sensitive and quick detection of target analytes ranging from small molecules to proteins. Signal generation in this class of sensor is linked to change in electron transfer efficiency upon binding-induced change in flexibility/conformation of the aptamer probe. Because of this signaling mechanism, signal gains of these sensors can be improved by employing a displacement-based recognition system, which links target binding with a large-scale flexibility/conformation shift from the aptamer-DNA duplex to the single-stranded DNA or the native aptamer. Despite the relatively large number of displacement-based E-AB sensor samples, little attention has been paid to the structure variation of the aptamer-DNA duplex probe. Here we detail the effects of complementary length and position of the aptamer-DNA duplex probe on the performance of a model displacement-based E-AB sensor for ATP. We find that, greater background suppression and signal gain are observed with longer complementary length of the aptamer-DNA duplex probe. However, sensor equilibration time slows monotonically with increasing complementary length; and with too many target binding sites in aptamer sequence being occupied by the complementary DNA, the aptamer-target binding does not occur and no signal gain observed. We also demonstrate that signal gain of the displacement-based E-AB sensor is strongly dependent on the complementary position of the aptamer-DNA duplex probe, with complementary position located at the electrode-attached or redox-tagged end of the duplex probe, larger background suppression and signal increase than that of the middle position are observed. These results highlight the importance of rational structure design of the aptamer-DNA duplex probe and provide new insights into the optimization of displacement-based E-AB sensors. Copyright

Displacement sensing system and method

Science.gov (United States)

VunKannon, Jr., Robert S

2006-08-08

A displacement sensing system and method addresses demanding requirements for high precision sensing of displacement of a shaft, for use typically in a linear electro-dynamic machine, having low failure rates over multi-year unattended operation in hostile environments. Applications include outer space travel by spacecraft having high-temperature, sealed environments without opportunity for servicing over many years of operation. The displacement sensing system uses a three coil sensor configuration, including a reference and sense coils, to provide a pair of ratio-metric signals, which are inputted into a synchronous comparison circuit, which is synchronously processed for a resultant displacement determination. The pair of ratio-metric signals are similarly affected by environmental conditions so that the comparison circuit is able to subtract or nullify environmental conditions that would otherwise cause changes in accuracy to occur.

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.

Science.gov (United States)

Borgogno, María V; Monti, Mariela R; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E; Pezza, Roberto J

2016-03-04

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Performance of Nb3Sn RRP strands and cables based on a 108/127 stack design

International Nuclear Information System (INIS)

Barzi, E.; Ambrosio, G.; Andreev, N.; Bossert, R.; Carcagno, R.; Feher, S.; Kashikhin, V.S.; Kashikhin, V.V.; Lamm, M.J.; Nobrega, F.; Novitski, I.; Pishalnikov, Y.; Sylvester, C.; Tartaglia, M.; Turrioni, D.; Yamada, R.; Zlobin, A.V.; Field, M.; Hong, S.; Parrell, J.; Zhang, Y.

2006-01-01

The high performance Nb 3 Sn strand produced by Oxford Superconducting Technology (OST) with the Restack Rod Process (RRP) is presently considered as a baseline conductor for the Fermilab's accelerator magnet R and D program. To improve the strand stability in the current and field range expected in magnet models, the number of subelements in the strand was increased by a factor of two (from 54 to 108), which resulted in a smaller effective filament size. The performance of the 1.0 and 0.7 mm strands of this design was studied using virgin and deformed strand samples. 27-strand Rutherford cables made of 1 mm strand were also tested using a superconducting transformer, small racetrack and 1-m shell-type dipole coils. This paper presents the RRP strand and cable parameters, and reports the results of strand, cable and coil testing

Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA

DEFF Research Database (Denmark)

Thresher, RJ; Christiansen, Gunna; Griffith, JD

1988-01-01

We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional Rec......M in the presence of 12 mM-Mg2+), and relatively low concentrations of SSB protein (1 monomer per 18 nucleotides). Assembly was depressed threefold when SSB protein was added to one monomer per nine nucleotides. These effects appeared to be exerted at the nucleation step. Following nucleation, RecA protein...... assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein...

Termination of DNA synthesis in vitro at apurinic sites but not at ethyl adducts of the template

Energy Technology Data Exchange (ETDEWEB)

Lockhart, M.L.; Deutsch, J.F.; Yamaura, I.; Cavalieri, L.F.; Rosenberg, B.H.

1982-01-01

The effects of DNA lesions produced by the carcinogenic alkylating agents ethylnitrosourea and diethylsulfate on the extent of DNA synthesis have been studied in a system utilizing circular single-stranded phi X174 DNA as template and a 392-base restriction fragment as primer with E. coli polymerase I (Klenow fragment). Apurinic sites produced by loss of unstable ethylated bases from the template terminate DNA synthesis at the first such site encountered, but ethyl adducts at most, if not all, locations permit readthrough. 22 references, 3 figures, 1 table.

Geomorphic and geologic evidence for slip along the San Bernardino strand of the San Andreas Fault System through the San Gorgonio Pass structural knot, southern California

Science.gov (United States)

Kendrick, K. J.; Matti, J. C.

2017-12-01

The San Gorgonio Pass (SGP) region of southern California represents an extraordinarily complex section of the San Andreas Fault (SAF) zone, often referred to as a structural knot. Complexity is expressed both structurally and geomorphically, and arises because multiple strands of the SAF have evolved here in Quaternary time. Our integration of geologic and geomorphic analyses led to recognition of multiple fault-bounded blocks characterized by crystalline rocks that have similar physical properties. Hence, any morphometric differences in hypsometric analysis, slope, slope distribution, texture, and stream-power measurements and discontinuities reflect landscape response to tectonic processes rather than differences in lithology. We propose that the differing morphometry of the two blocks on either side of the San Bernardino strand (SBS) of the SAF, the high-standing Kitching Peak block to the east and the lower, more subdued Pisgah Peak block to the west, strongly suggests that the blocks experienced different uplift histories. This difference in uplift histories, in turn suggests that dextral slip occurred over a long time interval on the SBS—despite long-lived controversy raised by the fact that, at the surface, a throughgoing trace of the SBS is not present at this location. A different tectonic history between the two blocks is consistent with the gravity data which indicate that low-density rocks underthrusting the Kitching Peak block are absent below the Pisgah Peak block (Langenheim et al., 2015). Throughgoing slip on the SBS implied by geomorphic differences between the two blocks is also consistent with displaced geologic and geomorphic features. We find compelling evidence for discrete offsets of between 0.6 and 6 km of dextral slip on the SBS, including offset of fluvial and landslide deposits, and beheaded drainages. Although we lack numerical age control for the offset features, the degree of soil development associated with displaced landforms

Effects of Fault Displacement on Emplacement Drifts

International Nuclear Information System (INIS)

Duan, F.

2000-01-01

The purpose of this analysis is to evaluate potential effects of fault displacement on emplacement drifts, including drip shields and waste packages emplaced in emplacement drifts. The output from this analysis not only provides data for the evaluation of long-term drift stability but also supports the Engineered Barrier System (EBS) process model report (PMR) and Disruptive Events Report currently under development. The primary scope of this analysis includes (1) examining fault displacement effects in terms of induced stresses and displacements in the rock mass surrounding an emplacement drift and (2 ) predicting fault displacement effects on the drip shield and waste package. The magnitude of the fault displacement analyzed in this analysis bounds the mean fault displacement corresponding to an annual frequency of exceedance of 10 -5 adopted for the preclosure period of the repository and also supports the postclosure performance assessment. This analysis is performed following the development plan prepared for analyzing effects of fault displacement on emplacement drifts (CRWMS M and O 2000). The analysis will begin with the identification and preparation of requirements, criteria, and inputs. A literature survey on accommodating fault displacements encountered in underground structures such as buried oil and gas pipelines will be conducted. For a given fault displacement, the least favorable scenario in term of the spatial relation of a fault to an emplacement drift is chosen, and the analysis is then performed analytically. Based on the analysis results, conclusions are made regarding the effects and consequences of fault displacement on emplacement drifts. Specifically, the analysis will discuss loads which can be induced by fault displacement on emplacement drifts, drip shield and/or waste packages during the time period of postclosure

Stranding of two sperm whales (Physeter macrocephalus) in the "North Sea trap" at Henne Strand, Denmark

DEFF Research Database (Denmark)

Hansen, Mette Sif; Alstrup, Aage K. O.; Hansen, Jørgen H.

2016-01-01

In February 2014 two male sperm whales (Physeter macrocephalus) stranded at Henne Strand, Denmark. One whale (MCE 1644) was found dead, while the other (MCE 1645) was still alive, but drowned during the high tide. To increase our knowledge of sperm whales, conduct forage investigations, post......-mortem and diagnostic examinations were carried out. The decay of the carcasses progressed quickly. The whales had large (MCE 1644) or moderate (MCE 1645) numbers of squid beaks (Gonatus fabricii) in the stomachventricles, but no evidence of recentfresh feeding. Both whales had acute dermatitis probably due to trauma...... severe localized or systemic infections. The finding of large volumes of bloody pleural fluid with large quantities of C. septicum suggests that MCE 1644 died of infection. However, reservations must be taken due to the pronounced decay of the carcass. Sperm whales have strong social bonds where...

Molecular mechanisms of induced mutagenesis. Replication in vivo of bacteriophage phiX174 single-stranded, ultraviolet light-irradiated DNA in intact and irradiated host cells

Energy Technology Data Exchange (ETDEWEB)

Caillet-Fauquet, P; Defais, M; Radman, M [Brussels Univ. (Belgium)

1977-11-25

Genetic analysis has revealed that radiation and many chemical mutagens induce in bacteria an error-prone DNA repair process which is responsible for their mutagenic effect. The biochemical mechanism of this inducible error-prone repair has been studied by analysis of the first round of DNA synthesis on ultraviolet light-irradiated phiX174 DNA in both intact and ultraviolet light-irradiated host cells. Intracellular phiX174 DNA was extracted, subjected to isopycnic CsCl density-gradient analysis, hydroxylapatite chromatography and digestion by single-strand-specific endonuclease S/sub 1/. Ultraviolet light-induced photolesions in viral DNA cause a permanent blockage of DNA synthesis in intact Escherichia coli cells. However, when host cells were irradiated and incubated to induce fully the error-prone repair system, a significant fraction of irradiated phiX174 DNA molecules can be fully replicated. Thus, inducible error-prone repair in E.coli is manifested by an increased capacity for DNA synthesis on damaged phiX174 DNA. Chloramphenicol (100 ..mu.. g/ml), which is an inhibitor of the inducible error-prone DNA repair, is also an inhibitor of this particular inducible DNA synthesis.

Strand critical current degradation in $Nb_{3}$ Sn Rutherford cables

CERN Document Server

Barzi, E; Higley, H C; Scanlan, R M; Yamada, R; Zlobin, A V

2001-01-01

Fermilab is developing 11 Tesla superconducting accelerator magnets based on Nb/sub 3/Sn superconductor. Multifilamentary Nb/sub 3/Sn strands produced using the modified jelly roll, internal tin, and powder-in-tube technologies were used for the development and test of the prototype cable. To optimize the cable geometry with respect to the critical current, short samples of Rutherford cable with packing factors in the 85 to 95% range were fabricated and studied. In this paper, the results of measurements of critical current, n-value and RRR made on the round virgin strands and on the strands extracted from the cable samples are presented. (5 refs).

Performance of Nb3Sn RRP strands and cables based on a 108/127 stack design

Energy Technology Data Exchange (ETDEWEB)

Barzi, E.; Ambrosio, G.; Andreev, N.; Bossert, R.; Carcagno, R.; Feher, S.; Kashikhin, V.S.; Kashikhin, V.V.; Lamm, M.J.; Nobrega, F.; Novitski, I.; /Fermilab /Oxford Supercond. Tech., Carteret

2006-08-01

The high performance Nb{sub 3}Sn strand produced by Oxford Superconducting Technology (OST) with the Restack Rod Process (RRP) is presently considered as a baseline conductor for the Fermilab's accelerator magnet R&D program. To improve the strand stability in the current and field range expected in magnet models, the number of subelements in the strand was increased by a factor of two (from 54 to 108), which resulted in a smaller effective filament size. The performance of the 1.0 and 0.7 mm strands of this design was studied using virgin and deformed strand samples. 27-strand Rutherford cables made of 1 mm strand were also tested using a superconducting transformer, small racetrack and 1-m shell-type dipole coils. This paper presents the RRP strand and cable parameters, and reports the results of strand, cable and coil testing.

Overproduction of the poly(ADP-ribose)polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.

NARCIS (Netherlands)

M. Molinete; W. Vermeulen (Wim); A. Bürkle; J. Mé nissier-de Murcia; J.H. Küpper; J.H.J. Hoeijmakers (Jan); G. de Murcia

1993-01-01

textabstractThe zinc-finger DNA-binding domain (DBD) of poly (ADP-ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA-damaging agents in eukaryotes. This, in turn, triggers the synthesis of polymers of ADP-ribose linked to nuclear proteins during

Displacement length and velocity of tagged logs in the tagliamento river

Directory of Open Access Journals (Sweden)

Diego Ravazzolo

2013-09-01

Full Text Available Large wood enhance the dynamics of geomorphic processes in river systems, increases the morphological complexity of the channel bed, and provides habitats for fish and invertebrates. On the other side, if transported during high-magnitude events, large wood pieces can increase flood risks in sensitive places such as bridges and narrow cross sections prone to outbank flows. However, the dynamics and mobility of logs in rivers is poorly understood, especially in wide gravel-bed rivers. Recent studies have employed fixed video cameras to assess logs velocity, but little evidence is still available about travel length during flood events of different magnitude. This study was conducted in a valley reach of the Tagliamento river, located in the North East of Italy. The Tagliamento river is approximately 800 m wide in the study area, and is characterized by relatively high natural conditions and complex fluvial dynamics. Log mobility have been studied from June 2010 to October 2011, a period characterized by a relatively high magnitude flood in November 2010. Log mobility and displacement during floods have been measured by implanting active radio transmitters (RFID in 113 logs and GPS track devices in 42 logs. The first devices allow to recover the log after flood events by using a portable antenna, and to derive the displacement length over the monitoring period, whereas the second devices allows to calculate instantaneous (1 sec and average log velocity of moving logs. Recovery rate of logs equipped with RFID and GPS was about 50% and 60%, respectively. A preliminary analysis of the data collected indicates that there is a positive relationship between displacement length and the peak of flood events, as well as a positive relationship between log velocity and the flood magnitude. Also, a critical flow rate over which logs stranded on active bars can be transported has been identified. The ability to predict wood mobility in gravel-bed rivers could

Possible roles of HIV-1 nucleocapsid protein in the specificity of proviral DNA synthesis and in its variability.

Science.gov (United States)

Lapadat-Tapolsky, M; Gabus, C; Rau, M; Darlix, J L

1997-05-02

Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it coats the dimeric RNA genome. Due to its nucleic acid binding and annealing activities, NC protein directs the annealing of the tRNA primer to the primer binding site and greatly facilitates minus strand DNA elongation and transfer while protecting the nucleic acids against nuclease degradation. To understand the role of NCp7 in viral DNA synthesis, we examined the influence of NCp7 on self-primed versus primer-specific reverse transcription. The results show that HIV-1 NCp7 can extensively inhibit self-primed reverse transcription of viral and cellular RNAs while promoting primer-specific synthesis of proviral DNA. The role of NCp7 vis-a-vis the presence of mutations in the viral DNA during minus strand elongation was examined. NCp7 maximized the annealing between a cDNA(-) primer containing one to five consecutive errors and an RNA representing the 3' end of the genome. The ability of reverse transcriptase (RT) in the presence of NCp7 to subsequently extend the mutated primers depended upon the position of the mismatch within the primer:template complex. When the mutations were at the polymerisation site, primer extension by RT in the presence of NCp7 was very high, about 40% for one mismatch and 3% for five consecutive mismatches. Mutations within the DNA primer or at its 5' end had little effect on the extension of viral DNA by RT. Taken together these results indicate that NCp7 plays major roles in proviral DNA synthesis within the virion core due to its ability to promote prime-specific proviral DNA synthesis while concurrently inhibiting non-specific reverse transcription of viral and cellular RNAs. Moreover, the observation that NCp7 enhances the incorporation of mutations during minus strand DNA elongation favours the notion that NCp7 is a factor contributing to the high mutation rate of HIV-1.

High-displacement spiral piezoelectric actuators

Science.gov (United States)

Mohammadi, F.; Kholkin, A. L.; Jadidian, B.; Safari, A.

1999-10-01

A high-displacement piezoelectric actuator, employing spiral geometry of a curved piezoelectric strip is described. The monolithic actuators are fabricated using a layered manufacturing technique, fused deposition of ceramics, which is capable of prototyping electroceramic components with complex shapes. The spiral actuators (2-3 cm in diameter) consisted of 4-5 turns of a lead zirconate titanate ceramic strip with an effective length up to 28 cm. The width was varied from 0.9 to 1.75 mm with a height of 3 mm. When driven by the electric field applied across the width of the spiral wall, the tip of the actuator was found to displace in both radial and tangential directions. The tangential displacement of the tip was about 210 μm under the field of 5 kV/cm. Both the displacement and resonant frequency of the spirals could be tailored by changing the effective length and wall width. The blocking force of the actuator in tangential direction was about 1 N under the field of 5 kV/cm. These properties are advantageous for high-displacement low-force applications where bimorph or monomorph actuators are currently employed.

DNA replication and the repair of DNA strand breaks in nuclei of Physarum polycephalum. Terminal report, August 1, 1978-March 31, 1980

International Nuclear Information System (INIS)

Brewer, E.N.; Evans, T.E.

1980-01-01

Nuclei isolated from Physarum are able to replicate approximately 15% of the total genome in a manner which is qualitatively similar to the DNA replication process occurring in the intact organism. Such nuclei, however, are defective in the joining of Okazaki intermediates in vitro. Two DNA polymerase species, isolated from nuclei or intact plasmodia of this organism, can be separated by sucrose density gradient centrifugation. Total DNA polymerase activity is low in nuclei isolated during mitosis. A heat-stable glycoprotein material present in aqueous nuclear extracts stimulates DNA synthesis in well-washed nuclei. A sub-nuclear preparation active in DNA synthesis in vitro has been obtained from isolated nuclei of Physarum. Radiation-induced DNA double-strand breaks are rejoined in intact plasmodia and isolated nuclei of Physarum in a cell cycle-dependent manner. This phenomenon does not appear to be due to an intrinsic difference in nuclear DNA endonuclease activity at different times of the mitotic cycle. DNA strand breaks and repair induced by the carcinogen 4-nitroquinoline-1-oxide is similar in several respects to that resulting from exposure of the organism to ionizing radiation. Temperature sensitive strains of Physarum have been constructed and preliminary genetical and biochemical characterizations have been carried out. Two of the strains appear to be conditionally defective in DNA metabolism. An isogenic ploidal series of amoebae has been prepared and characterized as to uv and ionizing radiation sensitivity (in terms of cell survival). There is a direct relationship between ploidy and resistance to uv whereas ploidal change does not appear to affect the response to ionizing radiation

Effects of the ssb-1 and ssb-113 mutations on survival and DNA repair in UV-irradiated delta uvrB strains of Escherichia coli K-12.

OpenAIRE

Wang, T C; Smith, K C

1982-01-01

The molecular defect in DNA repair caused by ssb mutations (single-strand binding protein) was studied by analyzing DNA synthesis and DNA double-strand break production in UV-irradiated Escherichia coli delta uvrB strains. The presence of the ssb-113 mutation produced a large inhibition of DNA synthesis and led to the formation of double-strand breaks, whereas the ssb-1 mutation produced much less inhibition of DNA synthesis and fewer double-strand breaks. We suggest that the single-strand bi...

Induction and repair of double- and single-strand DNA breaks in bacteriophage lambda superinfecting Escherichia coli

International Nuclear Information System (INIS)

Boye, E.; Krisch, R.E.

1980-01-01

Induction and repair of double-and single-strand DNA breaks have been measured after decays of 125 I and 3 H incorporated into the DNA and after external irradiation with 4 MeV electrons. For the decay experiments, cells of wild type Escherichia coli K-12 were superinfected with bacteriophage lambda DNA labelled with 5'-( 125 I)iodo-2'-deoxyuridine or with (methyl- 3 H)thymidine and frozen in liquid nitrogen. Aliquots were thawed at intervals and lysed at neutral pH, and the phage DNA was assayed for double- and single-strand breakage by neutral sucrose gradient centrifugation. The gradients used allowed measurements of both kinds of breaks in the same gradient. Decays of 125 I induced 0.39 single-strand breaks per double-strand break. No repair of either break type could be detected. Each 3 H disintegration caused 0.20 single-strand breaks and very few double-strand breaks. The single-strand breaks were rapidly rejoined after the cells were thawed. For irradiation with 4 MeV electrons, cells of wild type E. coli K-12 were superinfected with phage lambda and suspended in growth medium. Irradiation induced 42 single-strand breaks per double-strand break. The rates of break induction were 6.75 x 10 -14 (double-strand breaks) and 2.82 x 10 -12 (single-strand breaks) per rad and per dalton. The single-strand breaks were rapidly repaired upon incubation whereas the double-strand breaks seemed to remain unrepaired. It is concluded that double-strand breaks in superinfecting bacteriophage lambda DNA are repaired to a very small extent, if at all. (Author)

Mammalian DNA single-strand break repair: an X-ra(y)ted affair.

Science.gov (United States)

Caldecott, K W

2001-05-01

The genetic stability of living cells is continuously threatened by the presence of endogenous reactive oxygen species and other genotoxic molecules. Of particular threat are the thousands of DNA single-strand breaks that arise in each cell, each day, both directly from disintegration of damaged sugars and indirectly from the excision repair of damaged bases. If un-repaired, single-strand breaks can be converted into double-strand breaks during DNA replication, potentially resulting in chromosomal rearrangement and genetic deletion. Consequently, cells have adopted multiple pathways to ensure the rapid and efficient removal of single-strand breaks. A general feature of these pathways appears to be the extensive employment of protein-protein interactions to stimulate both the individual component steps and the overall repair reaction. Our current understanding of DNA single-strand break repair is discussed, and testable models for the architectural coordination of this important process are presented. Copyright 2001 John Wiley & Sons, Inc.

Investigating the dynamics of surface-immobilized DNA nanomachines

Science.gov (United States)

Dunn, Katherine E.; Trefzer, Martin A.; Johnson, Steven; Tyrrell, Andy M.

2016-07-01

Surface-immobilization of molecules can have a profound influence on their structure, function and dynamics. Toehold-mediated strand displacement is often used in solution to drive synthetic nanomachines made from DNA, but the effects of surface-immobilization on the mechanism and kinetics of this reaction have not yet been fully elucidated. Here we show that the kinetics of strand displacement in surface-immobilized nanomachines are significantly different to those of the solution phase reaction, and we attribute this to the effects of intermolecular interactions within the DNA layer. We demonstrate that the dynamics of strand displacement can be manipulated by changing strand length, concentration and G/C content. By inserting mismatched bases it is also possible to tune the rates of the constituent displacement processes (toehold-binding and branch migration) independently, and information can be encoded in the time-dependence of the overall reaction. Our findings will facilitate the rational design of surface-immobilized dynamic DNA nanomachines, including computing devices and track-based motors.

Repair and gamma radiation-induced single- and double-strand breaks in DNA of Escherichia coli

International Nuclear Information System (INIS)

Petrov, S.I.

1981-01-01

Studies in the kinetics of repair of γ-radiation-induced single- and double-strand breaks in DNA of E. coli cells showed that double-strand DNA breaks are rejoined by the following two ways. The first way is conditioned by repair of single-strand breaks and represents the repair of ''oblique'' double-strand breaks in DNA, whereas the second way is conditioned by functioning of the recombination mechanisms and, to all appearance, represents the repair of ''direct'' double-strand breaks in DNA

Magnetization, Low Field Instability and Quench of RHQT Nb(3)Al Strands

Energy Technology Data Exchange (ETDEWEB)

Yamada, R.; Wake, M.; Kikuchi, A.; Velev, V.; /Fermilab

2009-01-01

Since 2005, we made and tested three RHQT Nb{sub 3}Al strands, one with Nb matrix and two with Ta matrix, which are fully stabilized with Cu electroplating. We observed anomalously large magnetization curves extending beyond 1 to 1.5 Tesla with the F1 Nb matrix strand at 4.2 K, when we measured its magnetization with a balanced coil magnetometer. This problem was eliminated with the Ta matrix strands operating at 4.2 K. But with these strands a similar but smaller anomalous magnetization was observed at 1.9 K. We studied these phenomena with FEM. With the F1 Nb matrix strand, it is explained that at low external field, inter-filamentary coupling currents in the outer layers of sub-elements create a shielding effect. It reduces the inside field, keeps the inside Nb matrix superconductive, and stands against a higher outside field beyond the Hc of Nb. At an even higher external field, the superconductivity of the whole Nb matrix collapses and releases a large amount of energy, which may cause a big quench. Depending on the size of the energy in the strand or the cable, a magnet could quench, causing the low field instability. Some attempt to analyze the anomaly with FEM is presented.

The fidelity of reverse transcription differs in reactions primed with RNA versus DNA primers

NARCIS (Netherlands)

Oude Essink, B. B.; Berkhout, B.

1999-01-01

Reverse transcriptase enzymes (RT) convert single-stranded retroviral RNA genomes into double-stranded DNA. The RT enzyme can use both RNA and DNA primers, the former being used exclusively during initiation of minus- and plus-strand synthesis. Initiation of minus-strand DNA synthesis occurs by

Effects of carcinogen treatment on rat liver DNA synthesis in vivo and on nascent DNA synthesis and elongation in cultured hepatocytes

International Nuclear Information System (INIS)

Zurlo, J.; Mignano, J.E.; Eustice, D.C.; Poirier, M.C.; Yager, J.D.

1986-01-01

One objective of this study was to determine the effects of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) treatment on DNA synthesis in regenerating rat liver. It was found that N-OH-AAF caused a dose-dependent inhibition of [ 3 H]thymidine incorporation into liver DNA. This inhibition was followed by a gradual, but incomplete recovery. The second objective of the study was to determine the effects of DNA damage on the size distribution and elongation of nascent hepatocyte DNA. Hepatocytes, which have been shown to demonstrate a pattern of inhibition and subsequent recovery of DNA synthesis following UV irradiation similar to that seen in vivo upon treatment with N-OH-AAF were cultured. The size distribution of nascent DNA after UV irradiation was determined by pH step gradient alkaline elution analysis. The results show that UV irradiation caused a dose-dependent decrease in the size distribution of nascent DNA suggesting an inhibition of elongation. The results show that resumption of DNA synthesis and nascent strand elongation occur on damaged templates. These observations along with previous studies support the idea that DNA damage leading to inhibition of DNA synthesis may induce SOS-type processes which if mutagenic may play a role in the initiation of carcinogenesis. (Auth.)

Development of Nb3Sn strands for ITER in Japan

International Nuclear Information System (INIS)

Isono, T.; Nunoya, Y.; Matsui, K.; Nabara, Y.; Koizumi, N.; Takahashi, Y.; Okuno, K.

2007-01-01

Nb 3 Sn strands for ITER Toroidal Field (TF) coils and Central Solenoids (CS) are required to have both high current density (Jc) and low hysteresis loss. The required Jc at 12 T, 4.2 K and no external strain is lager than 700 A/mm 2 for bronze processed strand and lager than 800 A/mm 2 for internal tin processed one at 12 T, 4.2 K and no external strain. Upper limit of hysteresis loss is 1,000 mJ/cm 3 at 4.2 K and a cycle of ±3 T. Outer diameter is 0.82 mm and 0.83 mm for TF coils and CS, respectively. Area ratio of copper to non copper is one and outer surface is Cr plated. Japan will procure 25% of Nb 3 Sn for ITER TF coils and 100% for ITER CS. There are four Nb 3 Sn strand suppliers in Japan; three of them use bronze process and the other uses internal tin process. For bronze processed strand, increase Jc was achieved by using bronze with high tin content of 15 to 16%. To keep productivity while using the harder bronze with high tin content, Nb is used for barrier in place of Ta. The material is expected to have better adhesion to other materials such as bronze and copper. For internal tin process, size of modules, each of which has a tin rod and a lot of Nb filaments embedded in a copper cylinder, is reduced, while total amount of tin increase in order to satisfy both required high Jc and low hysteresis loss. Each supplier fabricated a strand of more than 0.1 ton for TF coils and succeeded to develop a strand satisfying the ITER requirements of Jc and hysteresis loss. (authors)

RNA signal amplifier circuit with integrated fluorescence output.

Science.gov (United States)

Akter, Farhima; Yokobayashi, Yohei

2015-05-15

We designed an in vitro signal amplification circuit that takes a short RNA input that catalytically activates the Spinach RNA aptamer to produce a fluorescent output. The circuit consists of three RNA strands: an internally blocked Spinach aptamer, a fuel strand, and an input strand (catalyst), as well as the Spinach aptamer ligand 3,5-difluoro-4-hydroxylbenzylidene imidazolinone (DFHBI). The input strand initially displaces the internal inhibitory strand to activate the fluorescent aptamer while exposing a toehold to which the fuel strand can bind to further displace and recycle the input strand. Under a favorable condition, one input strand was able to activate up to five molecules of the internally blocked Spinach aptamer in 185 min at 30 °C. The simple RNA circuit reported here serves as a model for catalytic activation of arbitrary RNA effectors by chemical triggers.

Peroneal tendon displacement accompanying intra-articular calcaneal fractures.

Science.gov (United States)

Toussaint, Rull James; Lin, Darius; Ehrlichman, Lauren K; Ellington, J Kent; Strasser, Nicholas; Kwon, John Y

2014-02-19

Peroneal tendon displacement (subluxation or dislocation) accompanying an intra-articular calcaneal fracture is often undetected and under-treated. The goals of this study were to determine (1) the prevalence of peroneal tendon displacement accompanying intra-articular calcaneal fractures, (2) the association of tendon displacement with fracture classifications, (3) the association of tendon displacement with heel width, and (4) the rate of missed diagnosis of the tendon displacement on radiographs and computed tomography (CT) scans and the resulting treatment rate. A retrospective radiographic review of all calcaneal fractures presenting at three institutions from June 30, 2006, to June 30, 2011, was performed. CT imaging of 421 intra-articular calcaneal fractures involving the posterior facet was available for review. The prevalence of peroneal tendon displacement was noted and its associations with fracture classification and heel width were evaluated. Peroneal tendon displacement was identified in 118 (28.0%) of the 421 calcaneal fracture cases. The presence of tendon displacement was significantly associated with joint-depression fractures compared with tongue-type fractures (p displacement had been identified in the radiology reports. Although sixty-five (55.1%) of the fractures with tendon displacement had been treated with internal fixation, the tendon displacement was treated surgically in only seven (10.8%) of these cases. Analysis of CT images showed a 28% prevalence of peroneal tendon displacement accompanying intra-articular calcaneal fractures. Surgeons and radiologists are encouraged to consider this association.

Influence of DNA conformation on radiation-induced single-strand breaks

International Nuclear Information System (INIS)

Barone, F.; Belli, M.; Mazzei, F.

1994-01-01

We performed experiments on two DNA fragments of about 300 bp having different conformation to test whether radiation-induced single-strand breakage is dependent on DNA conformation. Breakage analysis was carried out by denaturing polyacrylamide gel electrophoresis, which allows determination of the broken site at single nucleotide resolution. We found uniform cutting patterns in B-form regions. On the contrary, X- or γ-irradiation of curved fragments of kinetoplast DNA showed that the distribution of single-strand breaks was not uniform along the fragment, as the cleavage pattern was modulated in phase with the runs of A-T pairs. This modulation likely reflected the reduced accessibility of the sites which on hydroxyl-radical attack give rise to strand breaks. The cleavage pattern was phased with the runs of A-T pairs. Moreover, the overall yield of strand breaks was considerably lower in curved DNA fragments than in those with extended straight regions. The conformation effect found here indicates that the cleavage pattern reflects the fine structural features of DNA. (orig./MG)

Offshore Earthquakes Do Not Influence Marine Mammal Stranding Risk on the Washington and Oregon Coasts

Science.gov (United States)

Grant, Rachel A.; Savirina, Anna

2018-01-01

Simple Summary Marine mammals stranding on coastal beaches is not unusual. However, there appears to be no single cause for this, with several causes being probable, such as starvation, contact with humans (for example boat strike or entanglement with fishing gear), disease, and parasitism. We evaluated marine mammal stranding off the Washington and Oregon coasts and looked at offshore earthquakes as a possible contributing factor. Our analysis showed that offshore earthquakes did not make marine mammals more likely to strand. We also analysed a subset of data from the north of Washington State and found that non-adult animals made up a large proportion of stranded animals, and for dead animals the commonest cause of death was disease, traumatic injury, or starvation. Abstract The causes of marine mammals stranding on coastal beaches are not well understood, but may relate to topography, currents, wind, water temperature, disease, toxic algal blooms, and anthropogenic activity. Offshore earthquakes are a source of intense sound and disturbance and could be a contributing factor to stranding probability. We tested the hypothesis that the probability of marine mammal stranding events on the coasts of Washington and Oregon, USA is increased by the occurrence of offshore earthquakes in the nearby Cascadia subduction zone. The analysis carried out here indicated that earthquakes are at most, a very minor predictor of either single, or large (six or more animals) stranding events, at least for the study period and location. We also tested whether earthquakes inhibit stranding and again, there was no link. Although we did not find a substantial association of earthquakes with strandings in this study, it is likely that there are many factors influencing stranding of marine mammals and a single cause is unlikely to be responsible. Analysis of a subset of data for which detailed descriptions were available showed that most live stranded animals were pups, calves, or

Investigations of radiation-induced strand breaks of poly(U) in aqueous solutions

International Nuclear Information System (INIS)

Lemaire, D.G.E.

1984-01-01

DNA strand breaks induced by γ irradiation were studied in polyuridylic acid (Poly(U)), a single-strand model substance with a single base. Poly(U) in diluted, aqueous solution was irradiated in a Co-γ source, and the 100 eV yields of strand breaks (Cr values) were determined on the basis of the loss of molecular weight. The molecular weight was determined by small-angle laser light scattering. (orig./PW) [de

DNA single-strand breaks during repair of uv damage in human fibroblasts and abnormalities of repair in xeroderma pigmentosum

International Nuclear Information System (INIS)

Fornace, A.J. Jr.; Kohn, K.W.; Kann, H.E. Jr.

1976-01-01

The method of DNA alkaline elution was applied to a study of the formation and resealing of DNA single-strand breaks after irradiation of human fibroblasts with ultraviolet light (UV). The general features of the results were consistent with current concepts of DNA excision repair, in that breaks appeared rapidly after uv, and resealed slowly in normal fibroblasts, whereas breaks did not appear in those cells of patients with xeroderma pigmentosum (XP) that are known to have defects in DNA repair synthesis. The appearance of breaks required a short post-uv incubation, consistent with the expected action of an endonuclease. Cells of the variant form of XP characterized by normal DNA repair synthesis exhibited normal production of breaks after uv, but were slower than normal cells in resealing these breaks. This difference was enhanced by caffeine. A model is proposed to relate this finding with a previously described defect in post-replication repair in these XP variant cells. DNA crosslinking appears to cause an underestimate in the measurement of DNA breakage after uv

Project-induced displacement, secondary stressors, and health.

Science.gov (United States)

Cao, Yue; Hwang, Sean-Shong; Xi, Juan

2012-04-01

It has been estimated that about 15 million people are displaced by development projects around the world each year. Despite the magnitude of people affected, research on the health and other impacts of project-induced displacement is rare. This study extends existing knowledge by exploring the short-term health impact of a large scale population displacement resulting from China's Three Gorges Dam Project. The study is theoretically guided by the stress process model, but we supplement it with Cernea's impoverishment risks and reconstruction (IRR) model widely used in displacement literature. Our panel analysis indicates that the displacement is associated positively with relocatees' depression level, and negatively with their self-rated health measured against a control group. In addition, a path analysis suggests that displacement also affects depression and self-rated health indirectly by changing social integration, socioeconomic status, and community resources. The importance of social integration as a protective mechanism, a factor that has been overlooked in past studies of population displacement, is highlighted in this study. Published by Elsevier Ltd.

Electron attachment to DNA single strands: gas phase and aqueous solution.

Science.gov (United States)

Gu, Jiande; Xie, Yaoming; Schaefer, Henry F

2007-01-01

The 2'-deoxyguanosine-3',5'-diphosphate, 2'-deoxyadenosine-3',5'-diphosphate, 2'-deoxycytidine-3',5'-diphosphate and 2'-deoxythymidine-3',5'-diphosphate systems are the smallest units of a DNA single strand. Exploring these comprehensive subunits with reliable density functional methods enables one to approach reasonable predictions of the properties of DNA single strands. With these models, DNA single strands are found to have a strong tendency to capture low-energy electrons. The vertical attachment energies (VEAs) predicted for 3',5'-dTDP (0.17 eV) and 3',5'-dGDP (0.14 eV) indicate that both the thymine-rich and the guanine-rich DNA single strands have the ability to capture electrons. The adiabatic electron affinities (AEAs) of the nucleotides considered here range from 0.22 to 0.52 eV and follow the order 3',5'-dTDP > 3',5'-dCDP > 3',5'-dGDP > 3',5'-dADP. A substantial increase in the AEA is observed compared to that of the corresponding nucleic acid bases and the corresponding nucleosides. Furthermore, aqueous solution simulations dramatically increase the electron attracting properties of the DNA single strands. The present investigation illustrates that in the gas phase, the excess electron is situated both on the nucleobase and on the phosphate moiety for DNA single strands. However, the distribution of the extra negative charge is uneven. The attached electron favors the base moiety for the pyrimidine, while it prefers the 3'-phosphate subunit for the purine DNA single strands. In contrast, the attached electron is tightly bound to the base fragment for the cytidine, thymidine and adenosine nucleotides, while it almost exclusively resides in the vicinity of the 3'-phosphate group for the guanosine nucleotides due to the solvent effects. The comparatively low vertical detachment energies (VDEs) predicted for 3',5'-dADP(-) (0.26 eV) and 3',5'-dGDP(-) (0.32 eV) indicate that electron detachment might compete with reactions having high activation barriers

Controlled Folding, Motional, and Constitutional Dynamic Processes of Polyheterocyclic Molecular Strands.

Science.gov (United States)

Barboiu, Mihail; Stadler, Adrian-Mihail; Lehn, Jean-Marie

2016-03-18

General design principles have been developed for the control of the structural features of polyheterocyclic strands and their effector-modulated shape changes. Induced defined molecular motions permit designed enforcement of helical as well as linear molecular shapes. The ability of such molecular strands to bind metal cations allows the generation of coiling/uncoiling processes between helically folded and extended linear states. Large molecular motions are produced on coordination of metal ions, which may be made reversible by competition with an ancillary complexing agent and fueled by sequential acid/base neutralization energy. The introduction of hydrazone units into the strands confers upon them constitutional dynamics, whereby interconversion between different strand compositions is achieved through component exchange. These features have relevance for nanomechanical devices. We present a morphological and functional analysis of such systems developed in our laboratories. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Emittance growth rates for displaced beams

International Nuclear Information System (INIS)

Anderson, O.A.

1993-05-01

Emittance growth rates have been previously analyzed for nonuniform beams in linear channels and for initially uniform mismatched beams in nonlinear channels. These studies were for centered beams. Additional emittance growth can arise in cases where the beam is initially displaced. The purpose of this study is to obtain growth rates for displaced beams. This work differs from studies involving random displacement of electrodes. Our analysis assumes instead that the focusing system is perfectly aligned but that the beam is initially displaced with respect to the equilibrium axis. If the focusing force is slightly nonlinear, we find a gradual transfer of the potential energy of beam displacement into kinetic energy associated with emittance growth. We present explicit results for the emittance growth distance as a function of the nonlinearity of the channel. These results will have practical importance for designers of accelerators and transport systems when setting realistic tolerances for initial beam alignment. These tolerances will depend on the nonlinearity and the length of the system

Is Fibular Fracture Displacement Consistent with Tibiotalar Displacement?

NARCIS (Netherlands)

van den Bekerom, Michel P. J.; van Dijk, C. Niek

2010-01-01

We believed open reduction with internal fixation is required for supination-external rotation ankle fractures located at the level of the distal tibiofibular syndesmosis (Lauge-Hanssen SER II and Weber B) with 2 mm or more fibular fracture displacement. The rationale for surgery for these ankle

Phenomenon of displacement in Arabic language

Directory of Open Access Journals (Sweden)

2015-09-01

Full Text Available Displacement is one of the characteristics of language and common phenomena in the Arabic language. Not only is this phenomenon limited to Arabic poetry and prose, but it is also broadened, so we can see examples of this in the Qur'an. Because of this phenomenon extensively in Arabic literature and also because of its essence that leads to the transmission of the elements for the first visibility to the other visibility in the sentence and sometimes had to change the grammatical role of the words, its identify helps us in a better understanding of text and the correct translation of it and protects the reader from mistakes. This paper in the descriptive analytical approach tries studying of the phenomenon of the displacement in the Arabic language and bringing its instances in Arabic poetry and prose as well as verses contained in the Holy Quran, to show that through the types and characteristics in the Arabic language and to response to several questions, including: how important is the displacement and what is its types in rhetoric, and the reasons of the displacement, and etc... Of the most important results of this study may refer to the undeniable role of the displacement as a rhetorical method to better understanding of the texts including: one of the most important reasons of the displacement in the use of language is to improve speech verbally and morally, and violation of the standard language and create a poetic atmosphere, and the recognition of the occurrence of the phenomenon of displacement in the Arabic language that uphold different interpretations remote and estimates when faced with the displacement in the text and help us to understand it and etc...

Critical current scaling and the pivot-point in Nb3Sn strands

International Nuclear Information System (INIS)

Tsui, Y; Hampshire, D P

2012-01-01

Detailed measurements are provided of the engineering critical current density (J c ) and the index of transition (n-value) of two different types of advanced ITER Nb 3 Sn superconducting strand for fusion applications. The samples consist of one internal-tin strand (OST) and two bronze-route strands (BEAS I and BEAS II—reacted using different heat treatments). Tests on different sections of these wires show that prior to applying strain, J c is homogeneous to better than 2% along the length of each strand. J c data have been characterized as a function of magnetic field (B ≤ 14.5 T), temperature (4.2 K ≤ T ≤ 12 K) and applied axial strain ( − 1% ≤ ε A ≤ 0.8%). Strain-cycling tests demonstrate that the variable strain J c data are reversible to better than 2% when the applied axial strain is in the range of − 1% ≤ ε A ≤ 0.5%. The wires are damaged when the intrinsic strain (ε I ) is ε I ≥ 0.55% and ε I ≥ 0.23% for the OST and BEAS strands, respectively. The strain dependences of the normalized J c for each type of strand are similar to those of prototype strands of similar design measured in 2005 and 2008 to about 2% which makes them candidate strands for a round-robin interlaboratory comparison. The J c data are described by Durham, ITER and Josephson-junction parameterizations to an accuracy of about 4%. For all of these scaling laws, the percentage difference between the data and the parameterization is larger when J c is small, caused by high B, T or |ε I |. The n-values can be described by a modified power law of the form n=1+rI c s , where r and s are approximately constant and I c is the critical current. It has long been known that pivot-points (or cross-overs) in J c occur at high magnetic field and temperature. Changing the magnetic field or temperature from one side of the pivot-point to the other changes the highest J c sample to the lowest J c sample and vice versa. The pivot-point follows the B–T phase boundary

Earthquake related displacement fields near underground facilities

International Nuclear Information System (INIS)

Pratt, H.R.; Zandt, G.; Bouchon, M.

1979-04-01

Relative displacements of rock masses are evaluated in terms of geological evidence, seismological evidence, data from simulation experiments, and analytical predictive models. Numerical models have been developed to determine displacement fields as a function of depth, distance, and azimuth from an earthquake source. Computer calculations for several types of faults indicate that displacements decrease rapidly with distance from the fault, but that displacements can either increase or decrease as a function of depth depending on the type and geometry of the fault. For long shallow vertical strike-slip faults the displacement decreases markedly with depth. For square strike slip faults and for dip slip faults displacement does not decrease as markedly with depth. Geologic structure, material properties, and depth affect the seismic source spectrum. Amplification of the high frequencies of shear waves is larger by a factor of about 2 for layered geologic models than for an elastic half space

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

Science.gov (United States)

Park, Sojin; Choi, Seoyun; Ahn, Byungchan

2016-01-01

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents. PMID:26903030

Breaking DNA strands by extreme-ultraviolet laser pulses in vacuum

Czech Academy of Sciences Publication Activity Database

Nováková, Eva; Vyšín, Luděk; Burian, Tomáš; Juha, Libor; Davídková, Marie; Múčka, V.; Čuba, V.; Grisham, M. E.; Heinbuch, S.; Rocca, J.J.

2015-01-01

Roč. 91, č. 4 (2015), "042718-1"-"042718-8" ISSN 1539-3755 R&D Projects: GA ČR(CZ) GBP108/12/G108; GA ČR GA13-28721S Institutional support: RVO:68378271 ; RVO:61389005 Keywords : XUV * DNA damages * single- strand breaks (SSBs) * double- strand breaks (DSBs) Subject RIV: BO - Biophysics Impact factor: 2.288, year: 2014

A convenient procedure for the solid-phase synthesis of hydroxamic acids on PEGA resins

DEFF Research Database (Denmark)

Nandurkar, Nitin Subhash; Petersen, Rico; Qvortrup, Katrine

2011-01-01

An efficient method for the solid-phase synthesis of hydroxamic acids is described. The method comprises the nucleophilic displacement of esters immobilized on PEGA resins with hydroxylamine/sodium hydroxide in isopropanol. The hydroxyaminolysis protocol is compatible with a broad range of PEGA...

Long-Term Seasonal and Interannual Patterns of Marine Mammal Strandings in Subtropical Western South Atlantic

Science.gov (United States)

Prado, Jonatas H. F.; Mattos, Paulo H.; Silva, Kleber G.; Secchi, Eduardo R.

2016-01-01

Understanding temporal patterns of marine mammal occurrence is useful for establishing conservation strategies. We used a 38 yr-long dataset spanning 1976 to 2013 to describe temporal patterns and trends in marine mammal strandings along a subtropical stretch of the east coast of South America. This region is influenced by a transitional zone between tropical and temperate waters and is considered an important fishing ground off Brazil. Generalized Additive Models were used to evaluate the temporal stranding patterns of the most frequently stranded species. Forty species were documented in 12,540 stranding events. Franciscana (n = 4,574), South American fur seal, (n = 3,419), South American sea lion (n = 2,049), bottlenose dolphins (n = 293) and subantarctic fur seal (n = 219) were the most frequently stranded marine mammals. The seasonality of strandings of franciscana and bottlenose dolphin coincided with periods of higher fishing effort and strandings of South American and subantarctic fur seals with post-reproductive dispersal. For South American sea lion the seasonality of strandings is associated with both fishing effort and post-reproductive dispersal. Some clear seasonal patterns were associated with occurrence of cold- (e.g. subantarctic fur seal) and warm-water (e.g. rough-toothed dolphin) species in winter and summer, respectively. Inter-annual increases in stranding rate were observed for franciscana and South American fur seal and these are likely related to increased fishing effort and population growth, respectively. For subantarctic fur seal the stranding rate showed a slight decline while for bottlenose dolphin it remained steady. No significant year to year variation in stranding rate was observed for South American sea lion. The slight decrease in frequency of temperate/polar marine mammals and the increased occurrence of subtropical/tropical species since the late 1990s might be associated with environmental changes linked to climate change

Long-Term Seasonal and Interannual Patterns of Marine Mammal Strandings in Subtropical Western South Atlantic.

Directory of Open Access Journals (Sweden)

Jonatas H F Prado

Full Text Available Understanding temporal patterns of marine mammal occurrence is useful for establishing conservation strategies. We used a 38 yr-long dataset spanning 1976 to 2013 to describe temporal patterns and trends in marine mammal strandings along a subtropical stretch of the east coast of South America. This region is influenced by a transitional zone between tropical and temperate waters and is considered an important fishing ground off Brazil. Generalized Additive Models were used to evaluate the temporal stranding patterns of the most frequently stranded species. Forty species were documented in 12,540 stranding events. Franciscana (n = 4,574, South American fur seal, (n = 3,419, South American sea lion (n = 2,049, bottlenose dolphins (n = 293 and subantarctic fur seal (n = 219 were the most frequently stranded marine mammals. The seasonality of strandings of franciscana and bottlenose dolphin coincided with periods of higher fishing effort and strandings of South American and subantarctic fur seals with post-reproductive dispersal. For South American sea lion the seasonality of strandings is associated with both fishing effort and post-reproductive dispersal. Some clear seasonal patterns were associated with occurrence of cold- (e.g. subantarctic fur seal and warm-water (e.g. rough-toothed dolphin species in winter and summer, respectively. Inter-annual increases in stranding rate were observed for franciscana and South American fur seal and these are likely related to increased fishing effort and population growth, respectively. For subantarctic fur seal the stranding rate showed a slight decline while for bottlenose dolphin it remained steady. No significant year to year variation in stranding rate was observed for South American sea lion. The slight decrease in frequency of temperate/polar marine mammals and the increased occurrence of subtropical/tropical species since the late 1990s might be associated with environmental changes linked to

ZrC Ceramics Prepared by Self-propagating High-temperature Synthesis/Single Action Pressing

Directory of Open Access Journals (Sweden)

CHENG Yong

2017-01-01

Full Text Available ZrC ceramics were prepared by mechanical axial compression of self-propagating high-temperature synthesis/single action pressing (SHS/SAP.The effects of pressure on microstructure and densification of the products,as well as the relationship between displacement/variation of the load curve and SHS reaction,were studied.The structure and properties of the products were investigated by XRD and SEM.In addition,the density was measured by the drain away liquid method.Meanwhile,universal testing machine was used to record the displacement and load curve alternations.The results indicate that products are mainly composed of ZrC phase,the process of exhaust are accelerated as the increasing of pressure as well,leading to the smaller size of porosity and crystal particles.Density manifested as an increasing pattern by the elevated pressure with no longer change at 80MPa.Due to the strong attenuation of pressure at the peak of temperature,the density of the production is only 65.7% in 120MPa.The end point of the SHS reaction and the plastic time of the products can be monitored by displacement and load curve.The results provide evidence for the application of self-propagating high-temperature synthesis/pseudo-hot isostatic pressing to further improve the density of ceramics.

Necropsy report of a fin whale (Balaenoptera physalus) stranded in Denmark in 2010

DEFF Research Database (Denmark)

Alstrup, Aage K. O.; Hedayat, Abdi; Jensen, Trine Hammer

2013-01-01

There is little detailed information on stranded fin whales (Balaenoptera physalus) in the scientific literature (Notarbartolo di Sciara et al., 2003). In Denmark, at least eight fin whales stranded between the years 1603 and 1958 (Kinze, 1995). On 16 June 2010, a live subadult or adult male fin...... whale stranded in the Bay of Vejle (55º 69' N, 9º 58' E), Denmark. Despite several attempts, it was not possible to rescue the fin whale, which was only partially exposed by the water. The fin whale succumbed after 5 d stranded in shallow water. The dead fin whale was transported to a nearby pier...

Event-driven control of a speed varying digital displacement machine

DEFF Research Database (Denmark)

Pedersen, Niels Henrik; Johansen, Per; Andersen, Torben O.

2017-01-01

. The controller synthesis is carried out as a discrete optimal deterministic problem with full state feedback. Based on a linear analysis of the feedback control system, stability is proven in a pre-specified operation region. Simulation of a non-linear evaluation model with the controller implemented shows great...... be treated as a Discrete Linear Time Invariant control problem with synchronous sampling rate. To make synchronous linear control theory applicable for a variable speed digital displacement machine, a method based on event-driven control is presented. Using this method, the time domain differential equations...... are converted into the spatial (position) domain to obtain a constant sampling rate and thus allowing for use of classical control theory. The method is applied to a down scaled digital fluid power motor, where the motor speed is controlled at varying references under varying pressure and load torque conditions...

SOCIAL CAPITAL IN INVOLUNTARY DISPLACEMENT AND RESETTLEMENT

Directory of Open Access Journals (Sweden)

Melissa Quetulio-Navarra

2013-07-01

Full Text Available Social capital is often seen as a substitute for lack of other types of capital amongpoor people. Because of the recognized applicability of the social capital conceptand its correlation with the different dimensions of poverty, it has been used inevaluating the adaptation and integration of involuntarily displaced individualsinto their new environment. This paper presents insights based on a review of thefindings of studies that looked into the role of social capital in conflict- anddevelopment-induced displacement contexts. Althoughboth types of displace-ments are involuntary or forced in nature, they differ in terms of the role of socialcapital regarding its main sources, the formation pattern and its determinants.Social capital studies in forced resettlement appear to be relatively small innumber and are heavily concentrated on first worldcountries and conflict- anddevelopment-induced displacements. The conduct of similar studies in developingcountries and in a disaster-induced resettlement context, the third type ofinvoluntary displacement, should generate new and relevant findings regardingthe role of social capital in resettlement communities.

Statistical Assessment of Cetacean Stranding Events in Cape Cod (Massachusetts, USA) area.

Science.gov (United States)

Zellar, R.; Pulkkinen, A. A.; Moore, K.; Reeb, D.; Karakoylu, E.; Uritskaya, O.

2017-12-01

Cetacean (whales, dolphins and porpoises) mass strandings are a longstanding mystery in the field of marine biology that continue to be recorded in coastal environments around the world. For each of these events, anywhere from a few to several hundred otherwise healthy animals strand in onshore environments, often for no apparent reason. While the causes of these events remain unclear, anthropogenic and naturogenic mechanisms have been suggested. We present results of an inter-disciplinary study that draws expertise from space weather, marine mammal biology and ecology, and marine mammal stranding response. This study assessed 16 years of cetacean stranding events in the Cape Cod (Massachusetts, USA) area concurrently with a large dataset of meteorological, geophysical, biological, oceanographic and space weather data to produce inferences about possible causes for these unexplained events.

War, forced displacement and growth in Laotian adults.

Science.gov (United States)

Clarkin, Patrick F

2012-01-01

Evidence from several populations suggests that war negatively impacts civilian nutrition, physical growth and overall health. This effect is often enduring or permanent, particularly if experienced early in life. To assess whether the number of lifetime displacement experiences and being displaced in infancy were associated with adult height, sitting height, leg length and the sitting height ratio. Retrospective questionnaires on displacement and resettlement experiences and anthropometric data were collected from a sample of Laotian adult refugees (ethnic Hmong and Lao; n = 365). All were born in Laos or Thailand and had resettled in French Guiana or the US. Many had been displaced several times by military conflict in Laos. In bivariate analyses, being displaced in infancy and the number of lifetime displacement experiences one had were negatively associated with final adult height and leg length in both sexes. The association was stronger in females, particularly Hmong females. There was no significant association between total displacement experiences and the sitting height ratio. In multiple regression analyses, linear growth in males was negatively associated with being displaced in infancy; in females, the number of lifetime displacement experiences was a significant predictor. Forced displacement from war appears to have a lasting effect on final adult height, sitting height and leg length, although not necessarily on the sitting height ratio in this sample.

Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro

DEFF Research Database (Denmark)

Liu, Dekang; Frederiksen, Jane H.; Liberti, Sascha Emilie

2017-01-01

DNA mismatch repair (MMR) is a highly-conserved DNA repair mechanism, whose primary role is to remove DNA replication errors preventing them from manifesting as mutations, thereby increasing the overall genome stability. Defects in MMR are associated with increased cancer risk in humans and other...... organisms. Here, we characterize the interaction between MMR and a proofreading-deficient allele of the human replicative DNA polymerase delta, PolδD316A;E318A, which has a higher capacity for strand displacement DNA synthesis than wild type Polδ. Human cell lines overexpressing PolδD316A;E318A display...

Structural features in the HIV-1 repeat region facilitate strand transfer during reverse transcription

NARCIS (Netherlands)

Berkhout, B.; Vastenhouw, N. L.; Klasens, B. I.; Huthoff, H.

2001-01-01

Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer is facilitated by terminal repeat (R) elements in the viral genome. This strand-transfer reaction depends on base pairing between the cDNA of the 5'R and the 3'R. There

Displacement Ventilation

DEFF Research Database (Denmark)

Bjørn, Erik; Mattsson, Magnus; Sandberg, Mats

Full-scale experiments were made in a displacement ventilated room with two breathing thermal manikins to study the effect of movements and breathing on the vertical contaminant distribution, and on the personal exposure of occupants. Concentrations were measured with tracer gas equipment...

Strand Tension Control in Anchor Span for Suspension Bridge Using Dynamic Balance Theory

Directory of Open Access Journals (Sweden)

Da Wang

Full Text Available Abstract Strand tension control is essential in suspension bridge safety. However, few quantitative studies have examined the bending rigidity and boundary condition behavior of strands in the anchor span of suspension bridges because of their special structure and complex configuration. In this paper, a new calculation method for strand tension is explored by using dynamic balance theory to determine the effect of bending rigidity and boundary conditions. The accuracy and effectiveness of the proposed method are tested and confirmed with verification examples and application on Nanxi Yangtze Suspension Bridge in China. The results indicated that only low-order frequency calculation could be used to calculate the strand tension without considering the effect of bending rigidity to ensure control accuracy. The influence of bending rigidity on the control precision is related to the tension and the length of the strands, which is significantly determined by the specific value between the stress rigidity and the bending rigidity. The uncertain boundary conditions of the anchor span cable, which are fixed between consolidated and hinged, also have a major effect on the control accuracy. To improve the accuracy of strand tension control, the least squares method is proposed during the tension construction control of the anchor span. This approach can significantly improve the accuracy of the tension control of the main cable strand. Some recommendations for future bridge analysis are provided based on the results of this study.

Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii

OpenAIRE

Stroud, A. L.

2012-01-01

Replication protein A (RPA) is a single-stranded DNA-binding protein that is present in all three domains of life. The roles of RPA include stabilising and protecting single- stranded DNA from nuclease degradation during DNA replication and repair. To achieve this, RPA uses an oligosaccharide-binding fold (OB fold) to bind single- stranded DNA. Haloferax volcanii encodes three RPAs – RPA1, RPA2 and RPA3, of which rpa1 and rpa3 are in operons with genes encoding associated proteins (APs). ...

Genetic transformation of Neisseria gonorrhoeae shows a strand preference

OpenAIRE

Duffin, Paul M.; Seifert, H. Steven

2012-01-01

Natural transformation is the main means of horizontal genetic exchange in the obligate human pathogen Neisseria gonorrhoeae. Neisseria spp. have been shown to preferentially take up and transform their own DNA by recognizing a non-palindromic 10 or 12 nucleotide DNA uptake sequence (DUS10 or DUS12). We investigated the ability of the DUS12 to enhance single-stranded DNA (ssDNA) transformation. Given the non-palindromic nature of the DUS12, we tested whether both strands of the DUS equally en...

Using DNA origami nanostructures to determine absolute cross sections for UV photon-induced DNA strand breakage.

Science.gov (United States)

Vogel, Stefanie; Rackwitz, Jenny; Schürman, Robin; Prinz, Julia; Milosavljević, Aleksandar R; Réfrégiers, Matthieu; Giuliani, Alexandre; Bald, Ilko

2015-11-19

We have characterized ultraviolet (UV) photon-induced DNA strand break processes by determination of absolute cross sections for photoabsorption and for sequence-specific DNA single strand breakage induced by photons in an energy range from 6.50 to 8.94 eV. These represent the lowest-energy photons able to induce DNA strand breaks. Oligonucleotide targets are immobilized on a UV transparent substrate in controlled quantities through attachment to DNA origami templates. Photon-induced dissociation of single DNA strands is visualized and quantified using atomic force microscopy. The obtained quantum yields for strand breakage vary between 0.06 and 0.5, indicating highly efficient DNA strand breakage by UV photons, which is clearly dependent on the photon energy. Above the ionization threshold strand breakage becomes clearly the dominant form of DNA radiation damage, which is then also dependent on the nucleotide sequence.

Magnetization Losses of Roebel Cable Samples with 2G YBCO Coated Conductor Strands

CERN Document Server

Yang, Y.; Falorio, I.; Young, E.A.; Kario, A.; Goldacker, W.; Dhallé, M. M. J.; van Nugteren, J.; Kirby, G.; Bottura, L.; Ballarino, A.

2016-01-01

Roebel cable with 2G YBCO strands is one of the promising HTS solutions of fully transposed high current conductors for high field accelerator magnets. Following the considerable research effort on the manufacturing of Roebel cables in recent years, sample conductors are now available in useful lengths with reproducible performances to allow detailed characterizations beyond the standard critical current measurements. The ac loss and strands coupling are of significant interest for the field quality of the accelerator magnets. We report a set of systematic ac loss measurements on two different Roebel cable samples prepared for the EuCARD2 collaboration. The measurements were performed over a wide range of temperature between 5 K and 90 K and the results were analyzed in the context of strands architecture and coupling. The results show that the transposed bundles are partially decoupled and the strands in transposition sections behave as an isolated single tape if the strands are insulated.

Analytical model of the critical current of a bent Nb3Sn strand

International Nuclear Information System (INIS)

Koizumi, Norikiyo; Murakami, Haruyuki; Hemmi, Tsutomu; Nakajima, Hideo

2011-01-01

The critical current performance of a large Nb 3 Sn cable-in-conduit conductor (CICC) was degraded by periodic bending of strands due to a large transverse electromagnetic force. The degradation of each strand due to this bending should be evaluated in calculations of the critical current of a CICC, but a suitable model has not been developed yet. Therefore, the authors have developed a new analytical model which takes into account plastic deformation of copper and bronze and filament breakage. The calculated results were compared with test results for uniformly bent Nb 3 Sn bronze-route strands. The calculated results assuming a high transverse resistance model (HTRM) show good agreement with the test results, a finding which confirms the validity of the model. Because of a much shorter calculation time than for numerical simulation, the developed model seems much more practical for use in calculating the critical current performance of a Nb 3 Sn CICC. In addition, simulation results show that since the neutral axis of a bent strand shifts to the compressive side due to plastic deformation of the copper and bronze, and/or filament breakage, the strand is elongated by bending. This elongation may enhance the strand's critical current performance. Moreover, the calculated results indicate that the dependence of the critical current on the bending strain is affected by the bending history if the strand is excessively bent, especially when filaments are broken. In a real magnet, since a strand in a CICC is normally subject to the maximum electromagnetic force prior to an evaluation of its performance at a lower electromagnetic force, the effect of over-bending should be taken into account in calculations of its critical current performance, especially when filament breakage occurs.

Breaks in plasmid DNA strand induced by laser radiation at a wavelength of 193 nm

International Nuclear Information System (INIS)

Gurzadyan, G.G.; Shul'te Frolinde, D.

1996-01-01

DNA of plasmid pB322 irradiated with laser at a wavelength of 193 nm was treated with an extract containing proteins from E.coli K12 AB1157 (wild-type). The enzymes were found to produce single- and double-strand DNA breaks, which was interpreted as a transformation of a portion of cyclobutane pyrimidine dimers and (6-4) photoproducts into nonrepairable single-strand DNA breaks. The products resulted from ionization of DNA, in particular, single-strand breaks, transform to double-strand breaks. A comparison of these data with the data on survival of plasmid upon transformation of E.coli K12 AB1157 enables one to assess the biological significance of single- and double-strand breaks. The inactivation of the plasmid is mainly determined by the number of directly formed laser-induced single-strand breaks. 26 refs.; 2 figs

Tracking speckle displacement by double Kalman filtering

Institute of Scientific and Technical Information of China (English)

Donghui Li; Li Guo

2006-01-01

@@ A tracking technique using two sequentially-connected Kalman filter for tracking laser speckle displacement is presented. One Kalman filter tracks temporal speckle displacement, while another Kalman filter tracks spatial speckle displacement. The temporal Kalman filter provides a prior for the spatial Kalman filter, and the spatial Kalman filter provides measurements for the temporal Kalman filter. The contribution of a prior to estimations of the spatial Kalman filter is analyzed. An optical analysis system was set up to verify the double-Kalman-filter tracker's ability of tracking laser speckle's constant displacement.

Paul Strand y las paradojas de la modernidad americana

Directory of Open Access Journals (Sweden)

Vicente Sánchez Biosca

2010-06-01

Full Text Available As contribuições de Strand para o cinema estão estreitamente relacionadas com seus trabalhos fotográficos da época e mesmo com um olhar que, apesar de suas múltiplas diferenças, possui muitos pontos em comum. Se em Manhatta (1920, Strand (em colaboração com Sheeler oferece uma síntese da natureza e da modernidade, em Redes (1934 o que predomina, em contrapartida, é a experiência da arte muralista mexicana e a influência de Eisenstein (em suas andanças por México são os fatores que determinam seus esforços na construção de uma épica dos operários. Mas sem dúvida, o projeto mais ambicioso de Strand (ao mando da produtora Frontier Films foi Native Land, o filme que atendeu às demandas do American Popular Front e as moldou nas palavras dos fundadores da pátria americana.

IHF-independent assembly of the Tn10 strand transfer transpososome: implications for inhibition of disintegration

OpenAIRE

Stewart, Barry J.; Wardle, Simon J.; Haniford, David B.

2002-01-01

The frequency of DNA transposition in transposition systems that employ a strand transfer step may be significantly affected by the occurrence of a disintegration reaction, a reaction that reverses the strand transfer event. We have asked whether disintegration occurs in the Tn10 transposition system. We show that disintegration substrates (substrates constituting one half of the strand transfer product) are assembled into a transpososome that mimics the strand transfer intermediate. This str...

Chirality induction and protonation-induced molecular motions in helical molecular strands.

Science.gov (United States)

Kolomiets, Elena; Berl, Volker; Lehn, Jean-Marie

2007-01-01

The long oligopyridinedicarboxamide strand 9, containing 15 heterocyclic rings has been synthesized and its helical structure determined by X-ray crystallography. It was shown that the shorter analogue 6 displays induced circular dichroism and amplification of induced chirality upon dissolution in an optically active solvent, diethyl-L-tartrate. A novel class of helical foldamers was prepared, strands 14-16, based on two oligopyridine carboxamide segments linked through a L-tartaric acid derived spacer. These tartro strands display internal chirality induction as well as chirality amplification. NMR spectroscopy (on 8 and 9) and circular dichroism (on 16) studies show that the oligopyridine carboxamide strands undergo reversible unfolding/folding upon protonation. The protonation-induced unfolding has been confirmed by X-ray crystallographic determination of the molecular structure of the extended protonated heptameric form 8(+). The molecular-scale mechano-chemical motions of the protonation-induced structural switching consist of a change of the length of the molecule, from 6 angstroms (6, coiled form) to 29 angstroms (8(+), uncoiled form) for the heptamer and from 12.5 angstroms (9, coiled form, X-ray structure) to 57 angstroms (9(+), uncoiled form, from modeling) for the pentadecamer. Similar unfolding/folding motional processes take place in the L-tartro strands 15 and 16 upon protonation/deprotonation, with loss of helicity-induced circular dichroism on unfolding as shown for the protonated form 16(+).

Evolutionary implications of inversions that have caused intra-strand parity in DNA

Directory of Open Access Journals (Sweden)

Wei John

2007-06-01

Full Text Available Abstract Background Chargaff's rule of DNA base composition, stating that DNA comprises equal amounts of adenine and thymine (%A = %T and of guanine and cytosine (%C = %G, is well known because it was fundamental to the conception of the Watson-Crick model of DNA structure. His second parity rule stating that the base proportions of double-stranded DNA are also reflected in single-stranded DNA (%A = %T, %C = %G is more obscure, likely because its biological basis and significance are still unresolved. Within each strand, the symmetry of single nucleotide composition extends even further, being demonstrated in the balance of di-, tri-, and multi-nucleotides with their respective complementary oligonucleotides. Results Here, we propose that inversions are sufficient to account for the symmetry within each single-stranded DNA. Human mitochondrial DNA does not demonstrate such intra-strand parity, and we consider how its different functional drivers may relate to our theory. This concept is supported by the recent observation that inversions occur frequently. Conclusion Along with chromosomal duplications, inversions must have been shaping the architecture of genomes since the origin of life.

Time-dependent evolution of strand transfer length in pretensioned prestressed concrete members

Science.gov (United States)

Caro, L. A.; Martí-Vargas, J. R.; Serna, P.

2013-11-01

For design purposes, it is generally considered that prestressing strand transfer length does not change with time. However, some experimental studies on the effect of time on transfer lengths show contradictory results. In this paper, an experimental research to study transfer length changes over time is presented. A test procedure based on the ECADA testing technique to measure prestressing strand force variation over time in pretensioned prestressed concrete specimens has been set up. With this test method, an experimental program that varies concrete strength, specimen cross section, age of release, prestress transfer method, and embedment length has been carried out. Both the initial and long-term transfer lengths of 13-mm prestressing steel strands have been measured. The test results show that transfer length variation exists for some prestressing load conditions, resulting in increased transfer length over time. The applied test method based on prestressing strand force measurements has shown more reliable results than procedures based on measuring free end slips and longitudinal strains of concrete. An additional factor for transfer length models is proposed in order to include the time-dependent evolution of strand transfer length in pretensioned prestressed concrete members.

Displaced epithelium after liposuction for gynecomastia.

Science.gov (United States)

McLaughlin, Cristina S; Petrey, Chris; Grant, Shawn; Ransdell, Jill S; Reynolds, Carol

2011-08-01

The authors describe the case of a 36-year-old man with gynecomastia who was previously treated with liposuction of the breast for cosmetic purposes. Histologic examination of a subsequent excisional biopsy revealed nests of displaced epithelial cells in adipose tissue. Epithelial cell displacement is a well-known risk of core needle biopsies and fine-needle aspirations of breast lesions. However, to the authors' knowledge, epithelial displacement in gynecomastia after liposuction, mimicking invasive ductal carcinoma, has not previously been reported.

Synthesis of DNA

Science.gov (United States)

Mariella, Jr., Raymond P.

2008-11-18

A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

Single helically folded aromatic oligoamides that mimic the charge surface of double-stranded B-DNA

Science.gov (United States)

Ziach, Krzysztof; Chollet, Céline; Parissi, Vincent; Prabhakaran, Panchami; Marchivie, Mathieu; Corvaglia, Valentina; Bose, Partha Pratim; Laxmi-Reddy, Katta; Godde, Frédéric; Schmitter, Jean-Marie; Chaignepain, Stéphane; Pourquier, Philippe; Huc, Ivan

2018-05-01

Numerous essential biomolecular processes require the recognition of DNA surface features by proteins. Molecules mimicking these features could potentially act as decoys and interfere with pharmacologically or therapeutically relevant protein-DNA interactions. Although naturally occurring DNA-mimicking proteins have been described, synthetic tunable molecules that mimic the charge surface of double-stranded DNA are not known. Here, we report the design, synthesis and structural characterization of aromatic oligoamides that fold into single helical conformations and display a double helical array of negatively charged residues in positions that match the phosphate moieties in B-DNA. These molecules were able to inhibit several enzymes possessing non-sequence-selective DNA-binding properties, including topoisomerase 1 and HIV-1 integrase, presumably through specific foldamer-protein interactions, whereas sequence-selective enzymes were not inhibited. Such modular and synthetically accessible DNA mimics provide a versatile platform to design novel inhibitors of protein-DNA interactions.

Water displacement mercury pump

Science.gov (United States)

Nielsen, M.G.

1984-04-20

A water displacement mercury pump has a fluid inlet conduit and diffuser, a valve, a pressure cannister, and a fluid outlet conduit. The valve has a valve head which seats in an opening in the cannister. The entire assembly is readily insertable into a process vessel which produces mercury as a product. As the mercury settles, it flows into the opening in the cannister displacing lighter material. When the valve is in a closed position, the pressure cannister is sealed except for the fluid inlet conduit and the fluid outlet conduit. Introduction of a lighter fluid into the cannister will act to displace a heavier fluid from the cannister via the fluid outlet conduit. The entire pump assembly penetrates only a top wall of the process vessel, and not the sides or the bottom wall of the process vessel. This insures a leak-proof environment and is especially suitable for processing of hazardous materials.

Archaeal ribonuclease P proteins have potential for biotechnological applications where precise hybridization of nucleic acids is needed.

Science.gov (United States)

Miyanoshita, Mitsuru; Nakashima, Takashi; Kakuta, Yoshimitsu; Kimura, Makoto

2015-01-01

Fluorescence resonance energy transfer-based assay showed that archaeal ribonuclease P (RNase P) proteins significantly promoted DNA annealing and strand displacement. Moreover, we found that archaeal RNase P proteins could discriminate nucleotide exchanges in DNA chains via their activity accelerating DNA strand displacement, suggesting that they have potential for biotechnological application to genetic diagnosis.

Comparison Between Nb3Al and Nb3Sn Strands and Cables for High Field Accelerator Magnets

Energy Technology Data Exchange (ETDEWEB)

Yamada, R.; Kikuchi, A.; Barzi, E.; Chlachidze, G.; Rusy, A.; Takeuchi, T.; Tartaglia, M.; Turrioni, D.; Velev, V.; Wake, M.; Zlobin, A.V.; /Fermilab

2010-01-01

The Nb{sub 3}Al small racetrack magnet, SR07, has been successfully built and tested to its short sample limit beyond 10 Tesla without any training. Thus the practical application of Nb{sub 3}Al strands for high field accelerator magnets is established. The characteristics of the representative F4 strand and cable, are compared with the typical Nb{sub 3}Sn strand and cable. It is represented by the OST high current RRP Nb{sub 3}Sn strand with 108/127 configuration. The effects of Rutherford cabling to both type strands are explained and the inherent problem of the Nb{sub 3}Sn strand is discussed. Also the test results of two representative small racetrack magnets are compared from the stand point of Ic values, and training. The maximum current density of the Nb{sub 3}Al strands is still smaller than that of the Nb{sub 3}Sn strands, but if we take into account of the stress-strain characteristics, Nb{sub 3}Al strands become somewhat favorable in some applications.

Missing Strands? Dealing with Hair Loss

Science.gov (United States)

... 2017 Print this issue Missing Strands? Dealing with Hair Loss En español Send us your comments Hair loss is often associated with men and aging, but ... or their treatments, and many other things cause hair loss. The most common type of hair loss is ...

The stranding anomaly as population indicator: the case of harbour porpoise Phocoena phocoena in North-Western Europe.

Directory of Open Access Journals (Sweden)

Helene Peltier

Full Text Available Ecological indicators for monitoring strategies are expected to combine three major characteristics: ecological significance, statistical credibility, and cost-effectiveness. Strategies based on stranding networks rank highly in cost-effectiveness, but their ecological significance and statistical credibility are disputed. Our present goal is to improve the value of stranding data as population indicator as part of monitoring strategies by constructing the spatial and temporal null hypothesis for strandings. The null hypothesis is defined as: small cetacean distribution and mortality are uniform in space and constant in time. We used a drift model to map stranding probabilities and predict stranding patterns of cetacean carcasses under H0 across the North Sea, the Channel and the Bay of Biscay, for the period 1990-2009. As the most common cetacean occurring in this area, we chose the harbour porpoise Phocoena phocoena for our modelling. The difference between these strandings expected under H0 and observed strandings is defined as the stranding anomaly. It constituted the stranding data series corrected for drift conditions. Seasonal decomposition of stranding anomaly suggested that drift conditions did not explain observed seasonal variations of porpoise strandings. Long-term stranding anomalies increased first in the southern North Sea, the Channel and Bay of Biscay coasts, and finally the eastern North Sea. The hypothesis of changes in porpoise distribution was consistent with local visual surveys, mostly SCANS surveys (1994 and 2005. This new indicator could be applied to cetacean populations across the world and more widely to marine megafauna.

Prolonged displacement may compromise resilience in Eritrean mothers.

Science.gov (United States)

Almedom, Astier; Tesfamichael, Berhe; Mohammed, Zein; Mascie-Taylor, Nick; Muller, Jocelyn; Alemu, Zemui

2005-12-01

to assess the impact of prolonged displacement on the resilience of Eritrean mothers. an adapted SOC scale (short form) was administered. Complementary qualitative data were gathered from study participants' spontaneous reactions to and commentaries on the SOC scale. Displaced women's SOC scores were significantly less than those of the non-displaced: Mean = 54.84; SD = 6.48 in internally displaced person (IDP) camps, compared to non-displaced urban and rural/pastoralist: Mean = 48. 94, SD = 11.99; t = 3.831, p urban (non-displaced). Rural but traditionally mobile (pastoralist or transhumant) communities scored more or less the same as the urban non-displaced--i.e., significantly higher than those in IDP camps (p urban and pastoralist/transhumant groups were similar, while women in IDP camps were lower scoring--RR = .268, p < .001. The implications of these findings for health policy are critical. It is incumbent on the international health institutions including the World Health Organization and regional as well as local players to address the plight of internally displaced women, their families and communities in Eritrea and other places of dire conditions such as, for example Darfur in the Sudan.

Influence of oxidized purine processing on strand directionality of mismatch repair.

Science.gov (United States)

Repmann, Simone; Olivera-Harris, Maite; Jiricny, Josef

2015-04-17

Replicative DNA polymerases are high fidelity enzymes that misincorporate nucleotides into nascent DNA with a frequency lower than [1/10(5)], and this precision is improved to about [1/10(7)] by their proofreading activity. Because this fidelity is insufficient to replicate most genomes without error, nature evolved postreplicative mismatch repair (MMR), which improves the fidelity of DNA replication by up to 3 orders of magnitude through correcting biosynthetic errors that escaped proofreading. MMR must be able to recognize non-Watson-Crick base pairs and excise the misincorporated nucleotides from the nascent DNA strand, which carries by definition the erroneous genetic information. In eukaryotes, MMR is believed to be directed to the nascent strand by preexisting discontinuities such as gaps between Okazaki fragments in the lagging strand or breaks in the leading strand generated by the mismatch-activated endonuclease of the MutL homologs PMS1 in yeast and PMS2 in vertebrates. We recently demonstrated that the eukaryotic MMR machinery can make use also of strand breaks arising during excision of uracils or ribonucleotides from DNA. We now show that intermediates of MutY homolog-dependent excision of adenines mispaired with 8-oxoguanine (G(O)) also act as MMR initiation sites in extracts of human cells or Xenopus laevis eggs. Unexpectedly, G(O)/C pairs were not processed in these extracts and failed to affect MMR directionality, but extracts supplemented with exogenous 8-oxoguanine DNA glycosylase (OGG1) did so. Because OGG1-mediated excision of G(O) might misdirect MMR to the template strand, our findings suggest that OGG1 activity might be inhibited during MMR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Study in regularities in the formation of double stranded DNA breaks in irradiated rat thymocytes

International Nuclear Information System (INIS)

Ivannik, B.P.; ProskuryakoV, S.Ya.; Ryabchenko, N.I.

1979-01-01

Using low-gradient viscosimetry of neutral detergent nuclear lysates a study was made of postradiation changes in the molecular weight of double-stranded DNA of thymocytes. It was established that 375 eV are needed for one double-stranded break to appear, and a dose of 1 rad is required for 0.275 double-stranded break to occur at the site of DNA with m.w. 10 12 dalton. The repair of double-stranded breaks is only observed when rats are exposed to a dose of 500 R. It is assumed that the absence of repair of double-stranded DNA breaks and the presence of secondary postradiation degradation of DNA are responsible for thymocyte death

Velocity and processivity of helicase unwinding of double-stranded nucleic acids

International Nuclear Information System (INIS)

Betterton, M D; Juelicher, F

2005-01-01

Helicases are molecular motors which unwind double-stranded nucleic acids (dsNA) in cells. Many helicases move with directional bias on single-stranded (ss) nucleic acids, and couple their directional translocation to strand separation. A model of the coupling between translocation and unwinding uses an interaction potential to represent passive and active helicase mechanisms. A passive helicase must wait for thermal fluctuations to open dsNA base pairs before it can advance and inhibit NA closing. An active helicase directly destabilizes dsNA base pairs, accelerating the opening rate. Here we extend this model to include helicase unbinding from the nucleic-acid strand. The helicase processivity depends on the form of the interaction potential. A passive helicase has a mean attachment time which does not change between ss translocation and ds unwinding, while an active helicase in general shows a decrease in attachment time during unwinding relative to ss translocation. In addition, we describe how helicase unwinding velocity and processivity vary if the base-pair binding free energy is changed

DNA replication and the repair of DNA strand breaks in nuclei of Physarum polycephalum. Progress report, September 1, 1977--July 31, 1978

International Nuclear Information System (INIS)

Brewer, E.N.; Nygaard, O.F.; Kuncio, G.

1978-01-01

Isolated nuclei and intact plasmodia of Physarum contain a heat-stable stimulator of nuclear DNA replication. This substance has been purified extensively and found to contain both protein and carbohydrate. The molecular weight, estimated by gel filtration, is ca. 30,000 d. The purified material does not exhibit DNA polymerase or DNase activity, and does not stimulate DNA polymerase activity per se. In the presence of the stimulatory factor, DNA chain elongation occurs at an elevated rate, and continues for a longer time than in its absence, but G 2 nuclei are not stimulated to initiate DNA synthesis. Double-strand breaks in nuclear DNA of irradiated plasmodia are repaired in vitro to a greater extent following nuclear isolation during G 2 , and the DNA of unirradiated plasmodia is less susceptible to double-strand breakage during cell-free nuclear incubation, than is the DNA of S-phase nuclei. This correlation suggests a common basis for both observations, for example an increase in deoxyribonuclease activity or a decrease in DNA ligase activity during the S period. This, in turn, may account for the cell cycle-dependent sensitivity of this organism, in terms of mitotic delay, to ionizing radiation

Enzymatic induction of DNA double-strand breaks in γ-irradiated Escherichia coli K-12

International Nuclear Information System (INIS)

Bonura, T.; Smith, K.C.; Kaplan, H.S.

1975-01-01

The polA1 mutation increases the sensitivity of E. coli K-12 to killing by γ-irradiation in air by a factor of 2.9 and increases the yield of DNA double-strand breaks by a factor of 2.5. These additional DNA double-strand breaks appear to be due to the action of nucleases in the polA1 strain rather than to the rejoining of radiation-induced double-strand breaks in the pol + strain. This conclusion is based upon the observation that γ-irradiation at 3 0 did not affect the yield of DNA double-strand breaks in the pol + strain, but decreased the yield in the polA1 strain by a factor of 2.2. Irradiation of the polA1 strain at 3 0 followed by incubation at 3 0 for 20 min before plating resulted in approximately a 1.5-fold increase in the D 0 . The yield of DNA double-strand breaks was reduced by a factor of 1.5. The pol + strain, however, did not show the protective effect of the low temperature incubation upon either survival or DNA double-strand breakage. We suggest that the increased yield of DNA double-strand breaks in the polA 1 strain may be the result of the unsuccessful excision repair of ionizing radiation-induced dna base damage