Eukaryotic DNA Replication Fork.

Science.gov (United States)

Burgers, Peter M J; Kunkel, Thomas A

2017-06-20

This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

Data from Investigating Variation in Replicability: A “Many Labs” Replication Project

Directory of Open Access Journals (Sweden)

Richard A. Klein

2014-04-01

Full Text Available This dataset is from the Many Labs Replication Project in which 13 effects were replicated across 36 samples and over 6,000 participants. Data from the replications are included, along with demographic variables about the participants and contextual information about the environment in which the replication was conducted. Data were collected in-lab and online through a standardized procedure administered via an online link. The dataset is stored on the Open Science Framework website. These data could be used to further investigate the results of the included 13 effects or to study replication and generalizability more broadly.

Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress

Science.gov (United States)

García-Rodríguez, Néstor; Wong, Ronald P.; Ulrich, Helle D.

2016-01-01

Complete and faithful duplication of its entire genetic material is one of the essential prerequisites for a proliferating cell to maintain genome stability. Yet, during replication DNA is particularly vulnerable to insults. On the one hand, lesions in replicating DNA frequently cause a stalling of the replication machinery, as most DNA polymerases cannot cope with defective templates. This situation is aggravated by the fact that strand separation in preparation for DNA synthesis prevents common repair mechanisms relying on strand complementarity, such as base and nucleotide excision repair, from working properly. On the other hand, the replication process itself subjects the DNA to a series of hazardous transformations, ranging from the exposure of single-stranded DNA to topological contortions and the generation of nicks and fragments, which all bear the risk of inducing genomic instability. Dealing with these problems requires rapid and flexible responses, for which posttranslational protein modifications that act independently of protein synthesis are particularly well suited. Hence, it is not surprising that members of the ubiquitin family, particularly ubiquitin itself and SUMO, feature prominently in controlling many of the defensive and restorative measures involved in the protection of DNA during replication. In this review we will discuss the contributions of ubiquitin and SUMO to genome maintenance specifically as they relate to DNA replication. We will consider cases where the modifiers act during regular, i.e., unperturbed stages of replication, such as initiation, fork progression, and termination, but also give an account of their functions in dealing with lesions, replication stalling and fork collapse. PMID:27242895

Separation Techniques for Quantification of Radionuclides in Environmental Samples

Directory of Open Access Journals (Sweden)

Dusan Galanda

2009-01-01

Full Text Available The reliable and quantitative measurement of radionuclides is important in order to determine environmental quality and radiation safety, and to monitor regulatory compliance. We examined soil samples from Podunajske Biskupice, near the city of Bratislava in the Slovak Republic, for the presence of several natural (238U, 232Th, 40K and anthropogenic (137Cs, 90Sr, 239Pu, 240Pu, 241Am radionuclides. The area is adjacent to a refinery and hazardous waste processing center, as well as the municipal incinerator plant, and so might possess an unusually high level of ecotoxic metals. We found that the levels of both naturally occurring and anthropogenic radionuclides fell within the expected ranges, indicating that these facilities pose no radiological threat to the local environment. During the course of our analysis, we modified existing techniques in order to allow us to handle the unusually large and complex samples that were needed to determine the levels of 239Pu, 240Pu, and 241Am activity. We also rated three commercial techniques for the separation of 90Sr from aqueous solutions and found that two of them, AnaLig Sr-01 and Empore Extraction Disks, were suitable for the quantitative and reliable separation of 90Sr, while the third, Sr-Spec Resin, was less so. The main criterion in evaluating these methods was the chemical recovery of 90Sr, which was less than we had expected. We also considered speed of separation and additional steps needed to prepare the sample for separation.

Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase.

Science.gov (United States)

Lõoke, Marko; Maloney, Michael F; Bell, Stephen P

2017-02-01

Activation of the Mcm2-7 replicative DNA helicase is the committed step in eukaryotic DNA replication initiation. Although Mcm2-7 activation requires binding of the helicase-activating proteins Cdc45 and GINS (forming the CMG complex), an additional protein, Mcm10, drives initial origin DNA unwinding by an unknown mechanism. We show that Mcm10 binds a conserved motif located between the oligonucleotide/oligosaccharide fold (OB-fold) and A subdomain of Mcm2. Although buried in the interface between these domains in Mcm2-7 structures, mutations predicted to separate the domains and expose this motif restore growth to conditional-lethal MCM10 mutant cells. We found that, in addition to stimulating initial DNA unwinding, Mcm10 stabilizes Cdc45 and GINS association with Mcm2-7 and stimulates replication elongation in vivo and in vitro. Furthermore, we identified a lethal allele of MCM10 that stimulates initial DNA unwinding but is defective in replication elongation and CMG binding. Our findings expand the roles of Mcm10 during DNA replication and suggest a new model for Mcm10 function as an activator of the CMG complex throughout DNA replication. © 2017 Lõoke et al.; Published by Cold Spring Harbor Laboratory Press.

Soil separator and sampler and method of sampling

Science.gov (United States)

O'Brien, Barry H [Idaho Falls, ID; Ritter, Paul D [Idaho Falls, ID

2010-02-16

A soil sampler includes a fluidized bed for receiving a soil sample. The fluidized bed may be in communication with a vacuum for drawing air through the fluidized bed and suspending particulate matter of the soil sample in the air. In a method of sampling, the air may be drawn across a filter, separating the particulate matter. Optionally, a baffle or a cyclone may be included within the fluidized bed for disentrainment, or dedusting, so only the finest particulate matter, including asbestos, will be trapped on the filter. The filter may be removable, and may be tested to determine the content of asbestos and other hazardous particulate matter in the soil sample.

Accurate determination of trace amounts of phosphorus in geological samples by inductively coupled plasma atomic emission spectrometry with ion-exchange separation

International Nuclear Information System (INIS)

Asoh, Kazuya; Ebihara, Mitsuru

2013-01-01

Graphical abstract: -- Highlights: •We set up an effective ICP-AES procedure for determining trace P in rock samples. •Some certified values of P for reference rock samples were proved to be doubtful. •Accurate and reliable data were presented for a suite of geological reference rocks. -- Abstract: In order to determine trace amounts of phosphorus in geological and cosmochemical rock samples, simple as well as reliable analytical schemes using an ICP-AES instrument were investigated. A (conventional) ICP-AES procedure could determine phosphorus contents at the level of several 100 μg g −1 with a reasonable reproducibility ( −1 ; 1σ). An ICP-AES procedure coupled with matrix-separation using cation and anion exchange resins could lower the quantification level down to 1 μg g −1 or even lower under the present experimental conditions. The matrix-separation ICP-AES procedure developed in this study was applied to twenty-one geological reference samples issued by Geological Survey of Japan. Obtained values vary from 1250 μg g −1 for JB-3 (basalt) to 2.07 μg g −1 for JCt-1 (carbonate). Matrix-separation ICP-AES yielded reasonable reproducibility (less than 8.3%; 1σ) of three replicate analyses for all the samples analyzed. In comparison of our data with certificate values as well as literature or reported values, there appear to be an apparent (and large) discrepancy between our values and certificate/reported values regardless of phosphorus contents. Based on the reproducibility of our data and the analytical capability of the matrix-separation ICP-AES procedure developed in this study (in terms of quantification limit, recovery, selectivity of an analyte through pre-concentration process, etc.), it is concluded that certified values for several reference standard rocks should be reevaluated and revised accordingly. It may be further pointed that some phosphorus data reported in literatures should be critically evaluated when they are to be

Gender roles in relation to assertiveness and Eysenckian personality dimensions : Replication with a Spanish population sample

NARCIS (Netherlands)

Arrindell, W.A.; Vergara, A.I; Torres, B.; Caballo, V.E.; Sanderman, R.; Calvo, M G; Vanderende, J.; Oosterhof, L.; Castro, J

The objective of the present study was to evaluate the cross-national replicability of the usual pattern of associations observed in Anglo-Saxon samples between masculinity and femininity on the one hand and difficulty and distress in assertiveness and the major Eysenckian dimensions of personality

Direct separation of plutonium by thermochromatography from environmental samples

International Nuclear Information System (INIS)

Wacker, L.; Kraehenbuehl, U.

2002-01-01

A thermochromatographic separation was performed on plutonium from environmental soil samples. This procedure was investigated with the goal to measure low concentrations of plutonium by inductively coupled plasma-mass spectrometry (ICP-MS). The soil sample was chlorinated by thionylchloride as reactive gas at a temperature of 1400 K. The volatile chlorides were separated chromatographically and deposited in a temperature gradient tube filled with quartz grains. Results about the deposition behaviour of the elements were obtained. Two different formalisms based on the thermodynamic functions are used to describe the experimental data. One formula is used to describe the deposition behaviour of microscopic amounts of plutonium (adsorption), the other formula for macroamounts of the main matrix elements (desublimation). The calculated values are in a reasonable agreement with the experimental data. A determination of plutonium content was successfully made for a referenced sea sediment (IAEA-135) after the thermochromatographic sample preparation for ICP-MS. (orig.)

A Tumor-stroma Targeted Oncolytic Adenovirus Replicated in Human Ovary Cancer Samples and Inhibited Growth of Disseminated Solid Tumors in Mice

Science.gov (United States)

Lopez, M Veronica; Rivera, Angel A; Viale, Diego L; Benedetti, Lorena; Cuneo, Nicasio; Kimball, Kristopher J; Wang, Minghui; Douglas, Joanne T; Zhu, Zeng B; Bravo, Alicia I; Gidekel, Manuel; Alvarez, Ronald D; Curiel, David T; Podhajcer, Osvaldo L

2012-01-01

Targeting the tumor stroma in addition to the malignant cell compartment is of paramount importance to achieve complete tumor regression. In this work, we modified a previously designed tumor stroma-targeted conditionally replicative adenovirus (CRAd) based on the SPARC promoter by introducing a mutated E1A unable to bind pRB and pseudotyped with a chimeric Ad5/3 fiber (Ad F512v1), and assessed its replication/lytic capacity in ovary cancer in vitro and in vivo. AdF512v1 was able to replicate in fresh samples obtained from patients: (i) with primary human ovary cancer; (ii) that underwent neoadjuvant treatment; (iii) with metastatic disease. In addition, we show that four intraperitoneal (i.p.) injections of 5 × 1010 v.p. eliminated 50% of xenografted human ovary tumors disseminated in nude mice. Moreover, AdF512v1 replication in tumor models was enhanced 15–40-fold when the tumor contained a mix of malignant and SPARC-expressing stromal cells (fibroblasts and endothelial cells). Contrary to the wild-type virus, AdF512v1 was unable to replicate in normal human ovary samples while the wild-type virus can replicate. This study provides evidence on the lytic capacity of this CRAd and highlights the importance of targeting the stromal tissue in addition to the malignant cell compartment to achieve tumor regression. PMID:22948673

Development and application of an on-line sequential injection system for the separation of artificial and natural radionuclides in environmental samples

International Nuclear Information System (INIS)

Kim, C.-K.; Sansone, U.; Martin, P.; Kim, C.-S.

2007-02-01

The Chemistry Unit of the Physics, Chemistry and Instrumentation Laboratory in the IAEA's Seibersdorf Laboratory in Austria, has the programmatic responsibility to provide assistance to Member State laboratories in maintaining and improving the reliability of analytical measurement results, both in trace element and radionuclide determinations. This is accomplished through the provision of reference materials of terrestrial origin, validated analytical procedures, training in the implementation of internal quality control, and through the evaluation of measurement performance by organization of worldwide and regional interlaboratory comparison exercises. In this framework an on-line sequential injection (SI) system was developed, which can be widely used for the separation and preconcentration of target analytes from diverse environmental samples. The system enables the separation time to be shortened by maintaining a constant flow rate of solution and by avoiding clogging or bubbling in a chromatographic column. The SI system was successfully applied to the separation of Pu in IAEA reference material (IAEA Soil-6) and to the sequential separation of 210 Po and 210 Pb in phosphogypsum candidate reference material. The replicate analysis results of Pu in IAEA reference material (Soil-6) obtained with the SI system are in good agreement with the recommended value within 5% of standard deviation. The SI system enabled a halving in the separation time required for of radionuclides

Preconcentration of Arsenic in Water Samples Using the Composition-Induced Phase Separation Method and Determination by ETAAS

Directory of Open Access Journals (Sweden)

Güçoğlu M.

2013-04-01

Full Text Available A new phase transition microextraction method was developed for determination of trace amount of arsenic ions in water samples in this work. The method is based on the critical point of miscibility of solvents. In this method the mixed solution of sample and organic solvent is initially homogeneous but is separated into two phases by adding a secondary solvent (modifier. In acidic medium As(V was complexed with ammonium molybdate, this complex was quantitatively extracted to the mixture of organic solvent (acetonitrile/methyl isobutyl ketone before ETAAS determination. Total inorganic arsenic (III, V was extracted similarly after oxidation of As(III to As(V with nitric acid. Concentration of As(III was calculated by difference in the concentration between total arsenic and As(V. Optimization of the experimental conditions and instrumental parameters was investigated in detail. A detection limit of 0.05 μgL−1 with enrichment factor of 85 was achieved for only 5 mL of sample. The analytical curve was linear in the concentration range of 0.25-4.00 μgL−1. Relative standard deviation (RSD for 10 replicate determinations of 2.0 μgL−1 of As(V was 4,1%. The method was successfully applied to preconcentration and determination of arsenic in real water samples.

Outcomes of role stress: a multisample constructive replication.

Science.gov (United States)

Kemery, E R; Bedeian, A G; Mossholder, K W; Touliatos, J

1985-06-01

Responses from four separate samples of accountants and hospital employees provided a constructive replication of the Bedeian and Armenakis (1981) model of the causal nexus between role stress and selected outcome variables. We investigated the relationship between both role ambiguity and role conflict--as specific forms of role stress--and job-related tension, job satisfaction, and propensity to leave, using LISREL IV, a technique capable of providing statistical data for a hypothesized population model, as well as for specific causal paths. Results, which support the Bedeian and Armenakis model, are discussed in light of previous research.

Effector-Triggered Self-Replication in Coupled Subsystems.

Science.gov (United States)

Komáromy, Dávid; Tezcan, Meniz; Schaeffer, Gaël; Marić, Ivana; Otto, Sijbren

2017-11-13

In living systems processes like genome duplication and cell division are carefully synchronized through subsystem coupling. If we are to create life de novo, similar control over essential processes such as self-replication need to be developed. Here we report that coupling two dynamic combinatorial subsystems, featuring two separate building blocks, enables effector-mediated control over self-replication. The subsystem based on the first building block shows only self-replication, whereas that based on the second one is solely responsive toward a specific external effector molecule. Mixing the subsystems arrests replication until the effector molecule is added, resulting in the formation of a host-effector complex and the liberation of the building block that subsequently engages in self-replication. The onset, rate and extent of self-replication is controlled by the amount of effector present. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Addressing the "Replication Crisis": Using Original Studies to Design Replication Studies with Appropriate Statistical Power.

Science.gov (United States)

Anderson, Samantha F; Maxwell, Scott E

2017-01-01

Psychology is undergoing a replication crisis. The discussion surrounding this crisis has centered on mistrust of previous findings. Researchers planning replication studies often use the original study sample effect size as the basis for sample size planning. However, this strategy ignores uncertainty and publication bias in estimated effect sizes, resulting in overly optimistic calculations. A psychologist who intends to obtain power of .80 in the replication study, and performs calculations accordingly, may have an actual power lower than .80. We performed simulations to reveal the magnitude of the difference between actual and intended power based on common sample size planning strategies and assessed the performance of methods that aim to correct for effect size uncertainty and/or bias. Our results imply that even if original studies reflect actual phenomena and were conducted in the absence of questionable research practices, popular approaches to designing replication studies may result in a low success rate, especially if the original study is underpowered. Methods correcting for bias and/or uncertainty generally had higher actual power, but were not a panacea for an underpowered original study. Thus, it becomes imperative that 1) original studies are adequately powered and 2) replication studies are designed with methods that are more likely to yield the intended level of power.

Separation of Be and Al for AMS using single-step column chromatography

Energy Technology Data Exchange (ETDEWEB)

Binnie, Steven A., E-mail: sbinnie@uni-koeln.de [Institute for Geology und Mineralogy, University of Cologne, 4-6 Greinstrasse, Cologne D-50939 (Germany); Dunai, Tibor J.; Voronina, Elena; Goral, Tomasz [Institute for Geology und Mineralogy, University of Cologne, 4-6 Greinstrasse, Cologne D-50939 (Germany); Heinze, Stefan; Dewald, Alfred [University of Cologne, Institut für Kernphysik, Zülpicher Str. 77, Cologne D-50937 (Germany)

2015-10-15

With the aim of simplifying AMS target preparation procedures for TCN measurements we tested a new extraction chromatography approach which couples an anion exchange resin (WBEC) to a chelating resin (Beryllium resin) to separate Be and Al from dissolved quartz samples. Results show that WBEC–Beryllium resin stacks can be used to provide high purity Be and Al separations using a combination of hydrochloric/oxalic and nitric acid elutions. {sup 10}Be and {sup 26}Al concentrations from quartz samples prepared using more standard procedures are compared with results from replicate samples prepared using the coupled WBEC–Beryllium resin approach and show good agreement. The new column procedure is performed in a single step, reducing sample preparation times relative to more traditional methods of TCN target production.

Separation of Be and Al for AMS using single-step column chromatography

Science.gov (United States)

Binnie, Steven A.; Dunai, Tibor J.; Voronina, Elena; Goral, Tomasz; Heinze, Stefan; Dewald, Alfred

2015-10-01

With the aim of simplifying AMS target preparation procedures for TCN measurements we tested a new extraction chromatography approach which couples an anion exchange resin (WBEC) to a chelating resin (Beryllium resin) to separate Be and Al from dissolved quartz samples. Results show that WBEC-Beryllium resin stacks can be used to provide high purity Be and Al separations using a combination of hydrochloric/oxalic and nitric acid elutions. 10Be and 26Al concentrations from quartz samples prepared using more standard procedures are compared with results from replicate samples prepared using the coupled WBEC-Beryllium resin approach and show good agreement. The new column procedure is performed in a single step, reducing sample preparation times relative to more traditional methods of TCN target production.

Three Conceptual Replication Studies in Group Theory

Science.gov (United States)

Melhuish, Kathleen

2018-01-01

Many studies in mathematics education research occur with a nonrepresentative sample and are never replicated. To challenge this paradigm, I designed a large-scale study evaluating student conceptions in group theory that surveyed a national, representative sample of students. By replicating questions previously used to build theory around student…

Use of immunomagnetic separation for the detection of Desulfovibrio vulgaris from environmental samples

Energy Technology Data Exchange (ETDEWEB)

Chakraborty, R.; Hazen, T.C.; Joyner, D.C.; Kusel, K.; Singer, M.E.; Sitte, J.; Torok, T.

2011-04-15

Immunomagnetic separation (IMS) has proved highly efficient for recovering microorganisms from heterogeneous samples. Current investigation targeted the separation of viable cells of the sulfate-reducing bacterium, Desulfovibrio vulgaris. Streptavidin-coupled paramagnetic beads and biotin labeled antibodies raised against surface antigens of this microorganism were used to capture D. vulgaris cells in both bioreactor grown laboratory samples and from extremely low-biomass environmental soil and subsurface drilling samples. Initial studies on detection, recovery efficiency and viability for IMS were performed with laboratory grown D. vulgaris cells using various cell densities. Efficiency of cell isolation and recovery (i.e., release of the microbial cells from the beads following separation) was followed by microscopic imaging and acridine orange direct counts (AODC). Excellent recovery efficiency encouraged the use of IMS to capture Desulfovibrio spp. cells from low-biomass environmental samples. The environmental samples were obtained from a radionuclide-contaminated site in Germany and the chromium (VI)-contaminated Hanford site, an ongoing bioremediation project of the U.S. Department of Energy. Field deployable IMS technology may greatly facilitate environmental sampling and bioremediation process monitoring and enable transcriptomics and proteomics/metabolomics-based studies directly on cells collected from the field.

An electron-hadron separator for digital sampling calorimeters

International Nuclear Information System (INIS)

Winter, K. de; Geiregat, D.; Vilain, P.; Wilquet, G.; Bergsma, F.; Binder, U.; Burkard, H.; Capone, A.; Ereditato, A.; Flegel, W.; Grote, H.; Nieuwenhuis, C.; Oeveras, H.; Palladino, V.; Panman, J.; Piredda, G.; Winter, K.; Zacek, G.; Zacek, V.; Bauche, T.; Beyer, R.; Blobel, V.; Buesser, F.W.; Foos, C.; Gerland, L.; Niebergall, F.; Staehelin, P.; Tadsen, A.; Gorbunov, P.; Grigoriev, E.; Khovansky, V.; Maslennikov, A.; Rosanov, A.; Lippich, W.; Nathaniel, A.; Staude, A.; De Pedis, D.; Di Capua, E.; Dore, U.; Loverre, P.F.; Rambaldi-Frenkel, A.; Santacesaria, R.; Zanello, D.

1989-01-01

A fast and effective algorithm for electromagnetic and hadronic shower separation has been developed for the digital sampling calorimeter of the CHARM II experiment. It is based on a generalization of the minimal spanning tree concept and can be easily applied to other existing calorimeters. In this particular application, which requires the highest efficiency for retaining electromagnetic showers, one gets, for 99% efficiency, a rejection factor of the order of 100 for hadronic showers. (orig.)

[Separation anxiety disorder in a sample of children of divorce].

Science.gov (United States)

Orgilés Amorós, Mireia; Espada Sánchez, José Pedro; Méndez Carrillo, Xavier

2008-08-01

Anxiety in children through separation from parents is one of the most frequent psychological problems in the infantile population. Children of divorce are more vulnerable to suffer this disorder due to the abrupt separation from one of the parents after the break-up, which they may experience as a traumatic event that predisposes them to react anxiously in daily separations. The purpose of this study is to examine the presence of symptoms of separation anxiety and general anxiety in a Spanish sample of 95 students of ages between 8 and 12 years. They were compared to a group of children of similar ages and sex whose parents are not divorced. The results show that children of divorce present higher levels of separation anxiety than the children whose parents remain together. Moreover, they show significant levels of generalized anxiety, but similar to that of the other group of children (undivorced parents). The clinical implications of these findings are discussed, emphasizing the importance of cooperation and frequent contact of the children with both parents to promote their security and autonomy.

A large replication study and meta-analysis in European samples provides further support for association of AHI1 markers with schizophrenia

DEFF Research Database (Denmark)

Ingason, Andrés; Giegling, Ina; Cichon, Sven

2010-01-01

The Abelson helper integration site 1 (AHI1) gene locus on chromosome 6q23 is among a group of candidate loci for schizophrenia susceptibility that were initially identified by linkage followed by linkage disequilibrium mapping, and subsequent replication of the association in an independent sample....... Here, we present results of a replication study of AHI1 locus markers, previously implicated in schizophrenia, in a large European sample (in total 3907 affected and 7429 controls). Furthermore, we perform a meta-analysis of the implicated markers in 4496 affected and 18,920 controls. Both...... as the neighbouring phosphodiesterase 7B (PDE7B)-may be considered candidates for involvement in the genetic aetiology of schizophrenia....

Analysis of the temporal program of replication initiation in yeast chromosomes.

Science.gov (United States)

Friedman, K L; Raghuraman, M K; Fangman, W L; Brewer, B J

1995-01-01

The multiple origins of eukaryotic chromosomes vary in the time of their initiation during S phase. In the chromosomes of Saccharomyces cerevisiae the presence of a functional telomere causes nearby origins to delay initiation until the second half of S phase. The key feature of telomeres that causes the replication delay is the telomeric sequence (C(1-3)A/G(1-3)T) itself and not the proximity of the origin to a DNA end. A second group of late replicating origins has been found at an internal position on chromosome XIV. Four origins, spanning approximately 140 kb, initiate replication in the second half of S phase. At least two of these internal origins maintain their late replication time on circular plasmids. Each of these origins can be separated into two functional elements: those sequences that provide origin function and those that impose late activation. Because the assay for determining replication time is costly and laborious, it has not been possible to analyze in detail these 'late' elements. We report here the development of two new assays for determining replication time. The first exploits the expression of the Escherichia coli dam methylase in yeast and the characteristic period of hemimethylation that transiently follows the passage of a replication fork. The second uses quantitative hybridization to detect two-fold differences in the amount of specific restriction fragments as a function of progress through S phase. The novel aspect of this assay is the creation in vivo of a non-replicating DNA sequence by site-specific pop-out recombination. This non-replicating fragment acts as an internal control for copy number within and between samples. Both of these techniques are rapid and much less costly than the more conventional density transfer experiments that require CsCl gradients to detect replicated DNA. With these techniques it should be possible to identify the sequences responsible for late initiation, to search for other late replicating

Proteome profiling analysis of human ovarian cancer serum samples

International Nuclear Information System (INIS)

Cognetti, F.; Citro, G.

2009-01-01

Mass Spectrometry represents a powerful tool in cancer research to discovery of potential bio markers through peak identification from serum profiling. By using high resolution MALDITOF and bioinformatic analysis almost 400 serum sample homogeneously distributed between biopsy confirmed ovarian cancer and high risk serum samples were analyzed. Each serum sample run in duplicate and whole serum sample preparation procedure has been performed by Hamilton Star Robot in order to reduce bias and the replicates with a low Pearson coefficient are removed. After automated reverse phase magnetic beads separation the samples were tested in MALDI-TOF

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

Science.gov (United States)

Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J.; Schmidt, Kristina H.

2016-01-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186–212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks. PMID:27923055

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression.

Science.gov (United States)

Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J; Schmidt, Kristina H

2016-12-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.

Gene organization inside replication domains in mammalian genomes

Science.gov (United States)

Zaghloul, Lamia; Baker, Antoine; Audit, Benjamin; Arneodo, Alain

2012-11-01

We investigate the large-scale organization of human genes with respect to "master" replication origins that were previously identified as bordering nucleotide compositional skew domains. We separate genes in two categories depending on their CpG enrichment at the promoter which can be considered as a marker of germline DNA methylation. Using expression data in mouse, we confirm that CpG-rich genes are highly expressed in germline whereas CpG-poor genes are in a silent state. We further show that, whether tissue-specific or broadly expressed (housekeeping genes), the CpG-rich genes are over-represented close to the replication skew domain borders suggesting some coordination of replication and transcription. We also reveal that the transcription of the longest CpG-rich genes is co-oriented with replication fork progression so that the promoter of these transcriptionally active genes be located into the accessible open chromatin environment surrounding the master replication origins that border the replication skew domains. The observation of a similar gene organization in the mouse genome confirms the interplay of replication, transcription and chromatin structure as the cornerstone of mammalian genome architecture.

Atmospheric pressure surface sampling/ionization techniques for direct coupling of planar separations with mass spectrometry.

Science.gov (United States)

Pasilis, Sofie P; Van Berkel, Gary J

2010-06-18

Planar separations, which include thin layer chromatography and gel electrophoresis, are in widespread use as important and powerful tools for conducting separations of complex mixtures. To increase the utility of planar separations, new methods are needed that allow in situ characterization of the individual components of the separated mixtures. A large number of atmospheric pressure surface sampling and ionization techniques for use with mass spectrometry have emerged in the past several years, and several have been investigated as a means for mass spectrometric read-out of planar separations. In this article, we review the atmospheric pressure surface sampling and ionization techniques that have been used for the read-out of planar separation media. For each technique, we briefly explain the operational basics and discuss the analyte type for which it is appropriate and some specific applications from the literature. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Metal-organic frameworks for analytical chemistry: from sample collection to chromatographic separation.

Science.gov (United States)

Gu, Zhi-Yuan; Yang, Cheng-Xiong; Chang, Na; Yan, Xiu-Ping

2012-05-15

In modern analytical chemistry researchers pursue novel materials to meet analytical challenges such as improvements in sensitivity, selectivity, and detection limit. Metal-organic frameworks (MOFs) are an emerging class of microporous materials, and their unusual properties such as high surface area, good thermal stability, uniform structured nanoscale cavities, and the availability of in-pore functionality and outer-surface modification are attractive for diverse analytical applications. This Account summarizes our research on the analytical applications of MOFs ranging from sampling to chromatographic separation. MOFs have been either directly used or engineered to meet the demands of various analytical applications. Bulk MOFs with microsized crystals are convenient sorbents for direct application to in-field sampling and solid-phase extraction. Quartz tubes packed with MOF-5 have shown excellent stability, adsorption efficiency, and reproducibility for in-field sampling and trapping of atmospheric formaldehyde. The 2D copper(II) isonicotinate packed microcolumn has demonstrated large enhancement factors and good shape- and size-selectivity when applied to on-line solid-phase extraction of polycyclic aromatic hydrocarbons in water samples. We have explored the molecular sieving effect of MOFs for the efficient enrichment of peptides with simultaneous exclusion of proteins from biological fluids. These results show promise for the future of MOFs in peptidomics research. Moreover, nanosized MOFs and engineered thin films of MOFs are promising materials as novel coatings for solid-phase microextraction. We have developed an in situ hydrothermal growth approach to fabricate thin films of MOF-199 on etched stainless steel wire for solid-phase microextraction of volatile benzene homologues with large enhancement factors and wide linearity. Their high thermal stability and easy-to-engineer nanocrystals make MOFs attractive as new stationary phases to fabricate MOF

The Structure of The Extended Psychosis Phenotype in Early Adolescence—A Cross-sample Replication

Science.gov (United States)

Wigman, Johanna T. W.; Vollebergh, Wilma A. M.; Raaijmakers, Quinten A. W.; Iedema, Jurjen; van Dorsselaer, Saskia; Ormel, Johan; Verhulst, Frank C.; van Os, Jim

2011-01-01

The extended psychosis phenotype, or the expression of nonclinical positive psychotic experiences, is already prevalent in adolescence and has a dose-response risk relationship with later psychotic disorder. In 2 large adolescent general population samples (n = 5422 and n = 2230), prevalence and structure of the extended psychosis phenotype was investigated. Positive psychotic experiences, broadly defined, were reported by the majority of adolescents. Exploratory analysis with Structural Equation Modelling (Exploratory Factor Analysis followed by Confirmatory Factor Analysis [CFA]) in sample 1 suggested that psychotic experiences were best represented by 5 underlying dimensions; CFA in sample 2 provided a replication of this model. Dimensions were labeled Hallucinations, Delusions, Paranoia, Grandiosity, and Paranormal beliefs. Prevalences differed strongly, Hallucinations having the lowest and Paranoia having the highest rates. Girls reported more experiences on all dimensions, except Grandiosity, and from age 12 to 16 years rates increased. Hallucinations, Delusions, and Paranoia, but not Grandiosity and Paranormal beliefs, were associated with distress and general measures of psychopathology. Thus, only some of the dimensions of the extended psychosis phenotype in young people may represent a continuum with more severe psychopathology and predict later psychiatric disorder. PMID:20044595

Causation and the origin of life. Metabolism or replication first?

Science.gov (United States)

Pross, Addy

2004-06-01

The conceptual gulf that separates the 'metabolism first' and 'replication first' mechanisms for the emergence of life continues to cloud the origin of life debate. In the present paper we analyze this aspect of the origin of life problem and offer arguments in favor of the 'replication first' school. Utilizing Wicken's two-tier approach to causation we argue that a causal connection between replication and metabolism can only be demonstrated if replication would have preceded metabolism. In conjunction with existing empirical evidence and theoretical reasoning, our analysis concludes that there is no substantive evidence for a 'metabolism first' mechanism for life's emergence, while a coherent case can be made for the 'replication first' group of mechanisms. The analysis reaffirms our conviction that life is an extreme expression of kinetic control, and that the emergence of metabolic pathways can be understood by considering life as a manifestation of 'replicative chemistry'.

Volatile and non-volatile elements in grain-size separated samples of Apollo 17 lunar soils

International Nuclear Information System (INIS)

Giovanoli, R.; Gunten, H.R. von; Kraehenbuehl, U.; Meyer, G.; Wegmueller, F.; Gruetter, A.; Wyttenbach, A.

1977-01-01

Three samples of Apollo 17 lunar soils (75081, 72501 and 72461) were separated into 9 grain-size fractions between 540 and 1 μm mean diameter. In order to detect mineral fractionations caused during the separation procedures major elements were determined by instrumental neutron activation analyses performed on small aliquots of the separated samples. Twenty elements were measured in each size fraction using instrumental and radiochemical neutron activation techniques. The concentration of the main elements in sample 75081 does not change with the grain-size. Exceptions are Fe and Ti which decrease slightly and Al which increases slightly with the decrease in the grain-size. These changes in the composition in main elements suggest a decrease in Ilmenite and an increase in Anorthite with decreasing grain-size. However, it can be concluded that the mineral composition of the fractions changes less than a factor of 2. Samples 72501 and 72461 are not yet analyzed for the main elements. (Auth.)

Lack of association between digit ratio (2D:4D) and assertiveness: replication in a large sample.

Science.gov (United States)

Voracek, Martin

2009-12-01

Findings regarding within-sex associations of digit ratio (2D:4D), a putative pointer to long-lasting effects of prenatal androgen action, and sexually differentiated personality traits have generally been inconsistent or unreplicable, suggesting that effects in this domain, if any, are likely small. In contrast to evidence from Wilson's important 1983 study, a forerunner of modern 2D:4D research, two recent studies in 2005 and 2008 by Freeman, et al. and Hampson, et al. showed assertiveness, a presumably male-typed personality trait, was not associated with 2D:4D; however, these studies were clearly statistically underpowered. Hence this study examined this question anew, based on a large sample of 491 men and 627 women. Assertiveness was only modestly sexually differentiated, favoring men, and a positive correlate of age and education and a negative correlate of weight and Body Mass Index among women, but not men. Replicating the two prior studies, 2D:4D was throughout unrelated to assertiveness scores. This null finding was preserved with controls for correlates of assertiveness, also in nonparametric analysis and with tests for curvilinear relations. Discussed are implications of this specific null finding, now replicated in a large sample, for studies of 2D:4D and personality in general and novel research approaches to proceed in this field.

Radiochemical separation of actinides for their determination in environmental samples and waste products

Energy Technology Data Exchange (ETDEWEB)

Gleisberg, B [Nuclear Engineering and Analytics Rossendorf, Inc. (VKTA), Dresden (Germany)

1997-03-01

The determination of low level activities of actinides in environmental samples and waste products makes high demands on radiochemical separation methods. Artificial and natural actinides were analyzed in samples form the surrounding areas of NPP and of uranium mines, incorporation samples, solutions containing radioactive fuel, solutions and solids resutling from the process, and in wastes. The activities are measured by {alpha}-spectrometry and {gamma}-spectrometry. (DG)

Extraction chromatographic separation of iron from complex liquid samples and the determination of 55Fe

International Nuclear Information System (INIS)

Grahek, Z.; Rozmaric Macefat, M.

2006-01-01

Iron separation is described from liquid samples with a high concentration of ions that enables simple determination of 55 Fe. One of the described methods consists of iron precipitation from a large volume seawater by sodium hydroxide and/or ammonium carbonate and separation from other elements (Ca, Sr, Cu, Mg, etc.) on a TRU column with 4M HCl or 8M HNO 3 . In the other procedure iron is separated directly from a mixture of seawater samples and HCl on a TRU column. In both methods, the iron recovery is almost 100%. After separation, 55 Fe is determined by counting with a liquid scintillation counter. The binding of Fe and Zn on TEVA, U/TEVA and TRU resins from seawater solutions of HCl and HNO 3 depends on the type of the resin, concentration of acid and other ions. Iron and zinc can be separated from seawater on a U/TEVA column with 2M HCl. (author)

Separation techniques for low-level determination of actinides in soil samples

Energy Technology Data Exchange (ETDEWEB)

Eikenberg, J. [Paul Scherrer Institute, Department Logistics for Radiation Safety and Security, CH-5232 Villigen (Switzerland)], E-mail: jost.eikenberg@psi.ch; Jaeggi, M.; Beer, H.; Ruethi, M.; Zumsteg, I. [Paul Scherrer Institute, Department Logistics for Radiation Safety and Security, CH-5232 Villigen (Switzerland)

2009-05-15

The separation methods for soil samples applied at PSI are based on extraction chromatography and ion exchange. After sample leaching, the actinides are pre-concentrated via precipitation using oxalic acid. Besides the classical separation methods applying the extraction chromatographic resins U/TEVA{sup TM} (for U, Th), TRU{sup TM} (Pu, Am), new methods were recently implemented to increase the radiochemical recovery of particularly trivalent Am and Cm. These methods do not require initial reduction of Pu(IV) to Pu(III) but stabilize Pu on the tetravalent oxidation state using a mixture of NaNO{sub 2}/H{sub 2}O{sub 2} in strong acidic medium. The Pu-fraction is then fixed along with Th onto Dowex AG 1-X2 anion exchanger resin. Th is eluted via complexation with 10 M HCl, Pu via reduction with HI. The fractions of Am+Cm and U are loaded onto DGA{sup TM} resin. This resin shows extraordinary high distribution coefficients (k'-values) exceeding 10{sup 4} (for Am) in strong nitric acid medium. The separation between U and Am is obtained quantitatively by decreasing the HNO{sub 3} concentration from 3 to 0.25 M (stripping of the U-fraction) while Am can be easily eluted thereafter using 0.25 M HCl as complexation compound.

Prediction Interval: What to Expect When You're Expecting … A Replication.

Directory of Open Access Journals (Sweden)

Jeffrey R Spence

Full Text Available A challenge when interpreting replications is determining whether the results of a replication "successfully" replicate the original study. Looking for consistency between two studies is challenging because individual studies are susceptible to many sources of error that can cause study results to deviate from each other and the population effect in unpredictable directions and magnitudes. In the current paper, we derive methods to compute a prediction interval, a range of results that can be expected in a replication due to chance (i.e., sampling error, for means and commonly used indexes of effect size: correlations and d-values. The prediction interval is calculable based on objective study characteristics (i.e., effect size of the original study and sample sizes of the original study and planned replication even when sample sizes across studies are unequal. The prediction interval provides an a priori method for assessing if the difference between an original and replication result is consistent with what can be expected due to sample error alone. We provide open-source software tools that allow researchers, reviewers, replicators, and editors to easily calculate prediction intervals.

Sequencing of real-world samples using a microfabricated hybrid device having unconstrained straight separation channels.

Science.gov (United States)

Liu, Shaorong; Elkin, Christopher; Kapur, Hitesh

2003-11-01

We describe a microfabricated hybrid device that consists of a microfabricated chip containing multiple twin-T injectors attached to an array of capillaries that serve as the separation channels. A new fabrication process was employed to create two differently sized round channels in a chip. Twin-T injectors were formed by the smaller round channels that match the bore of the separation capillaries and separation capillaries were incorporated to the injectors through the larger round channels that match the outer diameter of the capillaries. This allows for a minimum dead volume and provides a robust chip/capillary interface. This hybrid design takes full advantage, such as sample stacking and purification and uniform signal intensity profile, of the unique chip injection scheme for DNA sequencing while employing long straight capillaries for the separations. In essence, the separation channel length is optimized for both speed and resolution since it is unconstrained by chip size. To demonstrate the reliability and practicality of this hybrid device, we sequenced over 1000 real-world samples from Human Chromosome 5 and Ciona intestinalis, prepared at Joint Genome Institute. We achieved average Phred20 read of 675 bases in about 70 min with a success rate of 91%. For the similar type of samples on MegaBACE 1000, the average Phred20 read is about 550-600 bases in 120 min separation time with a success rate of about 80-90%.

Solvent extraction method for rapid separation of strontium-90 in milk and food samples

International Nuclear Information System (INIS)

Hingorani, S.B.; Sathe, A.P.

1991-01-01

A solvent extraction method, using tributyl phosphate, for rapid separation of strontium-90 in milk and other food samples has been presented in this report in view of large number of samples recieved after Chernobyl accident for checking radioactive contamination. The earlier nitration method in use for the determination of 90 Sr through its daughter 90 Y takes over two weeks for analysis of a sample. While by this extraction method it takes only 4 to 5 hours for sample analysis. Complete estimation including initial counting can be done in a single day. The chemical recovery varies between 80-90% compared to nitration method which is 65-80%. The purity of the method has been established by following the decay of yttrium-90 separated. Some of the results obtained by adopting this chemical method for food analysis are included. The method is, thus, found to be rapid and convenient for accurate estimation of strontium-90 in milk and food samples. (author). 2 tabs., 1 fig

Detection of human parvovirus 4 viremia in the follow-up blood samples from seropositive individuals suggests the existence of persistent viral replication or reactivation of latent viral infection.

Science.gov (United States)

Chen, Mao-Yuan; Hung, Chien-Ching; Lee, Kuang-Lun

2015-06-19

The transmission routes for human parvovirus 4 (PARV4) infections in areas with high seroprevalence are not known. In the work described here, persistent PARV4 viral replication was investigated by conducting a longitudinal study. Ten healthcare workers each provided a blood sample at the beginning of the study (first sample) and 12 months later (second sample). The paired samples were tested for PARV4-positivity by immunoblotting analysis and nested polymerase chain reactions. IgG antibodies against PARV4 were detected in six participants, three of whom also had IgM antibodies against PARV4. The immunoblotting results did not vary over time. PARV4 DNA was detected in the first blood sample from one participant who had IgG antibodies against PARV4 and in the second blood samples from 2 participants who had IgG and IgM antibodies against PARV4. Detection of PARV4 DNA in the second blood samples from two seropositive participants suggests the existence of persistent PARV4 replication or reactivation of inactive virus in the tissues. The finding of persistent or intermittent PARV4 replication in individuals with past infections provides an important clue toward unraveling the non-parenteral transmission routes of PARV4 infection in areas where the virus is endemic.

Development of novel separation techniques for biological samples in capillary electrophoresis

Energy Technology Data Exchange (ETDEWEB)

Chang, Huan -Tsung [Iowa State Univ., Ames, IA (United States)

1994-07-27

This dissertation includes three different topics: general introduction of capillary electrophoresis (CE); gradient in CE and CE in biological separations; and capillary gel electrophoresis (CGE) for DNA separation. Factors such as temperature, viscosity, pH, and the surface of capillary walls affecting the separation performance are demonstrated. A pH gradient between 3.0 and 5.2 is useful to improve the resolution among eight different organic acids. A flow gradient due to the change in the concentration of surfactant, which is able to coat to the capillary wall to change the flow rate and its direction, is also shown as a good way to improve the resolution for organic compounds. A temperature gradient caused by joule heat is shown by voltage programming to enhance the resolution and shorten the separation time for several phenolic compounds. The author also shows that self-regulating dynamic control of electroosmotic flow in CE by simply running separation in different concentrations of surfactant has less matrix effect on the separation performance. One of the most important demonstrations in this dissertation is that the author proposes on-column reaction which gives several advantages including the use of a small amount of sample, low risk of contamination, and time saving and kinetic features. The author uses this idea with laser induced fluorescence (LIF) as a detection mode to detect an on-column digestion of sub-ng of protein. This technique also is applied to single cell analysis in the group.

Diverse replication-associated protein encoding circular DNA viruses in guano samples of Central-Eastern European bats.

Science.gov (United States)

Kemenesi, Gábor; Kurucz, Kornélia; Zana, Brigitta; Földes, Fanni; Urbán, Péter; Vlaschenko, Anton; Kravchenko, Kseniia; Budinski, Ivana; Szodoray-Parádi, Farkas; Bücs, Szilárd; Jére, Csaba; Csősz, István; Szodoray-Parádi, Abigél; Estók, Péter; Görföl, Tamás; Boldogh, Sándor; Jakab, Ferenc

2018-03-01

Circular replication-associated protein encoding single-stranded DNA (CRESS DNA) viruses are increasingly recognized worldwide in a variety of samples. Representative members include well-described veterinary pathogens with worldwide distribution, such as porcine circoviruses or beak and feather disease virus. In addition, numerous novel viruses belonging to the family Circoviridae with unverified pathogenic roles have been discovered in different human samples. Viruses of the family Genomoviridae have also been described as being highly abundant in different faecal and environmental samples, with case reports showing them to be suspected pathogens in human infections. In order to investigate the genetic diversity of these viruses in European bat populations, we tested guano samples from Georgia, Hungary, Romania, Serbia and Ukraine. This resulted in the detection of six novel members of the family Circoviridae and two novel members of the family Genomoviridae. Interestingly, a gemini-like virus, namely niminivirus, which was originally found in raw sewage samples in Nigeria, was also detected in our samples. We analyzed the nucleotide composition of members of the family Circoviridae to determine the possible host origins of these viruses. This study provides the first dataset on CRESS DNA viruses of European bats, and members of several novel viral species were discovered.

Replication of surface features from a master model to an amorphous metallic article

Science.gov (United States)

Johnson, William L.; Bakke, Eric; Peker, Atakan

1999-01-01

The surface features of an article are replicated by preparing a master model having a preselected surface feature thereon which is to be replicated, and replicating the preselected surface feature of the master model. The replication is accomplished by providing a piece of a bulk-solidifying amorphous metallic alloy, contacting the piece of the bulk-solidifying amorphous metallic alloy to the surface of the master model at an elevated replication temperature to transfer a negative copy of the preselected surface feature of the master model to the piece, and separating the piece having the negative copy of the preselected surface feature from the master model.

Properties influencing cracking and separation of palm nuts in a mechanical cracker cum separator

Directory of Open Access Journals (Sweden)

JOSHUA OLANREWAJU OLAOYE

2018-01-01

Full Text Available Experimental studies of some properties influencing cracking and separation of palm kernel from the shell was conducted in a palm kernel dual processing machine. A mechanical cracking cum separating machine was developed for the study. The cracking unit consists of feed hopper, impeller shaft, cracking drum and impeller blade. The nut falls by gravity through the hopper channel into the cracking drum where the cracking process takes place through the help of impeller blades that flip the palm nut against the walls of cylindrical cracking drum. The mass of cracked nut flows through the separating unit that separates the kernel from the shell. The separation is induced by high current of air mass generated by an axial fan. A dura palm variety was selected and a total sample of eighteen thousand (18000 palm nuts were obtained and divided into two groups (feed rates, A and B, of eight thousand and ten thousand palm nuts respectively. Sample groups A and B were further divided into five sub – groups of four hundred (400 and five hundred (500 palm kernel nuts. Each sub group (feed rate was replicated four times at different shaft speeds (600, 900, 1200, 1500 and 1800 rpm. Results showed that cracking efficiencies increased with respect to speed. Un-cracked nuts percentage ranged from 1.3 to 5.3% at 7.1% moisture content, and 1.6 to 4.5% for 400 and 500 feed rates, respectively. Cracking time for both feed rates decreased with shaft speed. Throughput capacity of 11.49 kg/h was observed to be the lowest at 600 rpm and moisture content of 7.1% for both 400 feed rates and the highest throughput capacity of 37.16 kg/h was recorded at 1800 rpm at moisture contents of 9.3% and 16.1%. The results of this study shows that moisture content, engine speed and feed rate are significant parameters that influence cracking of nuts and separation of palm kernel from the shell.

Speciation fingerprints of binary mixtures by the optimized sequential two-phase separation

International Nuclear Information System (INIS)

Macasek, F.

1995-01-01

The analysis of the separation methods suitable for chemical speciation of radionuclides and metals, and advantages of sequential (double) distribution technique were discussed. The equilibria are relatively easy to control and the method enables to minimize a matrix composition adjustment, and therefore it minimizes also the disturbance of original (native) state of elements. The technique may consist in the repeat solvent extraction of sample, or the replicate equilibration with sorbent. The common condition of applicability is a linear separation isotherm of the species, what is mostly a reasonable condition in case of trace concentrations. The equations used for simultaneous fitting were written in general form. 1 tab., 1 fig., 2 refs

Murine leukemia virus (MLV replication monitored with fluorescent proteins

Directory of Open Access Journals (Sweden)

Bittner Alexandra

2004-12-01

Full Text Available Abstract Background Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. Results We inserted the coding sequences for green fluorescent protein (GFP into the proline-rich region (PRR of the ecotropic envelope protein (Env and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. Conclusions Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.

Preconcentration and Separation of Mixed-Species Samples Near a Nano-Junction in a Convergent Microchannel

Directory of Open Access Journals (Sweden)

Ping-Hsien Chiu

2015-12-01

Full Text Available A fluidic microchip incorporating a convergent microchannel and a Nafion-nanoporous membrane is proposed for the preconcentration and separation of multi-species samples on a single platform. In the device, sample preconcentration is achieved by means of the ion concentration polarization effect induced at the micro/nano interface under the application of an external electric field, while species separation is achieved by exploiting the different electrophoretic mobilities of the sample components. The experimental results show that the device is capable of detecting C-reactive protein (CRP with an initial concentration as low as 9.50 × 10−6 mg/L given a sufficient preconcentration time and driving voltage. In addition, it is shown that a mixed-species sample consisting of three negatively-charged components (bovine serum albumin (BSA, tetramethylrhodamine(TAMRA isothiocyanate-Dextran and fluorescent polymer beads can be separated and preconcentrated within 20 min given a driving voltage of 100 V across 1 cm microchannel in length. In general, the present results confirm the feasibility of the device for the immunoassay or detection of various multi-species samples under low concentration in the biochemical and biomedical fields. The novel device can therefore improve the detection limit of traditional medical facilities.

The Betel Quid Dependence Scale: replication and extension in a Guamanian sample.

Science.gov (United States)

Herzog, Thaddeus A; Murphy, Kelle L; Little, Melissa A; Suguitan, Gil S; Pokhrel, Pallav; Kawamoto, Crissy T

2014-05-01

Betel quid is the fourth most commonly consumed psychoactive substance in the world. The Betel Quid Dependence Scale (BQDS) is the first instrument designed specifically to measure betel quid dependence. The three factor structure of the BQDS consists of "physical and psychological urgent need," "increasing dose," and "maladaptive use." The BQDS initially was validated in a sample of male prisoner ex-chewers in Taiwan. To replicate and extend the original validation research on the BQDS in a sample of male and female current betel quid chewers in Guam. A survey containing the BQDS was administered to 300 current betel quid chewers in Guam. Participants were compensated for their time with a gift card worth $25. Confirmatory factor analysis revealed an adequate fit with the hypothesized three-factor measurement model. ANOVAs and structural equations modeling revealed that betel quid dependence is associated with the inclusion of tobacco in the quid, number of chews per day, years of chewing, and education. The BQDS is valid for current English-speaking male and female chewers in Guam. Overall levels of betel quid dependence were high, and most chewers included tobacco in their betel quid. The results suggest that levels of dependence for betel quid are similar to those observed for nicotine dependence. Future research should explore other important psychological and behavioral aspects of betel quid chewing such as health risk perceptions and motivation to quit chewing. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Determination of 90Sr in environmental samples by microwave assisted digestion - chromatographic separation

International Nuclear Information System (INIS)

Torres, J.M.; Llaurado, M.; Rauret, G.

1998-01-01

The stages involved in the determination of 90 Sr in environmental samples are: sample attack, radiochemical separation (of both 90 Sr and its progeny 90 Y) and measurement. For the determination of 90 Sr, the introduction of microwave-assisted digestion methods has improved acid attack by drastically decreasing both digestion time and the volume of acidic reagents. Recent studies describe many applications of microwave-assisted methods for the determination of inorganic and organometallic compounds in several matrices. We have recently studied the microwave-assisted digestion of soils for the 90 Sr determination. The presented work extends the application of microwaves for the 90 Sr determination to other environmental samples such as sediments, vegetation and milk. An open-focused microwave system, which accepts large samples intakes usually required for radioanalytical chemistry due to the low level content of radionuclides in environmental samples, was used. This system can handle up to 10 g of sample intake which, in many cases, is enough to have acceptable limits of detection. Different digestion procedures are optimised for each matrix studied, paying special attention to the microwave power, the time of digestion and the volume of acidic reagents. Once the sample is in solution a new separation procedure using a specific resin -Sr.Spec- is applied and the measurement is performed by liquid scintillation. The results obtained are compared with a previously optimised method based on liquid-liquid extraction of 90 Y and Cerenkov radiation measurement

Separation and determination of 103Ru in samples of fission 99Mo

International Nuclear Information System (INIS)

Aghazarian, V.P.; Duran, Adrian P.; Mondino, Angel V.

2003-01-01

In Argentina 99 Mo is produced in the RA-3 reactor at the Ezeiza Atomic Center (CAE), by irradiation of miniplates of Al/U (90% 235 U) alloy. The 99 Mo separation is carried out at the Fission Radioisotopes Production Plant. Quality control is important to assure the quality of molybdenum that is produced in CAE. A new method to purify and on line quantify 103 Ru as an impurity present in 99 Mo samples was developed. This procedure is based in the RuO 4 volatilization and its dissolution in NaOH 6M. This is necessary due to the fact that 103 Ru cannot be detected in presence of high activities of 99 Mo without previous separation. This method allows a quantitative, specific, efficient, fast and reproducible separation of 103 Ru from 99 Mo. (author)

Fear of AIDS : are there replicable, invariant questionnaire dimensions?

NARCIS (Netherlands)

Arrindell, W.A.; Ross, M.W.; Bridges, K.Robert; van Hout, W.; Hofman, A.; Sanderman, R.

1989-01-01

Explored the dimensional structure of the 38-item Fear of acquired immune deficiency syndrome (AIDS) Schedule with 684 American students. Principal components analysis with VARIMAX rotation revealed 2 separate but related, internally consistent, and replicable dimensions of AIDS fear. These were (1)

DNA replication stress restricts ribosomal DNA copy number.

Science.gov (United States)

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

2017-09-01

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

DNA replication stress restricts ribosomal DNA copy number

Science.gov (United States)

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

2017-01-01

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

DNA replication stress restricts ribosomal DNA copy number.

Directory of Open Access Journals (Sweden)

Devika Salim

2017-09-01

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

Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication

Science.gov (United States)

On, Kin Fan; Beuron, Fabienne; Frith, David; Snijders, Ambrosius P; Morris, Edward P; Diffley, John F X

2014-01-01

Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of “firing factors.” Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins. PMID:24566989

Replication and robustness in developmental research.

Science.gov (United States)

Duncan, Greg J; Engel, Mimi; Claessens, Amy; Dowsett, Chantelle J

2014-11-01

Replications and robustness checks are key elements of the scientific method and a staple in many disciplines. However, leading journals in developmental psychology rarely include explicit replications of prior research conducted by different investigators, and few require authors to establish in their articles or online appendices that their key results are robust across estimation methods, data sets, and demographic subgroups. This article makes the case for prioritizing both explicit replications and, especially, within-study robustness checks in developmental psychology. It provides evidence on variation in effect sizes in developmental studies and documents strikingly different replication and robustness-checking practices in a sample of journals in developmental psychology and a sister behavioral science-applied economics. Our goal is not to show that any one behavioral science has a monopoly on best practices, but rather to show how journals from a related discipline address vital concerns of replication and generalizability shared by all social and behavioral sciences. We provide recommendations for promoting graduate training in replication and robustness-checking methods and for editorial policies that encourage these practices. Although some of our recommendations may shift the form and substance of developmental research articles, we argue that they would generate considerable scientific benefits for the field. (PsycINFO Database Record (c) 2014 APA, all rights reserved).

Quantitation of 11 alkylamines in atmospheric samples: separating structural isomers by ion chromatography

Science.gov (United States)

Place, Bryan K.; Quilty, Aleya T.; Di Lorenzo, Robert A.; Ziegler, Susan E.; VandenBoer, Trevor C.

2017-03-01

Amines are important drivers in particle formation and growth, which have implications for Earth's climate. In this work, we developed an ion chromatographic (IC) method using sample cation-exchange preconcentration for separating and quantifying the nine most abundant atmospheric alkylamines (monomethylamine (MMAH+), dimethylamine (DMAH+), trimethylamine (TMAH+), monoethylamine (MEAH+), diethylamine (DEAH+), triethylamine (TEAH+), monopropylamine (MPAH+), isomonopropylamine (iMPAH+), and monobutylamine (MBAH+)) and two alkyl diamines (1, 4-diaminobutane (DABH+) and 1, 5-diaminopentane (DAPH+)). Further, the developed method separates the suite of amines from five common atmospheric inorganic cations (Na+, NH4+, K+, Mg2+, Ca2+). All 16 cations are greater than 95 % baseline resolved and elute in a runtime of 35 min. This paper describes the first successful separation of DEAH+ and TMAH+ by IC and achieves separation between three sets of structural isomers, providing specificity not possible by mass spectrometry. The method detection limits for the alkylamines are in the picogram per injection range and the method precision (±1σ) analyzed over 3 months was within 16 % for all the cations. The performance of the IC method for atmospheric application was tested with biomass-burning (BB) particle extracts collected from two forest fire plumes in Canada. In extracts of a size-resolved BB sample from an aged plume, we detected and quantified MMAH+, DMAH+, TMAH+, MEAH+, DEAH+, and TEAH+ in the presence of Na+, NH4+, and K+ at molar ratios of amine to inorganic cation ranging from 1 : 2 to 1 : 1000. Quantities of DEAH+ and DMAH+ of 0.2-200 and 3-1200 ng m-3, respectively, were present in the extracts and an unprecedented amine-to-ammonium molar ratio greater than 1 was observed in particles with diameters spanning 56-180 nm. Extracts of respirable fine-mode particles (PM2. 5) from a summer forest fire in British Columbia in 2015 were found to contain iMPAH+, TMAH+, DEAH

Distributional Replication

OpenAIRE

Beare, Brendan K.

2009-01-01

Suppose that X and Y are random variables. We define a replicating function to be a function f such that f(X) and Y have the same distribution. In general, the set of replicating functions for a given pair of random variables may be infinite. Suppose we have some objective function, or cost function, defined over the set of replicating functions, and we seek to estimate the replicating function with the lowest cost. We develop an approach to estimating the cheapest replicating function that i...

Chromatin Immunoprecipitation of Replication Factors Moving with the Replication Fork

OpenAIRE

Rapp, Jordan B.; Ansbach, Alison B.; Noguchi, Chiaki; Noguchi, Eishi

2009-01-01

Replication of chromosomes involves a variety of replication proteins including DNA polymerases, DNA helicases, and other accessory factors. Many of these proteins are known to localize at replication forks and travel with them as components of the replisome complex. Other proteins do not move with replication forks but still play an essential role in DNA replication. Therefore, in order to understand the mechanisms of DNA replication and its controls, it is important to examine localization ...

Development of radiochemical separation method for determination of toxic elements in biological samples

International Nuclear Information System (INIS)

Maihara, V.A.; Vasconcellos, M.B.A.; Favaro, D.I.T.; Armelin, M.J.A.

1990-01-01

In order to determine Hg, Sb, As and Se in biological materials by neutron activation analysis, a radiochemical separation was developed. The chemical separation procedure used was based on the digestion of the irradiated sample in a mixture of H NO 3 and H 2 SO 4 in a teflon bomb, at 130 0 C for 1 to 4 hours. After the dissolution of organic matter, Hg and Sb were retained by a Dowex 2-X8 resin column in 6 M HCl. The effluent was passed through a TDO, tin dioxide column which retains As and Se in 3 M HCl medium. Radioactive tracers of these elements were used to determine the yields of the separation process. Certified reference materials were analyzed to check the precision and accuracy of the method. (author)

Development of a radiochemical separation method for toxic elements determination from biologic samples

International Nuclear Information System (INIS)

Malhara, V.A.; Vasconcellos, M.B.A.; Favaro, D.I.T.; Armelin, M.J.A.

1990-01-01

In order to determine Hg, Sb, As and Se in biological materials by neutron activation analysis, a radiochemical separation was developed. The chemical separation procedure used was based on the digestion of the irradiated sample in a mixture of HNO 3 and H 2 SO 4 in a teflon bomb, at 130 0 C for 1 to 4 hours. After the dissolution of organic matter, Hg and Sb were retained by a Dowex 2-XB resin column in 6M HCI. The efluent was passed through a TDO, tin dioxide column which retains As and Se in 3M HCI medium. Radioactive tracers of these elements were used to determine the yields of the separation process. Certified reference materials were analyzed to check the precision and accuracy of the method. (author)

Experimental toxicology: Issues of statistics, experimental design, and replication.

Science.gov (United States)

Briner, Wayne; Kirwan, Jeral

2017-01-01

The difficulty of replicating experiments has drawn considerable attention. Issues with replication occur for a variety of reasons ranging from experimental design to laboratory errors to inappropriate statistical analysis. Here we review a variety of guidelines for statistical analysis, design, and execution of experiments in toxicology. In general, replication can be improved by using hypothesis driven experiments with adequate sample sizes, randomization, and blind data collection techniques. Copyright © 2016 Elsevier B.V. All rights reserved.

Replication Catastrophe

DEFF Research Database (Denmark)

Toledo, Luis; Neelsen, Kai John; Lukas, Jiri

2017-01-01

Proliferating cells rely on the so-called DNA replication checkpoint to ensure orderly completion of genome duplication, and its malfunction may lead to catastrophic genome disruption, including unscheduled firing of replication origins, stalling and collapse of replication forks, massive DNA...... breakage, and, ultimately, cell death. Despite many years of intensive research into the molecular underpinnings of the eukaryotic replication checkpoint, the mechanisms underlying the dismal consequences of its failure remain enigmatic. A recent development offers a unifying model in which the replication...... checkpoint guards against global exhaustion of rate-limiting replication regulators. Here we discuss how such a mechanism can prevent catastrophic genome disruption and suggest how to harness this knowledge to advance therapeutic strategies to eliminate cancer cells that inherently proliferate under...

Method for the concentration and separation of actinides from biological and environmental samples

International Nuclear Information System (INIS)

Horwitz, E.P.; Dietz, M.L.

1989-01-01

A method and apparatus for the quantitative recover of actinide values from biological and environmental sample by passing appropriately prepared samples in a mineral acid solution through a separation column of a dialkyl(phenyl)-N,N-dialylcarbamoylmethylphosphine oxide dissolved in tri-n-butyl phosphate on an inert substrate which selectively extracts the actinide values. The actinide values can be eluted either as a group or individually and their presence quantitatively detected by alpha counting. 3 figs

Isolation of proteins involved in the replication of adenoviral DNA in vitro

International Nuclear Information System (INIS)

Lichy, J.H.; Nagata, K.; Friefeld, B.R.; Enomoto, T.; Field, J.; Guggenheimer, R.A.; Ikeda, J.E.; Horwitz, M.S.; Hurwitz, J.

1983-01-01

The simple mechanism of replication of adenoviral DNA has made adenovirus an especially useful model system for studies of eukaryotic replication mechanisms. The availability of this in vitro system that replicates exogenously added Ad DNA-pro has made it possible to characterize the factors involved in replication. The results presented in this paper summarize our further fractionation of the in vitro system. First, the properties of two factors purified from the uninfected nuclear extract are described. Second, the separation of the pTP/Ad Pol complex into subunits and the properties of the isolated subunits are presented. The 140K protein is shown to possess the Ad DNA polymerase activity. The results suggest that the only DNA polymerase required for adenoviral DNA replication in vitro is the 140K Ad DNA polymerase and that this enzyme is probably a viral gene product. 50 references, 10 figures, 3 tables

Optimization of a Pre-MEKC Separation SPE Procedure for Steroid Molecules in Human Urine Samples

Directory of Open Access Journals (Sweden)

Ilona Olędzka

2013-11-01

Full Text Available Many steroid hormones can be considered as potential biomarkers and their determination in body fluids can create opportunities for the rapid diagnosis of many diseases and disorders of the human body. Most existing methods for the determination of steroids are usually time- and labor-consuming and quite costly. Therefore, the aim of analytical laboratories is to develop a new, relatively low-cost and rapid implementation methodology for their determination in biological samples. Due to the fact that there is little literature data on concentrations of steroid hormones in urine samples, we have made attempts at the electrophoretic determination of these compounds. For this purpose, an extraction procedure for the optimized separation and simultaneous determination of seven steroid hormones in urine samples has been investigated. The isolation of analytes from biological samples was performed by liquid-liquid extraction (LLE with dichloromethane and compared to solid phase extraction (SPE with C18 and hydrophilic-lipophilic balance (HLB columns. To separate all the analytes a micellar electrokinetic capillary chromatography (MECK technique was employed. For full separation of all the analytes a running buffer (pH 9.2, composed of 10 mM sodium tetraborate decahydrate (borax, 50 mM sodium dodecyl sulfate (SDS, and 10% methanol was selected. The methodology developed in this work for the determination of steroid hormones meets all the requirements of analytical methods. The applicability of the method has been confirmed for the analysis of urine samples collected from volunteers—both men and women (students, amateur bodybuilders, using and not applying steroid doping. The data obtained during this work can be successfully used for further research on the determination of steroid hormones in urine samples.

Database Replication Prototype

OpenAIRE

Vandewall, R.

2000-01-01

This report describes the design of a Replication Framework that facilitates the implementation and com-parison of database replication techniques. Furthermore, it discusses the implementation of a Database Replication Prototype and compares the performance measurements of two replication techniques based on the Atomic Broadcast communication primitive: pessimistic active replication and optimistic active replication. The main contributions of this report can be split into four parts....

Autonomous model protocell division driven by molecular replication.

Science.gov (United States)

Taylor, J W; Eghtesadi, S A; Points, L J; Liu, T; Cronin, L

2017-08-10

The coupling of compartmentalisation with molecular replication is thought to be crucial for the emergence of the first evolvable chemical systems. Minimal artificial replicators have been designed based on molecular recognition, inspired by the template copying of DNA, but none yet have been coupled to compartmentalisation. Here, we present an oil-in-water droplet system comprising an amphiphilic imine dissolved in chloroform that catalyses its own formation by bringing together a hydrophilic and a hydrophobic precursor, which leads to repeated droplet division. We demonstrate that the presence of the amphiphilic replicator, by lowering the interfacial tension between droplets of the reaction mixture and the aqueous phase, causes them to divide. Periodic sampling by a droplet-robot demonstrates that the extent of fission is increased as the reaction progresses, producing more compartments with increased self-replication. This bridges a divide, showing how replication at the molecular level can be used to drive macroscale droplet fission.Coupling compartmentalisation and molecular replication is essential for the development of evolving chemical systems. Here the authors show an oil-in-water droplet containing a self-replicating amphiphilic imine that can undergo repeated droplet division.

Separation of selenium compounds by CE-ICP-MS in dynamically coated capillaries applied to selenized yeast samples

DEFF Research Database (Denmark)

Bendahl, Lars; Gammelgaard, Bente

2004-01-01

The selenium species in nutritional supplement tablets, based on selenized yeast, were separated by capillary zone electrophoresis using capillaries coated dynamically with poly(vinyl sulfonate) and detected by ICP-MS. Sample pre-treatment consisted of cold-water extraction by sonication and subs......The selenium species in nutritional supplement tablets, based on selenized yeast, were separated by capillary zone electrophoresis using capillaries coated dynamically with poly(vinyl sulfonate) and detected by ICP-MS. Sample pre-treatment consisted of cold-water extraction by sonication...

The Replication Recipe: What makes for a convincing replication?

NARCIS (Netherlands)

Brandt, M.J.; IJzerman, H.; Dijksterhuis, A.J.; Farach, F.J.; Geller, J.; Giner-Sorolla, R.; Grange, J.A.; Perugini, M.; Spies, J.R.; Veer, A. van 't

2014-01-01

Psychological scientists have recently started to reconsider the importance of close replications in building a cumulative knowledge base; however, there is no consensus about what constitutes a convincing close replication study. To facilitate convincing close replication attempts we have developed

The replication recipe : What makes for a convincing replication?

NARCIS (Netherlands)

Brandt, M.J.; IJzerman, H.; Dijksterhuis, Ap; Farach, Frank J.; Geller, Jason; Giner-Sorolla, Roger; Grange, James A.; Perugini, Marco; Spies, Jeffrey R.; van 't Veer, Anna

Psychological scientists have recently started to reconsider the importance of close replications in building a cumulative knowledge base; however, there is no consensus about what constitutes a convincing close replication study. To facilitate convincing close replication attempts we have developed

Open Probe fast GC-MS - combining ambient sampling ultra-fast separation and in-vacuum ionization for real-time analysis.

Science.gov (United States)

Keshet, U; Alon, T; Fialkov, A B; Amirav, A

2017-07-01

An Open Probe inlet was combined with a low thermal mass ultra-fast gas chromatograph (GC), in-vacuum electron ionization ion source and a mass spectrometer (MS) of GC-MS for obtaining real-time analysis with separation. The Open Probe enables ambient sampling via sample vaporization in an oven that is open to room air, and the ultra-fast GC provides ~30-s separation, while if no separation is required, it can act as a transfer line with 2 to 3-s sample transfer time. Sample analysis is as simple as touching the sample, pushing the sample holder into the Open Probe oven and obtaining the results in 30 s. The Open Probe fast GC was mounted on a standard Agilent 7890 GC that was coupled with an Agilent 5977A MS. Open Probe fast GC-MS provides real-time analysis combined with GC separation and library identification, and it uses the low-cost MS of GC-MS. The operation of Open Probe fast GC-MS is demonstrated in the 30-s separation and 50-s full analysis cycle time of tetrahydrocannabinol and cannabinol in Cannabis flower, sub 1-min analysis of trace trinitrotoluene transferred from a finger onto a glass surface, vitamin E in canola oil, sterols in olive oil, polybrominated flame retardants in plastics, alprazolam in Xanax drug pill and free fatty acids and cholesterol in human blood. The extrapolated limit of detection for pyrene is Open Probe fast GC-MS is demonstrated in the analysis of heroin in its street drug powder. The use of Open Probe with the fast GC acting as a transfer line is demonstrated in <10-s analysis without separation of ibuprofen and estradiol. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Speculations on the initiation of chromosome replication in Escherichia coli: the dualism hypothesis.

Science.gov (United States)

Norris, Vic

2011-05-01

The exact nature of the mechanism that triggers initiation of chromosome replication in the best understood of all organisms, Escherichia coli, remains mysterious. Here, I suggest that this mechanism evolved in response to the problems that arise if chromosome replication does not occur. E. coli is now known to be highly structured. This leads me to propose a mechanism for initiation of replication based on the dynamics of large assemblies of molecules and macromolecules termed hyperstructures. In this proposal, hyperstructures and their constituents are put into two classes, non-equilibrium and equilibrium, that spontaneously separate and that are appropriate for life in either good or bad conditions. Maintaining the right ratio(s) of non-equilibrium to equilibrium hyperstructures is therefore a major challenge for cells. I propose that this maintenance entails a major transfer of material from equilibrium to non-equilibrium hyperstructures once per cell and I further propose that this transfer times the cell cycle. More specifically, I speculate that the dialogue between hyperstructures involves the structuring of water and the condensation of cations and that one of the outcomes of ion condensation on ribosomal hyperstructures and decondensation from the origin hyperstructure is the separation of strands at oriC responsible for triggering initiation of replication. The dualism hypothesis that comes out of these speculations may help integrate models for initiation of replication, chromosome segregation and cell division with the 'prebiotic ecology' scenario of the origins of life. Copyright © 2011 Elsevier Ltd. All rights reserved.

Separation and Preconcentration of Trace Amounts of Nickel from Aqueous Samples

Directory of Open Access Journals (Sweden)

Reyhaneh Rahnama

2018-05-01

Full Text Available In this paper, a new method for preconcentration and measurement of trace amounts of nickel in aqueous samples by magnetic solid phase extraction (MSPE via magnetic carbon nanotubes (Mag-CNTs was developed. In order to increase selectivity, α-Furildioxime was used as chelating agent. In order to do extraction, optimum amount of ligand was added to the nickel sample and pH was set on 9, then 7 ml. of adsorbent was added and stirred for 15 minutes. After that, aqueous phase and adsorbent were separated by a strong magnet. Finally, the absorption was measured via flame atomic absorption spectrometry by analyte elution from the absorbent with an appropriate solution. Parameters affecting the extraction and preconcentration of nickel were investigated and optimized. Under optimum conditions, the calibration curve was linear in concentration range from 2.5 to 375 µg L-1 and the detection limit was 0.8 µg L-1 of nickel. The method was applied for determination of nickel in aqueous samples. The relative efficiency values of nickel measurement in aqueous samples were from 98.7% to 102.1%.  Results indicated that Mag-CNTs can be used as an effective and inexpensive absorbent for preconcentration and extraction of nickel from actual samples.

Performance and separation occurrence of binary probit regression estimator using maximum likelihood method and Firths approach under different sample size

Science.gov (United States)

Lusiana, Evellin Dewi

2017-12-01

The parameters of binary probit regression model are commonly estimated by using Maximum Likelihood Estimation (MLE) method. However, MLE method has limitation if the binary data contains separation. Separation is the condition where there are one or several independent variables that exactly grouped the categories in binary response. It will result the estimators of MLE method become non-convergent, so that they cannot be used in modeling. One of the effort to resolve the separation is using Firths approach instead. This research has two aims. First, to identify the chance of separation occurrence in binary probit regression model between MLE method and Firths approach. Second, to compare the performance of binary probit regression model estimator that obtained by MLE method and Firths approach using RMSE criteria. Those are performed using simulation method and under different sample size. The results showed that the chance of separation occurrence in MLE method for small sample size is higher than Firths approach. On the other hand, for larger sample size, the probability decreased and relatively identic between MLE method and Firths approach. Meanwhile, Firths estimators have smaller RMSE than MLEs especially for smaller sample sizes. But for larger sample sizes, the RMSEs are not much different. It means that Firths estimators outperformed MLE estimator.

Method of separate determination of high-ohmic sample resistance and contact resistance

Directory of Open Access Journals (Sweden)

Vadim A. Golubiatnikov

2015-09-01

Full Text Available A method of separate determination of two-pole sample volume resistance and contact resistance is suggested. The method is applicable to high-ohmic semiconductor samples: semi-insulating gallium arsenide, detector cadmium-zinc telluride (CZT, etc. The method is based on near-contact region illumination by monochromatic radiation of variable intensity from light emitting diodes with quantum energies exceeding the band gap of the material. It is necessary to obtain sample photo-current dependence upon light emitting diode current and to find the linear portion of this dependence. Extrapolation of this linear portion to the Y-axis gives the cut-off current. As the bias voltage is known, it is easy to calculate sample volume resistance. Then, using dark current value, one can determine the total contact resistance. The method was tested for n-type semi-insulating GaAs. The contact resistance value was shown to be approximately equal to the sample volume resistance. Thus, the influence of contacts must be taken into account when electrophysical data are analyzed.

Rapid filtration separation-based sample preparation method for Bacillus spores in powdery and environmental matrices.

Science.gov (United States)

Isabel, Sandra; Boissinot, Maurice; Charlebois, Isabelle; Fauvel, Chantal M; Shi, Lu-E; Lévesque, Julie-Christine; Paquin, Amélie T; Bastien, Martine; Stewart, Gale; Leblanc, Eric; Sato, Sachiko; Bergeron, Michel G

2012-03-01

Authorities frequently need to analyze suspicious powders and other samples for biothreat agents in order to assess environmental safety. Numerous nucleic acid detection technologies have been developed to detect and identify biowarfare agents in a timely fashion. The extraction of microbial nucleic acids from a wide variety of powdery and environmental samples to obtain a quality level adequate for these technologies still remains a technical challenge. We aimed to develop a rapid and versatile method of separating bacteria from these samples and then extracting their microbial DNA. Bacillus atrophaeus subsp. globigii was used as a simulant of Bacillus anthracis. We studied the effects of a broad variety of powdery and environmental samples on PCR detection and the steps required to alleviate their interference. With a benchmark DNA extraction procedure, 17 of the 23 samples investigated interfered with bacterial lysis and/or PCR-based detection. Therefore, we developed the dual-filter method for applied recovery of microbial particles from environmental and powdery samples (DARE). The DARE procedure allows the separation of bacteria from contaminating matrices that interfere with PCR detection. This procedure required only 2 min, while the DNA extraction process lasted 7 min, for a total of sample preparation procedure allowed the recovery of cleaned bacterial spores and relieved detection interference caused by a wide variety of samples. Our procedure was easily completed in a laboratory facility and is amenable to field application and automation.

Determination of gold and platinum in biological materials by radiochemical neutron activation analysis using electrolytic separation of gold

International Nuclear Information System (INIS)

Reitz, B.; Heydorn, K.

1993-01-01

A new method is presented for the determination of Au and Pt in biological materials based on neutron activation analysis with radiochemical separation of gold. Separation of gold by electrolytic deposition on a niobium cathode ascertains thee highest radiochemical purity without any interference from calcium or other major elements. With 199 Au as indicator for platinum the gold content of the sample not only strongly affects the limit of detection, but also causes interference by double neutron capture. Replicate analyses of BCR Certified Reference Materials No. 184, 185 and 186 were carried out. (author) 18 refs.; 3 figs.; 2 tabs

Cadmium determination in biological samples using neutron activation analysis with radiochemical separations

International Nuclear Information System (INIS)

Munoz A, Luis; Gras R, Nuri

2005-01-01

Chile has 7500 km of coastline on the Southern Pacific ocean,with about 4500 km of continental coastline that contains a variety of different geographical zones.This variety means that there is a great diversity of marine resources such as fish, shellfish and seaweeds. The utilization of these resources has been increasing in recent years making this sector an economically important one. The catch as of May 2002 came to 1.9 million tons and exports of the different species amounted to US$611.5 million as of April.But this important economic resource is being threatened by the technical demands imposed by importing countries, mainly the specific requirements for sanitary certification for fishery export products, depending on the markets of destination. The chemical element cadmium is one of the most strictly controlled elements due some shellfish accumulate a large amount of this element and to its high toxicity. The Chilean standard's analytical procedures for cadmium determination in hydro biological products, which must be met by laboratories that certify and control these products for export, are now being evaluated. Through its Chemical Metrology Unit, the Chilean Nuclear Energy Commission is strongly supporting this sector by preparing the secondary reference or control materials, and it has developed and implemented nuclear analytical methods for the certification of these materials, which will be used mostly in collaborative studies. This work describes the methodology developed for the determination of cadmium in biological samples, particularly in shellfish and fish. The method is based on neutron activation analysis with radiochemical separations, using the radioisotopes 115 Cd and 115m In, generated in the samples by bombarding with neutrons in a nuclear reactor. The samples were digested at 110 o C with H 2 SO 4 and H 2 O 2 and then the radioactive cadmium element was separated from the other elements present in the samples using a Bio Rad AG 2-X8

The determination of radioactive strontium in environmental samples by preferential ion exchange separation from calcium

International Nuclear Information System (INIS)

Fukushima, Hiroto; Nonaka, Nobuhiro; Honda, Tetsutaro; Kimura, Toshimasa; Higuchi, Hideo

1979-01-01

The determination of radioactive strontium in environmental samples involves a difficulty in the separation and purification of strontium from large amounts of calcium. The principal technique employed in this study is based on a cation exchange separation after preferential chelation of calcium over strontium by EDTA. The alkaline earth fraction separated from samples is dissolved in 1.2 M HCl and an equivalent amount of EDTA is added. The solution is passed through a Dowex 50W-X8 ion exchange column at a flow rate of 20 ml/min after the pH has been adjusted to 5.1. A fraction of calcium retained on the resin is eluted out with 1.8 l of 2 w/v% EDTA solution at pH 5.1. Then the strontium is eluted with 450 ml of 2 M HCl. The strontium carbonate is precipitated, and the chemical yield is determined. After standing for 14 days to reach the radioactive equilibrium of 90 Sr - 90 Y, the activity of 89 Sr + 90 Sr + 90 Y is measured. Then the activity of 90 Sr + 90 Y is measured by milking the 90 Y. The proposed procedure has been applied to sea water, soil and various ash samples of vegetables, seaweeds and daily foods. The results obtained are in good agreement with those by a classical fuming nitric acid precipitation method. The detection limits are 0.1 pCi/l for 10 l of sea water, 10 pCi/kg for 100 g of soil and 1 pCi/kg-fresh for 20 g ash samples, respectively. (author)

Comparison of alkali fusion and acid digestion methods for radiochemical separation of Uranium from dietary samples

International Nuclear Information System (INIS)

Kamesh Viswanathan, B.; Arunachalam, Kantha D.; Sathesh Kumar, A.; Jayakrishana, K.; Shanmugamsundaram, H.; Rao, D.D.

2014-01-01

Several methods exist for separation and measurement of uranium in dietary samples such as neutron activation analysis (NAA), alpha spectrometric determination, inductively coupled plasma mass spectrometry (ICP-MS) and fluorimetry. For qualitative determination of activity, NAA and alpha spectrometry are said to be superior to evaluate the isotopes of uranium ( 238 U, 234 U and 235 U). In case of alpha spectrometry, the samples have to undergo radiochemical analysis for separation from other elements for uranium detection. In our studies, uranium was determined in food matrices by acid digestion (AD) and alkali fusion (AF) methods. The recovery yield of uranium in food matrices was compared in order to get consistent yield. The average activity levels of 238 U and 234 U in food samples were calculated based on recovery yield of 232 U in the samples. The average recovery of 232 U in AD method was 22 ± 8% and in AF method, it was 14.9 ± 1.3%. The spread is more in AD method than the AF method from their mean. The lowest recovery of 232 U was found in AF method. This is due to the interference of other elements in the sample during electroplating. Experimental results showed that the uranium separation by AD method has better recovery than the AF method. The consistency in recovery of 232 U was better for AF method, which was lower than the AD method. However, overall for both the methods, the recovery can be termed as poor and need rigorous follow up studies for consistently higher recoveries (>50%) in these type of biological samples. There are reports indicating satisfactory recoveries of around 80% with 232 U as tracer in the food matrices

Variation: Use It or Misuse It--Replication and Its Variants

Science.gov (United States)

Drummond, Gordon B.; Vowler, Sarah L.

2012-01-01

In this article, the authors talk about variation and how variation between measurements may be reduced if sampling is not random. They also talk about replication and its variants. A replicate is a repeated measurement from the same experimental unit. An experimental unit is the smallest part of an experiment or a study that can be subject to a…

Fast Separation and Determination of Flavonoids in Honey Samples by Capillary Zone Electrophoresis

Directory of Open Access Journals (Sweden)

Jian-Qiu Tu

2017-03-01

Full Text Available Flavonoids have crucial applications in the biological and physiological fields. Honey, as an important sweet food made by bees, is rich in flavonoids. In this paper, the analytical method for flavonoids determination in different sorts of honey from different geographical locations was developed by capillary zone electrophoresis with direct ultraviolet detection. With a running buffer (borate, 20 mmol l−1 at pH of 8.4, four typical flavonoids, rutin, quercetin, kaempferol and myricetin, were separated in five minutes under a applied potential of 25 kV. A linear relationship within the range of 2.0 – 500 mg l−1 was found for these four kinds of flavonoids. Moreover, the detection limits ranged from 1.17 to 1.76 mg l−1. The recoveries lie in the range between 80 % – 107 %. The developed method was then used in the separation and determination of flavonoids in real honey samples collected from 12 geographical locations in the Henan Province of China. Rutin was detected in six, and quercetin in eight honey samples, which may be the markers for the identification of honey from different geographical origins.

Replicative Intermediates of Human Papillomavirus Type 11 in Laryngeal Papillomas: Site of Replication Initiation and Direction of Replication

Science.gov (United States)

Auborn, K. J.; Little, R. D.; Platt, T. H. K.; Vaccariello, M. A.; Schildkraut, C. L.

1994-07-01

We have examined the structures of replication intermediates from the human papillomavirus type 11 genome in DNA extracted from papilloma lesions (laryngeal papillomas). The sites of replication initiation and termination utilized in vivo were mapped by using neutral/neutral and neutral/alkaline two-dimensional agarose gel electrophoresis methods. Initiation of replication was detected in or very close to the upstream regulatory region (URR; the noncoding, regulatory sequences upstream of the open reading frames in the papillomavirus genome). We also show that replication forks proceed bidirectionally from the origin and converge 180circ opposite the URR. These results demonstrate the feasibility of analysis of replication of viral genomes directly from infected tissue.

A rapid and simple separation method of Tc-99 in water samples

International Nuclear Information System (INIS)

Uchida, Shigeo; Tagami, Keiko

1999-01-01

Technetium-99 ( 99 Tc) is one of the important elements on safety evaluation of reprocessing plant and waste disposal plant, because it is a long half-life nuclide and large fission yield (about 6%) from 235 U and 239 Pu. We studied in this work a rapid and simple separation method of 99 Tc in water samples by ICP-MS. Tc in 2 litre of inland water or 1.5 litre of sea water sample solutions was condensed and separated by mini-column resin (TEVA·Spec resin) (Eichrom Co., Ltd.). The experimental results showed the best conditions as followed that 1) after controlling 0.1M nitric acid sample solution, passed it through the column, 2) then the inhibitory elements such as Ru are removed by 40 ml of 2M nitric acid solution, and 3) Tc was recovered by 4 ml of 8M nitric acid solution. 95% or more recovery of Tc was obtained. 100% Ru, an inhibited element of ICP-MS, was removed. The elute obtained from 4 ml of 8M nitric acid solution was diluted ten times with pure water and 99 Tc was measured by ICP-MS. The limit of detection of 2 litre inland water and 1.5 litre sea water were 3 mBql -1 and 4 mBql -1 , respectively. The organic compounds were not removed before running to column, because the concentration of organic compounds was not so large. When large amounts of them are contained, they should be removed. If suspended materials absorb Tc, Tc should be changed to TcO 4 - . (S.Y.)

DNA replication and cancer: From dysfunctional replication origin activities to therapeutic opportunities.

Science.gov (United States)

Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

2016-06-01

A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways to promote genome integrity during DNA replication. This includes suppressing new replication origin firing, stabilization of replicating forks, and the safe restart of forks to prevent any loss of genetic information. Here, we describe mechanisms by which oncogenes can interfere with DNA replication thereby causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

BinQuasi: a peak detection method for ChIP-sequencing data with biological replicates.

Science.gov (United States)

Goren, Emily; Liu, Peng; Wang, Chao; Wang, Chong

2018-04-19

ChIP-seq experiments that are aimed at detecting DNA-protein interactions require biological replication to draw inferential conclusions, however there is no current consensus on how to analyze ChIP-seq data with biological replicates. Very few methodologies exist for the joint analysis of replicated ChIP-seq data, with approaches ranging from combining the results of analyzing replicates individually to joint modeling of all replicates. Combining the results of individual replicates analyzed separately can lead to reduced peak classification performance compared to joint modeling. Currently available methods for joint analysis may fail to control the false discovery rate at the nominal level. We propose BinQuasi, a peak caller for replicated ChIP-seq data, that jointly models biological replicates using a generalized linear model framework and employs a one-sided quasi-likelihood ratio test to detect peaks. When applied to simulated data and real datasets, BinQuasi performs favorably compared to existing methods, including better control of false discovery rate than existing joint modeling approaches. BinQuasi offers a flexible approach to joint modeling of replicated ChIP-seq data which is preferable to combining the results of replicates analyzed individually. Source code is freely available for download at https://cran.r-project.org/package=BinQuasi, implemented in R. pliu@iastate.edu or egoren@iastate.edu. Supplementary material is available at Bioinformatics online.

Selenium speciation in human urine samples by LC- and CE-ICP-MS-separation and identification of selenosugars

DEFF Research Database (Denmark)

Gammelgaard, Bente; Bendahl, L.

2004-01-01

at ambient temperature and methanol extraction. A pre-concentration factor of 10 was achieved with this procedure. On occasions when a pre-concentration factor of 100 was obtained by lyophilsation and methanol extraction, at least 10 selenium compounds were separated in the urine sample. Urine samples were...

Effectiveness of strategies to increase the validity of findings from association studies: size vs. replication

Directory of Open Access Journals (Sweden)

Kallischnigg Gerd

2010-05-01

Full Text Available Abstract Background The capacity of multiple comparisons to produce false positive findings in genetic association studies is abundantly clear. To address this issue, the concept of false positive report probability (FPRP measures "the probability of no true association between a genetic variant and disease given a statistically significant finding". This concept involves the notion of prior probability of an association between a genetic variant and a disease, making it difficult to achieve acceptable levels for the FPRP when the prior probability is low. Increasing the sample size is of limited efficiency to improve the situation. Methods To further clarify this problem, the concept of true report probability (TRP is introduced by analogy to the positive predictive value (PPV of diagnostic testing. The approach is extended to consider the effects of replication studies. The formula for the TRP after k replication studies is mathematically derived and shown to be only dependent on prior probability, alpha, power, and number of replication studies. Results Case-control association studies are used to illustrate the TRP concept for replication strategies. Based on power considerations, a relationship is derived between TRP after k replication studies and sample size of each individual study. That relationship enables study designers optimization of study plans. Further, it is demonstrated that replication is efficient in increasing the TRP even in the case of low prior probability of an association and without requiring very large sample sizes for each individual study. Conclusions True report probability is a comprehensive and straightforward concept for assessing the validity of positive statistical testing results in association studies. By its extension to replication strategies it can be demonstrated in a transparent manner that replication is highly effective in distinguishing spurious from true associations. Based on the generalized TRP

The Nature of Stability in Replicating Systems

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Addy Pross

2011-02-01

Full Text Available We review the concept of dynamic kinetic stability, a type of stability associated specifically with replicating entities, and show how it differs from the well-known and established (static kinetic and thermodynamic stabilities associated with regular chemical systems. In the process we demonstrate how the concept can help bridge the conceptual chasm that continues to separate the physical and biological sciences by relating the nature of stability in the animate and inanimate worlds, and by providing additional insights into the physicochemical nature of abiogenesis.

Repair replication in replicating and nonreplicating DNA after irradiation with uv light

Energy Technology Data Exchange (ETDEWEB)

Slor, H.; Cleaver, J.E.

1978-06-01

Ultraviolet light induces more pyrimidine dimers and more repair replication in DNA that replicates within 2 to 3 h of irradiation than in DNA that does not replicate during this period. This difference may be due to special conformational changes in DNA and chromatin that might be associated with semiconservative DNA replication.

Examining the Validity of Cyclothymic Disorder in a Youth Sample: Replication and Extension

Science.gov (United States)

Van Meter, Anna; Youngstrom, Eric A.; Demeter, Christine; Findling, Robert L.

2013-01-01

DSM-IV-TR defines four subtypes of bipolar disorder (BP): bipolar I, bipolar II, cyclothymic disorder and bipolar not otherwise specified (NOS). However, cyclothymic disorder in children is rarely researched, or often subsumed in an "NOS" category. The present study tests the replicability of findings from an earlier study, and expands on the…

Bayesian analysis of finite population sampling in multivariate co-exchangeable structures with separable covariance matric

OpenAIRE

Shaw, Simon C.; Goldstein, Michael

2017-01-01

We explore the effect of finite population sampling in design problems with many variables cross-classified in many ways. In particular, we investigate designs where we wish to sample individuals belonging to different groups for which the underlying covariance matrices are separable between groups and variables. We exploit the generalised conditional independence structure of the model to show how the analysis of the full model can be reduced to an interpretable series of lower dimensional p...

Identification of cadmium in biological samples using neutron activation analysis with radiochemistry separations

International Nuclear Information System (INIS)

Munoz A, Luis; Gras R, Nuri

2002-01-01

Chile's 7500 km coastline of the Southern Pacific ocean, with about 4500 km of continental coastline that contains a variety of different geographical zones. This variety means that there is a great diversity of marine resources such as fish, shellfish and seaweeds. The utilization of these resources has been increasing in recent years making this sector an economically important one. The catch as of May 2002 came to 1.9 million tons and exports of the different species amounted to US$611.5 million as of April. But this important economic resource is being threatened by the technical demands imposed by importing countries, mainly the specific requirements for sanitary certification for fishery export products, depending on the markets of destination. The chemical element cadmium is one of the most strictly controlled elements due some shellfish accumulate a large amount of this element and to its high toxicity. The Chilean standard's analytical procedures for cadmium determination in hydro biological products, which must be met by laboratories that certify and control these products for export, are now being evaluated. Through its Chemical Metrology Unit, the Chilean Nuclear Energy Commission is strongly supporting this sector by preparing the secondary reference or control materials, and it has developed and implemented nuclear analytical methods for the certification of these materials, which will be used mostly in collaborative studies. This work describes the methodology developed for the determination of cadmium in biological samples, particularly in shellfish and fish. The method is based on neutron activation analysis with radiochemical separations, using the radioisotopes 115 Cd and 115m In, generated in the samples by bombarding with neutrons in a nuclear reactor. The samples were digested at 110 o C with H 2 SO 4 and H 2 O 2 and then the radioactive cadmium element was separated from the other elements present in the samples using a Bio Rad AG 2-X8 resin

Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication.

Science.gov (United States)

Zou, Wei; Wang, Zekun; Xiong, Min; Chen, Aaron Yun; Xu, Peng; Ganaie, Safder S; Badawi, Yomna; Kleiboeker, Steve; Nishimune, Hiroshi; Ye, Shui Qing; Qiu, Jianming

2018-03-01

Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication. IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly

MOF Suppresses Replication Stress and Contributes to Resolution of Stalled Replication Forks.

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Singh, Dharmendra Kumar; Pandita, Raj K; Singh, Mayank; Chakraborty, Sharmistha; Hambarde, Shashank; Ramnarain, Deepti; Charaka, Vijaya; Ahmed, Kazi Mokim; Hunt, Clayton R; Pandita, Tej K

2018-03-15

The human MOF (hMOF) protein belongs to the MYST family of histone acetyltransferases and plays a critical role in transcription and the DNA damage response. MOF is essential for cell proliferation; however, its role during replication and replicative stress is unknown. Here we demonstrate that cells depleted of MOF and under replicative stress induced by cisplatin, hydroxyurea, or camptothecin have reduced survival, a higher frequency of S-phase-specific chromosome damage, and increased R-loop formation. MOF depletion decreased replication fork speed and, when combined with replicative stress, also increased stalled replication forks as well as new origin firing. MOF interacted with PCNA, a key coordinator of replication and repair machinery at replication forks, and affected its ubiquitination and recruitment to the DNA damage site. Depletion of MOF, therefore, compromised the DNA damage repair response as evidenced by decreased Mre11, RPA70, Rad51, and PCNA focus formation, reduced DNA end resection, and decreased CHK1 phosphorylation in cells after exposure to hydroxyurea or cisplatin. These results support the argument that MOF plays an important role in suppressing replication stress induced by genotoxic agents at several stages during the DNA damage response. Copyright © 2018 American Society for Microbiology.

The Anisotropy of Replicated Aluminum Foams

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Eugeny L. Furman

2014-01-01

Full Text Available The replication casting process gives the open-cell aluminum foams that can be used in many industrial applications as well as in filtering technology. The essential requirement for filters is the uniformity of filtering degree which is defined by the minimal pore size. However the structure of replication castings is often inhomogeneous and the minimal pore radius is decreasing in the direction of melt infiltration. The objective of this investigation is to study the dynamics of melt impregnation of the porous medium by vacuum suction to identify the possibility of reducing the anisotropy. Theoretical data illustrate the processes at the boundary between melt and gas medium. The experiments were carried out using the replication aluminum samples produced according to commercial technology. It was found that the permeability coefficient varies throughout the height of castings. A method for estimation of pressure on the line of melt movement was proposed. The resistance of NaCl layer and circular vents of the mold causes the inhomogeneity of castings. Finally the ways of minimizing the anisotropy were offered.

Database Replication

CERN Document Server

Kemme, Bettina

2010-01-01

Database replication is widely used for fault-tolerance, scalability and performance. The failure of one database replica does not stop the system from working as available replicas can take over the tasks of the failed replica. Scalability can be achieved by distributing the load across all replicas, and adding new replicas should the load increase. Finally, database replication can provide fast local access, even if clients are geographically distributed clients, if data copies are located close to clients. Despite its advantages, replication is not a straightforward technique to apply, and

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

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Caroline M Li

Full Text Available We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE. In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40 origin of DNA replication and the viral large tumor antigen (T-antigen protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA, DNA topoisomerase I (topo I, DNA polymerase δ (Pol δ, DNA polymerase ɛ (Pol ɛ, replication protein A (RPA and replication factor C (RFC. Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

Science.gov (United States)

Li, Caroline M; Miao, Yunan; Lingeman, Robert G; Hickey, Robert J; Malkas, Linda H

2016-01-01

We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

X-irradiation affects all DNA replication intermediates when inhibiting replication initiation

International Nuclear Information System (INIS)

Loenn, U.; Karolinska Hospital, Stockholm

1982-01-01

When a human melanoma line was irradiated with 10 Gy, there was, after 30 to 60 min, a gradual reduction in the DNA replication rate. Ten to twelve hours after the irradiation, the DNA replication had returned to near normal rate. The results showed tht low dose-rate X-irradiation inhibits preferentially the formation of small DNA replication intermediates. There is no difference between the inhibition of these replication intermediates formed only in the irradiated cells and those formed also in untreated cells. (U.K.)

Chromatin Structure and Replication Origins: Determinants Of Chromosome Replication And Nuclear Organization

Science.gov (United States)

Smith, Owen K.; Aladjem, Mirit I.

2014-01-01

The DNA replication program is, in part, determined by the epigenetic landscape that governs local chromosome architecture and directs chromosome duplication. Replication must coordinate with other biochemical processes occurring concomitantly on chromatin, such as transcription and remodeling, to insure accurate duplication of both genetic and epigenetic features and to preserve genomic stability. The importance of genome architecture and chromatin looping in coordinating cellular processes on chromatin is illustrated by two recent sets of discoveries. First, chromatin-associated proteins that are not part of the core replication machinery were shown to affect the timing of DNA replication. These chromatin-associated proteins could be working in concert, or perhaps in competition, with the transcriptional machinery and with chromatin modifiers to determine the spatial and temporal organization of replication initiation events. Second, epigenetic interactions are mediated by DNA sequences that determine chromosomal replication. In this review we summarize recent findings and current models linking spatial and temporal regulation of the replication program with epigenetic signaling. We discuss these issues in the context of the genome’s three-dimensional structure with an emphasis on events occurring during the initiation of DNA replication. PMID:24905010

Prelife catalysts and replicators

OpenAIRE

Ohtsuki, Hisashi; Nowak, Martin A.

2009-01-01

Life is based on replication and evolution. But replication cannot be taken for granted. We must ask what there was prior to replication and evolution. How does evolution begin? We have proposed prelife as a generative system that produces information and diversity in the absence of replication. We model prelife as a binary soup of active monomers that form random polymers. ‘Prevolutionary’ dynamics can have mutation and selection prior to replication. Some sequences might have catalytic acti...

Development of analytical methods for the separation of plutonium, americium, curium and neptunium from environmental samples

Energy Technology Data Exchange (ETDEWEB)

Salminen, S.

2009-07-01

In this work, separation methods have been developed for the analysis of anthropogenic transuranium elements plutonium, americium, curium and neptunium from environmental samples contaminated by global nuclear weapons testing and the Chernobyl accident. The analytical methods utilized in this study are based on extraction chromatography. Highly varying atmospheric plutonium isotope concentrations and activity ratios were found at both Kurchatov (Kazakhstan), near the former Semipalatinsk test site, and Sodankylae (Finland). The origin of plutonium is almost impossible to identify at Kurchatov, since hundreds of nuclear tests were performed at the Semipalatinsk test site. In Sodankylae, plutonium in the surface air originated from nuclear weapons testing, conducted mostly by USSR and USA before the sampling year 1963. The variation in americium, curium and neptunium concentrations was great as well in peat samples collected in southern and central Finland in 1986 immediately after the Chernobyl accident. The main source of transuranium contamination in peats was from global nuclear test fallout, although there are wide regional differences in the fraction of Chernobyl-originated activity (of the total activity) for americium, curium and neptunium. The separation methods developed in this study yielded good chemical recovery for the elements investigated and adequately pure fractions for radiometric activity determination. The extraction chromatographic methods were faster compared to older methods based on ion exchange chromatography. In addition, extraction chromatography is a more environmentally friendly separation method than ion exchange, because less acidic waste solutions are produced during the analytical procedures. (orig.)

Replicability of time-varying connectivity patterns in large resting state fMRI samples.

Science.gov (United States)

Abrol, Anees; Damaraju, Eswar; Miller, Robyn L; Stephen, Julia M; Claus, Eric D; Mayer, Andrew R; Calhoun, Vince D

2017-12-01

The past few years have seen an emergence of approaches that leverage temporal changes in whole-brain patterns of functional connectivity (the chronnectome). In this chronnectome study, we investigate the replicability of the human brain's inter-regional coupling dynamics during rest by evaluating two different dynamic functional network connectivity (dFNC) analysis frameworks using 7 500 functional magnetic resonance imaging (fMRI) datasets. To quantify the extent to which the emergent functional connectivity (FC) patterns are reproducible, we characterize the temporal dynamics by deriving several summary measures across multiple large, independent age-matched samples. Reproducibility was demonstrated through the existence of basic connectivity patterns (FC states) amidst an ensemble of inter-regional connections. Furthermore, application of the methods to conservatively configured (statistically stationary, linear and Gaussian) surrogate datasets revealed that some of the studied state summary measures were indeed statistically significant and also suggested that this class of null model did not explain the fMRI data fully. This extensive testing of reproducibility of similarity statistics also suggests that the estimated FC states are robust against variation in data quality, analysis, grouping, and decomposition methods. We conclude that future investigations probing the functional and neurophysiological relevance of time-varying connectivity assume critical importance. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Evidence for an asthma risk locus on chromosome Xp: a replication linkage study

DEFF Research Database (Denmark)

Brasch-Andersen, C; Møller, M U; Haagerup, A

2008-01-01

replication sample as used in the present study. The aim of the study was to replicate linkage to candidate regions for asthma in an independent Danish sample. METHODS: We performed a replication study investigating linkage to candidate regions for asthma on chromosomes 1p36.31-p36.21, 5q15-q23.2, 6p24.3-p22...... studies have been carried out the results are still conflicting and call for replication experiments. A Danish genome-wide scan has prior reported evidence for candidate regions for asthma susceptibility genes on chromosomes 1p, 5q, 6p, 12q and Xp. Linkage to chromosome 12q was later confirmed in the same.......3, and Xp22.31-p11.4 using additional markers in an independent set of 136 Danish asthmatic sib pair families. RESULTS: Nonparametric multipoint linkage analyses yielded suggestive evidence for linkage to asthma to chromosome Xp21.2 (MLS 2.92) but failed to replicate linkage to chromosomes 1p36.31-p36.21, 5...

Replicated Risk Nicotinic Cholinergic Receptor Genes for Nicotine Dependence

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Lingjun Zuo

2016-11-01

Full Text Available It has been hypothesized that the nicotinic acetylcholine receptors (nAChRs play important roles in nicotine dependence (ND and influence the number of cigarettes smoked per day (CPD in smokers. We compiled the associations between nicotinic cholinergic receptor genes (CHRNs and ND/CPD that were replicated across different studies, reviewed the expression of these risk genes in human/mouse brains, and verified their expression using independent samples of both human and mouse brains. The potential functions of the replicated risk variants were examined using cis-eQTL analysis or predicted using a series of bioinformatics analyses. We found replicated and significant associations for ND/CPD at 19 SNPs in six genes in three genomic regions (CHRNB3-A6, CHRNA5-A3-B4 and CHRNA4. These six risk genes are expressed in at least 18 distinct areas of the human/mouse brain, with verification in our independent human and mouse brain samples. The risk variants might influence the transcription, expression and splicing of the risk genes, alter RNA secondary or protein structure. We conclude that the replicated associations between CHRNB3-A6, CHRNA5-A3-B4, CHRNA4 and ND/CPD are very robust. More research is needed to examine how these genetic variants contribute to the risk for ND/CPD.

Separation and characterization of nanoparticles in complex food and environmental samples by field-flow fractionation

DEFF Research Database (Denmark)

Kammer, Frank von der; Legros, Samuel; Hofmann, Thilo

2011-01-01

The thorough analysis of natural nanoparticles (NPs) and engineered NPs involves the sequence of detection, identification, quantification and, if possible, detailed characterization. In a complex or heterogeneous sample, each step of this sequence is an individual challenge, and, given suitable...... has been applied for separation of various types of NP (e.g., organic macromolecules, and carbonaceous or inorganic NPs) in different types of media (e.g., natural waters, soil extracts or food samples).FFF can be coupled to different types of detectors that offer additional information...... conditions on all types of NP in the sample. A holistic methodological approach is preferable to a technique-focused one....

Separation and recovery of Cm from Cm-Pu mixed oxide samples containing Am impurity

International Nuclear Information System (INIS)

Hirokazu Hayashi; Hiromichi Hagiya; Mitsuo Akabori; Yasuji Morita; Kazuo Minato

2013-01-01

Curium was separated and recovered as an oxalate from a Cm-Pu mixed oxide which had been a 244 Cm oxide sample prepared more than 40 years ago and the ratio of 244 Cm to 240 Pu was estimated to 0.2:0.8. Radiochemical analyses of the solution prepared by dissolving the Cm-Pu mixed oxide in nitric acid revealed that the oxide contained about 1 at% of 243 Am impurity. To obtain high purity curium solution, plutonium and americium were removed from the solution by an anion exchange method and by chromatographic separation using tertiary pyridine resin embedded in silica beads with nitric acid/methanol mixed solution, respectively. Curium oxalate, a precursor compound of curium oxide, was prepared from the purified curium solution. 11.9 mg of Cm oxalate having some amounts of impurities, which are 243 Am (5.4 at%) and 240 Pu (0.3 at%) was obtained without Am removal procedure. Meanwhile, 12.0 mg of Cm oxalate (99.8 at% over actinides) was obtained with the procedure including Am removals. Both of the obtained Cm oxalate sample were supplied for the syntheses and measurements of the thermochemical properties of curium compounds. (author)

A new digestion and chemical separation technique for rapid and highly reproducible determination of Lu/Hf and Hf isotope ratios in geological materials by mc-ICP-MS

DEFF Research Database (Denmark)

Bizzarro, Martin; Baker, J.A.; Ulfbeck, D.

2003-01-01

for isotopic analysis of the Sm-Nd system and/or the other HFSE (Ti, Zr). Total procedural blanks for this technique are 5 g) samples. We present data from replicate digestions of international rock reference materials which....... The relative simplicity of this technique, coupled with the ease of digestion (and samplespike equilibration) of large difficult-to-dissolve samples, and the speed (2 days) with which samples can be digested and processed through the chemical separation scheme makes it an attractive new method for preparing...

Nanocapillaries for Open Tubular Chromatographic Separations of Proteins in Femtoliter to Picoliter Samples

Science.gov (United States)

Wang, Xiayan; Cheng, Chang; Wang, Shili; Zhao, Meiping; Dasgupta, Purnendu K.; Liu, Shaorong

2009-01-01

We have recently examined the potential of bare nanocapillaries for free solution DNA separations and demonstrated efficiencies exceeding 106 theoretical plates/m. In the present work, we demonstrate the use of bare and hydroxypropylcellulose (HPC) coated open tubular nanocapillaries for protein separations. Using 1.5 μm inner diameter (i.d.) capillary columns, hydrodynamically injecting femto to picoliter (fL-pL) volumes of fluorescent or fluorescent dye labeled protein samples, utilizing a pneumatically pressurized chamber containing 1.0 mM sodium tetraborate solution eluent (typ. 200 psi) as the pump and performing on-column detection using a simple laser-induced fluorescence detector, we demonstrate efficiencies of close to a million theoretical plates/m while generating single digit μL volumes of waste for a complete chromatographic run. We achieve baseline resolution for a protein mixture consisting of transferrin, α-lactalbumin, insulin, and α -2-macroglobulin. PMID:19663450

A Chip-Capillary Hybrid Device for Automated Transfer of Sample Pre-Separated by Capillary Isoelectric Focusing to Parallel Capillary Gel Electrophoresis for Two-Dimensional Protein Separation

Science.gov (United States)

Lu, Joann J.; Wang, Shili; Li, Guanbin; Wang, Wei; Pu, Qiaosheng; Liu, Shaorong

2012-01-01

In this report, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to re-route the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed. PMID:22830584

Rapid determination of environmental plutonium in large water samples by means of manganese dioxide Co-precipitation and extraction chromatographic separation

International Nuclear Information System (INIS)

Sidhu, R.S.; Hoff, P.

1999-01-01

Plutonium activity determinations in environmental water samples are routinely performed in many laboratories. Due to the low plutonium concentrations and the complexity of the plutonium aqueous chemistry, these analysis involve cumbersome preconcentration and separation procedures and long measurement times. We describe a procedure where MnO 2 (s) is used as scavenger to preconcentrate Pu prior to separation by the transuranium specific extraction chromatographic resin TRU-Resin. The ability of MnO 4 - /MnO 2 (s) to destroy organic matter and oxidise plutonium is combined with the specific behaviour of the TRU-resin for transuranium elements. The method facilitates a rapid preconcentration and separation of plutonium. With the use of this procedure, the laboratory work on one sample can be performed in one day with minimum attention. The overall yields from a 200 litre fresh- or seawater sample to a measurable Pu source are between 70-85%. (orig.)

NACSA Charter School Replication Guide: The Spectrum of Replication Options. Authorizing Matters. Replication Brief 1

Science.gov (United States)

O'Neill, Paul

2010-01-01

One of the most important and high-profile issues in public education reform today is the replication of successful public charter school programs. With more than 5,000 failing public schools in the United States, there is a tremendous need for strong alternatives for parents and students. Replicating successful charter school models is an…

Rapid separation method for {sup 237}Np and Pu isotopes in large soil samples

Energy Technology Data Exchange (ETDEWEB)

Maxwell, Sherrod L., E-mail: sherrod.maxwell@srs.go [Savannah River Nuclear Solutions, LLC, Building 735-B, Aiken, SC 29808 (United States); Culligan, Brian K.; Noyes, Gary W. [Savannah River Nuclear Solutions, LLC, Building 735-B, Aiken, SC 29808 (United States)

2011-07-15

A new rapid method for the determination of {sup 237}Np and Pu isotopes in soil and sediment samples has been developed at the Savannah River Site Environmental Lab (Aiken, SC, USA) that can be used for large soil samples. The new soil method utilizes an acid leaching method, iron/titanium hydroxide precipitation, a lanthanum fluoride soil matrix removal step, and a rapid column separation process with TEVA Resin. The large soil matrix is removed easily and rapidly using these two simple precipitations with high chemical recoveries and effective removal of interferences. Vacuum box technology and rapid flow rates are used to reduce analytical time.

rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data.

Science.gov (United States)

Shen, Shihao; Park, Juw Won; Lu, Zhi-xiang; Lin, Lan; Henry, Michael D; Wu, Ying Nian; Zhou, Qing; Xing, Yi

2014-12-23

Ultra-deep RNA sequencing (RNA-Seq) has become a powerful approach for genome-wide analysis of pre-mRNA alternative splicing. We previously developed multivariate analysis of transcript splicing (MATS), a statistical method for detecting differential alternative splicing between two RNA-Seq samples. Here we describe a new statistical model and computer program, replicate MATS (rMATS), designed for detection of differential alternative splicing from replicate RNA-Seq data. rMATS uses a hierarchical model to simultaneously account for sampling uncertainty in individual replicates and variability among replicates. In addition to the analysis of unpaired replicates, rMATS also includes a model specifically designed for paired replicates between sample groups. The hypothesis-testing framework of rMATS is flexible and can assess the statistical significance over any user-defined magnitude of splicing change. The performance of rMATS is evaluated by the analysis of simulated and real RNA-Seq data. rMATS outperformed two existing methods for replicate RNA-Seq data in all simulation settings, and RT-PCR yielded a high validation rate (94%) in an RNA-Seq dataset of prostate cancer cell lines. Our data also provide guiding principles for designing RNA-Seq studies of alternative splicing. We demonstrate that it is essential to incorporate biological replicates in the study design. Of note, pooling RNAs or merging RNA-Seq data from multiple replicates is not an effective approach to account for variability, and the result is particularly sensitive to outliers. The rMATS source code is freely available at rnaseq-mats.sourceforge.net/. As the popularity of RNA-Seq continues to grow, we expect rMATS will be useful for studies of alternative splicing in diverse RNA-Seq projects.

Association between Spouse/Child Separation and Migration-Related Stress among a Random Sample of Rural-to-Urban Migrants in Wuhan, China.

Science.gov (United States)

Guo, Yan; Chen, Xinguang; Gong, Jie; Li, Fang; Zhu, Chaoyang; Yan, Yaqiong; Wang, Liang

2016-01-01

Millions of people move from rural areas to urban areas in China to pursue new opportunities while leaving their spouses and children at rural homes. Little is known about the impact of migration-related separation on mental health of these rural migrants in urban China. Survey data from a random sample of rural-to-urban migrants (n = 1113, aged 18-45) from Wuhan were analyzed. The Domestic Migration Stress Questionnaire (DMSQ), an instrument with four subconstructs, was used to measure migration-related stress. The relationship between spouse/child separation and stress was assessed using survey estimation methods to account for the multi-level sampling design. 16.46% of couples were separated from their spouses (spouse-separation only), 25.81% of parents were separated from their children (child separation only). Among the participants who married and had children, 5.97% were separated from both their spouses and children (double separation). Spouse-separation only and double separation did not scored significantly higher on DMSQ than those with no separation. Compared to parents without child separation, parents with child separation scored significantly higher on DMSQ (mean score = 2.88, 95% CI: [2.81, 2.95] vs. 2.60 [2.53, 2.67], p separation type and by gender indicated that the association was stronger for child-separation only and for female participants. Child-separation is an important source of migration-related stress, and the effect is particularly strong for migrant women. Public policies and intervention programs should consider these factors to encourage and facilitate the co-migration of parents with their children to mitigate migration-related stress.

Association between Spouse/Child Separation and Migration-Related Stress among a Random Sample of Rural-to-Urban Migrants in Wuhan, China.

Directory of Open Access Journals (Sweden)

Yan Guo

Full Text Available Millions of people move from rural areas to urban areas in China to pursue new opportunities while leaving their spouses and children at rural homes. Little is known about the impact of migration-related separation on mental health of these rural migrants in urban China.Survey data from a random sample of rural-to-urban migrants (n = 1113, aged 18-45 from Wuhan were analyzed. The Domestic Migration Stress Questionnaire (DMSQ, an instrument with four subconstructs, was used to measure migration-related stress. The relationship between spouse/child separation and stress was assessed using survey estimation methods to account for the multi-level sampling design.16.46% of couples were separated from their spouses (spouse-separation only, 25.81% of parents were separated from their children (child separation only. Among the participants who married and had children, 5.97% were separated from both their spouses and children (double separation. Spouse-separation only and double separation did not scored significantly higher on DMSQ than those with no separation. Compared to parents without child separation, parents with child separation scored significantly higher on DMSQ (mean score = 2.88, 95% CI: [2.81, 2.95] vs. 2.60 [2.53, 2.67], p < .05. Stratified analysis by separation type and by gender indicated that the association was stronger for child-separation only and for female participants.Child-separation is an important source of migration-related stress, and the effect is particularly strong for migrant women. Public policies and intervention programs should consider these factors to encourage and facilitate the co-migration of parents with their children to mitigate migration-related stress.

Two-Dimensional Capillary Electrophoresis with On-Line Sample Preparation and Cyclodextrin Separation Environment for Direct Determination of Serotonin in Human Urine.

Science.gov (United States)

Piešťanský, Juraj; Maráková, Katarína; Mikuš, Peter

2017-10-07

An advanced two-dimensional capillary electrophoresis method, based on on-line combination of capillary isotachophoresis and capillary zone electrophoresis with cyclodextrin additive in background electrolyte, was developed for effective determination of serotonin in human urine. Hydrodynamically closed separation system and large bore capillaries (300-800 µm) were chosen for the possibility to enhance the sample load capacity, and, by that, to decrease limit of detection. Isotachophoresis served for the sample preseparation, defined elimination of sample matrix constituents (sample clean up), and preconcentration of the analyte. Cyclodextrin separation environment enhanced separation selectivity of capillary zone electrophoresis. In this way, serotonin could be successfully separated from the rest of the sample matrix constituents migrating in capillary zone electrophoresis step so that human urine could be directly (i.e., without any external sample preparation) injected into the analyzer. The proposed method was successfully validated, showing favorable parameters of sensitivity (limit of detection for serotonin was 2.32 ng·mL -1 ), linearity (regression coefficient higher than 0.99), precision (repeatability of the migration time and peak area were in the range of 0.02-1.17% and 5.25-7.88%, respectively), and recovery (ranging in the interval of 90.0-93.6%). The developed method was applied for the assay of the human urine samples obtained from healthy volunteers. The determined concentrations of serotonin in such samples were in the range of 12.4-491.2 ng·mL -1 that was in good agreement with literature data. This advanced method represents a highly effective, reliable, and low-cost alternative for the routine determination of serotonin as a biomarker in human urine.

DNA Replication in Engineered Escherichia coli Genomes with Extra Replication Origins.

Science.gov (United States)

Milbredt, Sarah; Farmani, Neda; Sobetzko, Patrick; Waldminghaus, Torsten

2016-10-21

The standard outline of bacterial genomes is a single circular chromosome with a single replication origin. From the bioengineering perspective, it appears attractive to extend this basic setup. Bacteria with split chromosomes or multiple replication origins have been successfully constructed in the last few years. The characteristics of these engineered strains will largely depend on the respective DNA replication patterns. However, the DNA replication has not been investigated systematically in engineered bacteria with multiple origins or split replicons. Here we fill this gap by studying a set of strains consisting of (i) E. coli strains with an extra copy of the native replication origin (oriC), (ii) E. coli strains with an extra copy of the replication origin from the secondary chromosome of Vibrio cholerae (oriII), and (iii) a strain in which the E. coli chromosome is split into two linear replicons. A combination of flow cytometry, microarray-based comparative genomic hybridization (CGH), and modeling revealed silencing of extra oriC copies and differential timing of ectopic oriII copies compared to the native oriC. The results were used to derive construction rules for future multiorigin and multireplicon projects.

Replicability and generalizability of PTSD networks

DEFF Research Database (Denmark)

Eiko I., Fried; Eidhof, Marloes B.; Palic, Sabina

2018-01-01

The growing literature conceptualizing mental disorders like Posttraumatic Stress Disorder (PTSD) as networks of interacting symptoms faces three key challenges. Prior studies predominantly used (a) small samples with low power for precise estimation, (b) non-clinical samples, and (c) single...... samples. This renders network structures in clinical data, and the extent to which networks replicate across datasets, unknown. To overcome these limitations, the present cross-cultural multisite study estimated regularized partial correlation networks of 16 PTSD symptoms across four datasets...... of traumatized patients receiving treatment for PTSD (total N=2,782). Despite differences in culture, trauma-type and severity of the samples, considerable similarities emerged, with moderate to high correlations between symptom profiles (0.43-0.82), network structures (0.62-0.74), and centrality estimates (0...

Hydroxyurea-Induced Replication Stress

Directory of Open Access Journals (Sweden)

Kenza Lahkim Bennani-Belhaj

2010-01-01

Full Text Available Bloom's syndrome (BS displays one of the strongest known correlations between chromosomal instability and a high risk of cancer at an early age. BS cells combine a reduced average fork velocity with constitutive endogenous replication stress. However, the response of BS cells to replication stress induced by hydroxyurea (HU, which strongly slows the progression of replication forks, remains unclear due to publication of conflicting results. Using two different cellular models of BS, we showed that BLM deficiency is not associated with sensitivity to HU, in terms of clonogenic survival, DSB generation, and SCE induction. We suggest that surviving BLM-deficient cells are selected on the basis of their ability to deal with an endogenous replication stress induced by replication fork slowing, resulting in insensitivity to HU-induced replication stress.

Rapid instrumental and separation methods for monitoring radionuclides in food and environmental samples. Progress report

International Nuclear Information System (INIS)

Bhat, I.S.; Shukla, V.K.; Singh, A.N.; Nair, C.K.G.; Hingorani, S.B.; Dey, N.N.; Jha, S.K.; Rao, D.D.

1995-01-01

When activity levels are low, the direct gamma counting of milk and water samples take very long time, initial concentration step increases the sensitivity. 131 I in aqueous samples can be concentrated by absorption on AgCI in acidic condition. In case of milk, initial treatment with TCA, separation of precipitated casin and stirring the acidified (dil. HNO 3 ) clear solution with about 500 mg AgCI gives all the 131 I (more than 95%) picked up by AgCI which can be counted in a well crystal gamma spectrometer. In case of water samples acidification and direct stirring with AgCI all 131 I gets absorbed on to AgCI. About half an hour stirring has been found sufficient to give reproducible result. The total time required will be about 3 hrs. In case of 137 Cs, the aqueous solution should be stirred with ammonium phosphomolybdate (AMP) after acidification with HNO 3 . After an hour of AMP settling time, decantation, filtration and centrifuging one can get the AMP ready for counting in a gamma spectrometer having a well type detector. The analysis can be completed within 2 hrs. AgCI concentration of 131 I and AMP concentration of 137 Cs reduces the counting time significantly. These methods have been used for sea water and milk samples analysis. Methods are being standardised for solvent extraction separation of Pu, Am and Cm from preconcentrated environmental samples and direct counting of organic extract by liquid scintillation counting. For Pu determination, solvent extraction by TTA, back extraction and reextraction to 5% D2EHPA and direct liquid scintillation counting of Pu-alphas is planned. This will reduce the time required for Pu analysis to a significant extent. After bringing the sample to solution, this separation step can be carried out within 1 1/2 to 2 hrs. With Instagel scintillator cocktail in the packard 1550 LSS, Pu-239 counting had 70% efficiency with 5.3 cpm background. Pu-239 estimated in a few sediment sample gave results by both LSS method and Si

Gambling, Risk-Taking, and Antisocial Behavior: A Replication Study Supporting the Generality of Deviance.

Science.gov (United States)

Mishra, Sandeep; Lalumière, Martin L; Williams, Robert J

2017-03-01

Research suggests that high frequency gambling is a component of the "generality of deviance", which describes the observation that various forms of risky and antisocial behavior tend to co-occur among individuals. Furthermore, risky and antisocial behaviors have been associated with such personality traits as low self-control, and impulsivity, and sensation-seeking. We conducted a replication (and extension) of two previous studies examining whether high frequency gambling is part of the generality of deviance using a large and diverse community sample (n = 328). This study was conducted as a response to calls for more replication studies in the behavioral and psychological sciences (recent systematic efforts suggest that a significant proportion of psychology studies do not replicate). The results of the present study largely replicate those previously found, and in many cases, we observed stronger associations among measures of gambling, risk-taking, and antisocial behavior in this diverse sample. Together, this study provides evidence for the generality of deviance inclusive of gambling (and, some evidence for the replicability of research relating to gambling and individual differences).

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.

Cdc7 is required throughout the yeast S phase to activate replication origins.

Science.gov (United States)

Donaldson, A D; Fangman, W L; Brewer, B J

1998-02-15

The long-standing conclusion that the Cdc7 kinase of Saccharomyces cerevisiae is required only to trigger S phase has been challenged by recent data that suggests it acts directly on individual replication origins. We tested the possibility that early- and late-activated origins have different requirements for Cdc7 activity. Cells carrying a cdc7(ts) allele were first arrested in G1 at the cdc7 block by incubation at 37 degrees C, and then were allowed to enter S phase by brief incubation at 23 degrees C. During the S phase, after return to 37 degrees C, early-firing replication origins were activated, but late origins failed to fire. Similarly, a plasmid with a late-activated origin was defective in replication. As a consequence of the origin activation defect, duplication of chromosomal sequences that are normally replicated from late origins was greatly delayed. Early-replicating regions of the genome duplicated at approximately their normal time. The requirements of early and late origins for Cdc7 appear to be temporally rather than quantitatively different, as reducing overall levels of Cdc7 by growth at semi-permissive temperature reduced activation at early and late origins approximately equally. Our results show that Cdc7 activates early and late origins separately, with late origins requiring the activity later in S phase to permit replication initiation.

Detection of foot-and-mouth disease virus RNA in pharyngeal epithelium biopsy samples obtained from infected cattle: Investigation of possible sites of virus replication and persistence

DEFF Research Database (Denmark)

Stenfeldt, Anna Carolina; Belsham, Graham

2012-01-01

measurements of the levels of FMDV RNA in the DSP as well as mandibular and retropharyngeal lymph nodes beyond 28 days after infection. Results indicated only low levels of FMDV RNA present in samples of pharyngeal epithelia during both early and persistent phases of infection with significantly higher levels...... of virus detected in pharyngeal excretions. It is concluded that the targeted area for sampling within the DSP does not harbour significant levels of virus replication during acute or persistent FMDV infection in cattle. Furthermore, the DSP and the mandibular and retropharyngeal lymph nodes cannot...

Modeling inhomogeneous DNA replication kinetics.

Directory of Open Access Journals (Sweden)

Michel G Gauthier

Full Text Available In eukaryotic organisms, DNA replication is initiated at a series of chromosomal locations called origins, where replication forks are assembled proceeding bidirectionally to replicate the genome. The distribution and firing rate of these origins, in conjunction with the velocity at which forks progress, dictate the program of the replication process. Previous attempts at modeling DNA replication in eukaryotes have focused on cases where the firing rate and the velocity of replication forks are homogeneous, or uniform, across the genome. However, it is now known that there are large variations in origin activity along the genome and variations in fork velocities can also take place. Here, we generalize previous approaches to modeling replication, to allow for arbitrary spatial variation of initiation rates and fork velocities. We derive rate equations for left- and right-moving forks and for replication probability over time that can be solved numerically to obtain the mean-field replication program. This method accurately reproduces the results of DNA replication simulation. We also successfully adapted our approach to the inverse problem of fitting measurements of DNA replication performed on single DNA molecules. Since such measurements are performed on specified portion of the genome, the examined DNA molecules may be replicated by forks that originate either within the studied molecule or outside of it. This problem was solved by using an effective flux of incoming replication forks at the model boundaries to represent the origin activity outside the studied region. Using this approach, we show that reliable inferences can be made about the replication of specific portions of the genome even if the amount of data that can be obtained from single-molecule experiments is generally limited.

Mechanisms of DNA replication termination.

Science.gov (United States)

Dewar, James M; Walter, Johannes C

2017-08-01

Genome duplication is carried out by pairs of replication forks that assemble at origins of replication and then move in opposite directions. DNA replication ends when converging replication forks meet. During this process, which is known as replication termination, DNA synthesis is completed, the replication machinery is disassembled and daughter molecules are resolved. In this Review, we outline the steps that are likely to be common to replication termination in most organisms, namely, fork convergence, synthesis completion, replisome disassembly and decatenation. We briefly review the mechanism of termination in the bacterium Escherichia coli and in simian virus 40 (SV40) and also focus on recent advances in eukaryotic replication termination. In particular, we discuss the recently discovered E3 ubiquitin ligases that control replisome disassembly in yeast and higher eukaryotes, and how their activity is regulated to avoid genome instability.

Replication of a Modified Factor Structure for the Eating Disorder Examination-Questionnaire: Extension to Clinical Eating Disorder and Non-clinical Samples in Portugal.

Science.gov (United States)

Machado, Paulo P P; Grilo, Carlos M; Crosby, Ross D

2018-01-01

Psychometric investigations of the Eating Disorder Examination-Questionnaire (EDE-Q) have generally not supported the original scale structure. The present study tested an alternative brief factor structure in two large Portuguese samples: (1) a non-clinical sample of N = 4117 female students and (2) a treatment-seeking sample of N = 609 patients diagnosed with eating disorders. Confirmatory factor analysis revealed a poor fit for the original EDE-Q structure in both the non-clinical and the clinical samples but revealed a good fit for the alternative 7-item 3-factor structure (dietary restraint, shape/weight overvaluation and body dissatisfaction). Factor loadings were invariant across samples and across the different specific eating disorder diagnoses in the clinical sample. These confirmatory factor analysis findings, which replicate findings from studies with diverse predominately overweight/obese samples, supported a modified 7-item, 3-factor structure for the EDE-Q. The reliable findings across different non-clinical and clinical eating disorder groups provide confidence regarding the potential utility of this brief version. Copyright © 2017 John Wiley & Sons, Ltd and Eating Disorders Association. Copyright © 2017 John Wiley & Sons, Ltd and Eating Disorders Association.

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

Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks

DEFF Research Database (Denmark)

Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia

2016-01-01

Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose...... RAD52 facilitates repair of collapsed DNA replication forks in cancer cells....

The impact of acute stress on the neural processing of food cues in bulimia nervosa: Replication in two samples.

Science.gov (United States)

Collins, Brittany; Breithaupt, Lauren; McDowell, Jennifer E; Miller, L Stephen; Thompson, James; Fischer, Sarah

2017-07-01

The impact of acute stress on the neural processing of food cues in bulimia nervosa (BN) is unknown, despite theory that acute stress decreases cognitive control over food and hence increases vulnerability to environmental triggers for binge eating. Thus, the goals of this manuscript were to explore the impact of acute stress on the neural processing of food cues in BN. In Study 1, 10 women with Diagnostic and Statistical Manual of Mental Disorders (5th ed.; DSM-5; American Psychiatric Association, 2013) BN and 10 healthy controls participated in an fMRI paradigm examining the neural correlates of visual food cue processing pre and post an acute stress induction. Whole brain analysis indicated that women with BN exhibited significant decreases in activation in the precuneus, associated with self-referential processing, the paracingulate gyrus, and the anterior vermis of the cerebellum. Healthy controls exhibited increased activation in these regions in response to food cues poststress. In Study 2, 17 women with DSM-5 BN or otherwise specified feeding and eating disorder with BN symptoms participated in the same paradigm. A region of interest analysis replicated findings from Study 1. Replication of imaging findings in 2 different samples suggests the potential importance of these regions in relation to BN. Decreased activation in the precuneus, specifically, is consistent with models of BN that posit that binge eating serves as a concrete distraction from aversive internal stimuli. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Fabrication of All Glass Bifurcation Microfluidic Chip for Blood Plasma Separation

Directory of Open Access Journals (Sweden)

Hyungjun Jang

2017-02-01

Full Text Available An all-glass bifurcation microfluidic chip for blood plasma separation was fabricated by a cost-effective glass molding process using an amorphous carbon (AC mold, which in turn was fabricated by the carbonization of a replicated furan precursor. To compensate for the shrinkage during AC mold fabrication, an enlarged photoresist pattern master was designed, and an AC mold with a dimensional error of 2.9% was achieved; the dimensional error of the master pattern was 1.6%. In the glass molding process, a glass microchannel plate with negligible shape errors (~1.5% compared to AC mold was replicated. Finally, an all-glass bifurcation microfluidic chip was realized by micro drilling and thermal fusion bonding processes. A separation efficiency of 74% was obtained using the fabricated all-glass bifurcation microfluidic chip.

Acoustofluidic bacteria separation

International Nuclear Information System (INIS)

Li, Sixing; Huang, Tony Jun; Ma, Fen; Zeng, Xiangqun; Bachman, Hunter; Cameron, Craig E

2017-01-01

Bacterial separation from human blood samples can help with the identification of pathogenic bacteria for sepsis diagnosis. In this work, we report an acoustofluidic device for label-free bacterial separation from human blood samples. In particular, we exploit the acoustic radiation force generated from a tilted-angle standing surface acoustic wave (taSSAW) field to separate Escherichia coli from human blood cells based on their size difference. Flow cytometry analysis of the E. coli separated from red blood cells shows a purity of more than 96%. Moreover, the label-free electrochemical detection of the separated E. coli displays reduced non-specific signals due to the removal of blood cells. Our acoustofluidic bacterial separation platform has advantages such as label-free separation, high biocompatibility, flexibility, low cost, miniaturization, automation, and ease of in-line integration. The platform can be incorporated with an on-chip sensor to realize a point-of-care sepsis diagnostic device. (paper)

Acoustofluidic bacteria separation

Science.gov (United States)

Li, Sixing; Ma, Fen; Bachman, Hunter; Cameron, Craig E.; Zeng, Xiangqun; Huang, Tony Jun

2017-01-01

Bacterial separation from human blood samples can help with the identification of pathogenic bacteria for sepsis diagnosis. In this work, we report an acoustofluidic device for label-free bacterial separation from human blood samples. In particular, we exploit the acoustic radiation force generated from a tilted-angle standing surface acoustic wave (taSSAW) field to separate Escherichia coli from human blood cells based on their size difference. Flow cytometry analysis of the E. coli separated from red blood cells shows a purity of more than 96%. Moreover, the label-free electrochemical detection of the separated E. coli displays reduced non-specific signals due to the removal of blood cells. Our acoustofluidic bacterial separation platform has advantages such as label-free separation, high biocompatibility, flexibility, low cost, miniaturization, automation, and ease of in-line integration. The platform can be incorporated with an on-chip sensor to realize a point-of-care sepsis diagnostic device.

REPLICATION TOOL AND METHOD OF PROVIDING A REPLICATION TOOL

DEFF Research Database (Denmark)

2016-01-01

The invention relates to a replication tool (1, 1a, 1b) for producing a part (4) with a microscale textured replica surface (5a, 5b, 5c, 5d). The replication tool (1, 1a, 1b) comprises a tool surface (2a, 2b) defining a general shape of the item. The tool surface (2a, 2b) comprises a microscale...... energy directors on flange portions thereof uses the replication tool (1, 1a, 1b) to form an item (4) with a general shape as defined by the tool surface (2a, 2b). The formed item (4) comprises a microscale textured replica surface (5a, 5b, 5c, 5d) with a lateral arrangement of polydisperse microscale...

Development of a fully automated open-column chemical-separation system—COLUMNSPIDER—and its application to Sr-Nd-Pb isotope analyses of igneous rock samples

Science.gov (United States)

Miyazaki, Takashi; Vaglarov, Bogdan Stefanov; Takei, Masakazu; Suzuki, Masahiro; Suzuki, Hiroaki; Ohsawa, Kouzou; Chang, Qing; Takahashi, Toshiro; Hirahara, Yuka; Hanyu, Takeshi; Kimura, Jun-Ichi; Tatsumi, Yoshiyuki

A fully automated open-column resin-bed chemical-separation system, named COLUMNSPIDER, has been developed. The system consists of a programmable micropipetting robot that dispenses chemical reagents and sample solutions into an open-column resin bed for elemental separation. After the initial set up of resin columns, chemical reagents, and beakers for the separated chemical components, all separation procedures are automated. As many as ten samples can be eluted in parallel in a single automated run. Many separation procedures, such as radiogenic isotope ratio analyses for Sr and Nd, involve the use of multiple column separations with different resin columns, chemical reagents, and beakers of various volumes. COLUMNSPIDER completes these separations using multiple runs. Programmable functions, including the positioning of the micropipetter, reagent volume, and elution time, enable flexible operation. Optimized movements for solution take-up and high-efficiency column flushing allow the system to perform as precisely as when carried out manually by a skilled operator. Procedural blanks, examined for COLUMNSPIDER separations of Sr, Nd, and Pb, are low and negligible. The measured Sr, Nd, and Pb isotope ratios for JB-2 and Nd isotope ratios for JB-3 and BCR-2 rock standards all fall within the ranges reported previously in high-accuracy analyses. COLUMNSPIDER is a versatile tool for the efficient elemental separation of igneous rock samples, a process that is both labor intensive and time consuming.

Recommendations for Replication Research in Special Education: A Framework of Systematic, Conceptual Replications

Science.gov (United States)

Coyne, Michael D.; Cook, Bryan G.; Therrien, William J.

2016-01-01

Special education researchers conduct studies that can be considered replications. However, they do not often refer to them as replication studies. The purpose of this article is to consider the potential benefits of conceptualizing special education intervention research within a framework of systematic, conceptual replication. Specifically, we…

DNA replication and cancer

DEFF Research Database (Denmark)

Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

2016-01-01

A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways...... causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy....

Late-replicating X-chromosome: replication patterns in mammalian females

Directory of Open Access Journals (Sweden)

Tunin Karen

2002-01-01

Full Text Available The GTG-banding and 5-BrdU incorporation patterns of the late-replicating X-chromosome were studied in female dogs and cattle, and compared to human female patterns. The replication patterns of the short arm of the X-chromosomes did not show any difference between human, dog and cattle females. As to the long arm, some bands showed differences among the three studied species regarding the replication kinetics pattern. These differences were observed in a restricted region of the X-chromosome, delimited by Xq11 -> q25 in humans, by Xq1 -> q8 in dogs, and by Xq12 -> q32 in cattle. In an attempt to find out if these differences in the replication kinetics could be a reflection of differences in the localization of genes in that region of the X-chromosome, we used the probe for the human androgen receptor gene (AR localized at Xq12, which is in the region where we observed differences among the three studied species. We did not, however, observe hybridization signals. Our study goes on, using other human probes for genes located in the region Xq11 -> Xq25.

Robust Replication Control Is Generated by Temporal Gaps between Licensing and Firing Phases and Depends on Degradation of Firing Factor Sld2

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Karl-Uwe Reusswig

2016-10-01

Full Text Available Temporal separation of DNA replication initiation into licensing and firing phases ensures the precise duplication of the genome during each cell cycle. Cyclin-dependent kinase (CDK is known to generate this separation by activating firing factors and at the same time inhibiting licensing factors but may not be sufficient to ensure robust separation at transitions between both phases. Here, we show that a temporal gap separates the inactivation of firing factors from the re-activation of licensing factors during mitosis in budding yeast. We find that gap size critically depends on phosphorylation-dependent degradation of the firing factor Sld2 mediated by CDK, DDK, Mck1, and Cdc5 kinases and the ubiquitin-ligases Dma1/2. Stable mutants of Sld2 minimize the gap and cause increased genome instability in an origin-dependent manner when combined with deregulation of other replication regulators or checkpoint mechanisms. Robust separation of licensing and firing phases therefore appears indispensable to safeguard genome stability.

A Replication by Any Other Name: A Systematic Review of Replicative Intervention Studies

Science.gov (United States)

Cook, Bryan G.; Collins, Lauren W.; Cook, Sara C.; Cook, Lysandra

2016-01-01

Replication research is essential to scientific knowledge. Reviews of replication studies often electronically search for "replicat*" as a textword, which does not identify studies that replicate previous research but do not self-identify as such. We examined whether the 83 intervention studies published in six non-categorical research…

International Expansion through Flexible Replication

DEFF Research Database (Denmark)

Jonsson, Anna; Foss, Nicolai Juul

2011-01-01

Business organizations may expand internationally by replicating a part of their value chain, such as a sales and marketing format, in other countries. However, little is known regarding how such “international replicators” build a format for replication, or how they can adjust it in order to ada......, etc.) are replicated in a uniform manner across stores, and change only very slowly (if at all) in response to learning (“flexible replication”). We conclude by discussing the factors that influence the approach to replication adopted by an international replicator.......Business organizations may expand internationally by replicating a part of their value chain, such as a sales and marketing format, in other countries. However, little is known regarding how such “international replicators” build a format for replication, or how they can adjust it in order to adapt...

Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants.

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Xu, Kai; Nagy, Peter D

2017-04-01

Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication have been gaining full attention only recently. Previous work with Tomato bushy stunt tombusvirus (TBSV) in model host yeast has revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we showed that the TBSV p33 and p92 replication proteins could bind to sterols in vitro The sterol binding by p33 is supported by cholesterol recognition/interaction amino acid consensus (CRAC) and CARC-like sequences within the two transmembrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also showed the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis on both the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The data support the notion that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Together, the results obtained in this study and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication. IMPORTANCE One intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly, or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are

A replication of a factor analysis of motivations for trapping

Science.gov (United States)

Schroeder, Susan; Fulton, David C.

2015-01-01

Using a 2013 sample of Minnesota trappers, we employed confirmatory factor analysis to replicate an exploratory factor analysis of trapping motivations conducted by Daigle, Muth, Zwick, and Glass (1998).  We employed the same 25 items used by Daigle et al. and tested the same five-factor structure using a recent sample of Minnesota trappers. We also compared motivations in our sample to those reported by Daigle et el.

Can concurrent memory load reduce distraction? A replication study and beyond.

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Gil-Gómez de Liaño, Beatriz; Stablum, Franca; Umiltà, Carlo

2016-01-01

The effects of concurrent working memory load in attentional processes have been 1 of the most puzzling issues in cognitive psychology. Studies have shown detrimental effects, no effects, and even beneficial effects of working memory load in different attentional tasks. In the present study we attempted to replicate Kim, Kim, and Chun's (2005, Experiment 3b) findings of beneficial effects of concurrent working memory load in a spatial Stroop-like task. In 3 experiments in which our sample was 3 times larger than that in the original Kim et al. study, we could not replicate their findings. The results are discussed in terms of what may have produced the conflicting results, trying to shed light on how working memory load affects attentional tasks. Also, we emphasize the importance of using adequately large samples in cognitive research. Although we acknowledge the relevance of meta-analyses to analyze conflicting results, in the present article we stress (perhaps more important) the power of an essential trademark in science for research development: replicability. (c) 2015 APA, all rights reserved).

The influence of mortality and socioeconomic status on risk and delayed rewards: a replication with British participants

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Gillian V. Pepper

2017-07-01

Full Text Available Here, we report three attempts to replicate a finding from an influential psychological study (Griskevicius et al., 2011b. The original study found interactions between childhood SES and experimental mortality-priming condition in predicting risk acceptance and delay discounting outcomes. The original study used US student samples. We used British university students (replication 1 and British online samples (replications 2 and 3 with a modified version of the original priming material, which was tailored to make it more credible to a British audience. We did not replicate the interaction between childhood SES and mortality-priming condition in any of our three experiments. The only consistent trend of note was an interaction between sex and priming condition for delay discounting. We note that psychological priming effects are considered fragile and often fail to replicate. Our failure to replicate the original finding could be due to demographic differences in study participants, alterations made to the prime, or other study limitations. However, it is also possible that the previously reported interaction is not a robust or generalizable finding.

Power and precision of replicated helicopter surveys in mixed bushveld

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B.K. Reilly

1998-07-01

Full Text Available It is well known that aerial game counts in South Africa are often applied in a non-standardised, unreplicated fashion. They contribute to poor management decisions based on their results as they may be subject to large statistical Type I and II errors. Replicate counts of large herbivores were conducted in a 8 500 ha sample site in the Loskop Dam Nature Reserve in July 1991. These data were used to estimate precision of the counts and estimate statistical power to detect population changes for different combinations of replications and significance levels.

Replication Rate, Framing, and Format Affect Attitudes and Decisions about Science Claims.

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Barnes, Ralph M; Tobin, Stephanie J; Johnston, Heather M; MacKenzie, Noah; Taglang, Chelsea M

2016-01-01

A series of five experiments examined how the evaluation of a scientific finding was influenced by information about the number of studies that had successfully replicated the initial finding. The experiments also tested the impact of frame (negative, positive) and numeric format (percentage, natural frequency) on the evaluation of scientific findings. In Experiments 1 through 4, an attitude difference score served as the dependent measure, while a measure of choice served as the dependent measure in Experiment 5. Results from a diverse sample of 188 non-institutionalized U.S. adults (Experiment 2) and 730 undergraduate college students (Experiments 1, 3, and 4) indicated that attitudes became more positive as the replication rate increased and attitudes were more positive when the replication information was framed positively. The results also indicate that the manner in which replication rate was framed had a greater impact on attitude than the replication rate itself. The large effect for frame was attenuated somewhat when information about replication was presented in the form of natural frequencies rather than percentages. A fifth study employing 662 undergraduate college students in a task in which choice served as the dependent measure confirmed the framing effect and replicated the replication rate effect in the positive frame condition, but provided no evidence that the use of natural frequencies diminished the effect.

Replication Rate, Framing, and Format Affect Attitudes and Decisions about Science Claims

Directory of Open Access Journals (Sweden)

Ralph M. Barnes

2016-11-01

Full Text Available A series of five experiments examined how the evaluation of a scientific finding was influenced by information about the number of studies that had successfully replicated the initial finding. The experiments also tested the impact of frame (negative, positive and numeric format (percentage, natural frequency on the evaluation of scientific findings. In Experiments 1 through 4, an attitude difference score served as the dependent measure, while a measure of choice served as the dependent measure in Experiment 5. Results from a diverse sample of 188 non-institutionalized U.S. adults (Experiment 2 and 730 undergraduate college students (Experiments 1, 3, and 4 indicated that attitudes became more positive as the replication rate increased and attitudes were more positive when the replication information was framed positively. The results also indicate that the manner in which replication rate was framed had a greater impact on attitude than the replication rate itself. The large effect for frame was attenuated somewhat when information about replication was presented in the form of natural frequencies rather than percentages. A fifth study employing 662 undergraduate college students in a task in which choice served as the dependent measure confirmed the framing effect and replicated the replication rate effect in the positive frame condition, but provided no evidence that the use of natural frequencies diminished the effect.

Replication Rate, Framing, and Format Affect Attitudes and Decisions about Science Claims

Science.gov (United States)

Barnes, Ralph M.; Tobin, Stephanie J.; Johnston, Heather M.; MacKenzie, Noah; Taglang, Chelsea M.

2016-01-01

A series of five experiments examined how the evaluation of a scientific finding was influenced by information about the number of studies that had successfully replicated the initial finding. The experiments also tested the impact of frame (negative, positive) and numeric format (percentage, natural frequency) on the evaluation of scientific findings. In Experiments 1 through 4, an attitude difference score served as the dependent measure, while a measure of choice served as the dependent measure in Experiment 5. Results from a diverse sample of 188 non-institutionalized U.S. adults (Experiment 2) and 730 undergraduate college students (Experiments 1, 3, and 4) indicated that attitudes became more positive as the replication rate increased and attitudes were more positive when the replication information was framed positively. The results also indicate that the manner in which replication rate was framed had a greater impact on attitude than the replication rate itself. The large effect for frame was attenuated somewhat when information about replication was presented in the form of natural frequencies rather than percentages. A fifth study employing 662 undergraduate college students in a task in which choice served as the dependent measure confirmed the framing effect and replicated the replication rate effect in the positive frame condition, but provided no evidence that the use of natural frequencies diminished the effect. PMID:27920743

Replication of associations between LRP5 and ESRRA variants and bone density in premenopausal women.

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Giroux, S; Elfassihi, L; Cole, D E C; Rousseau, F

2008-12-01

Replication is a critical step to validate positive genetic associations. In this study, we tested two previously reported positive associations. The low density lipoprotein receptor-related protein 5 (LRP5) Val667Met and lumbar spine bone density are replicated. This result is in line with results from large consortiums such as Genomos. However, the estrogen-related receptor alpha (ESRRA) repeat in the promoter is not replicated although the polymorphism studied was functional and could have been a causative variant. We sought to validate associations previously reported between LRP5 V667M polymorphism and lumbar spine (LS, p = 0.013) and femoral neck (FN, p = 0.0002) bone mineral density (BMD), and between ESRRA 23 base pair repeat polymorphism and LS BMD (p = 0.0036) in a sample of premenopausal Caucasian women using an independent sample. For the replication sample, we recruited 673 premenopausal women from the Toronto metropolitan area. All women were Caucasian and had BMD measured. LRP5 V667M was genotyped by allele-specific PCR and ESRRA repeats by sizing of PCR products on agarose gels. We reproduced the same association as we reported previously between LRP5 V667M and LS BMD (p = 0.015) but not with FN BMD (p = 0.254). The combined data from the two populations indicate an effect size of 0.28SD for LS BMD (p = 0.00048) and an effect size of 0.26 SD for FN BMD (p = 0.00037). In contrast, the association we reported earlier between ESRRA repeats and LS BMD was not replicated in the sample from Toronto (p = 0.645). The association between LRP5 V667M and LS BMD is confirmed but not that between ESRRA repeats and LS BMD. This result indicates that it is imperative to validate any positive association in an independent sample.

Who Needs Replication?

Science.gov (United States)

Porte, Graeme

2013-01-01

In this paper, the editor of a recent Cambridge University Press book on research methods discusses replicating previous key studies to throw more light on their reliability and generalizability. Replication research is presented as an accepted method of validating previous research by providing comparability between the original and replicated…

Mechanisms of bacterial DNA replication restart

Science.gov (United States)

Windgassen, Tricia A; Wessel, Sarah R; Bhattacharyya, Basudeb

2018-01-01

Abstract Multi-protein DNA replication complexes called replisomes perform the essential process of copying cellular genetic information prior to cell division. Under ideal conditions, replisomes dissociate only after the entire genome has been duplicated. However, DNA replication rarely occurs without interruptions that can dislodge replisomes from DNA. Such events produce incompletely replicated chromosomes that, if left unrepaired, prevent the segregation of full genomes to daughter cells. To mitigate this threat, cells have evolved ‘DNA replication restart’ pathways that have been best defined in bacteria. Replication restart requires recognition and remodeling of abandoned replication forks by DNA replication restart proteins followed by reloading of the replicative DNA helicase, which subsequently directs assembly of the remaining replisome subunits. This review summarizes our current understanding of the mechanisms underlying replication restart and the proteins that drive the process in Escherichia coli (PriA, PriB, PriC and DnaT). PMID:29202195

What Should Researchers Expect When They Replicate Studies? A Statistical View of Replicability in Psychological Science.

Science.gov (United States)

Patil, Prasad; Peng, Roger D; Leek, Jeffrey T

2016-07-01

A recent study of the replicability of key psychological findings is a major contribution toward understanding the human side of the scientific process. Despite the careful and nuanced analysis reported, the simple narrative disseminated by the mass, social, and scientific media was that in only 36% of the studies were the original results replicated. In the current study, however, we showed that 77% of the replication effect sizes reported were within a 95% prediction interval calculated using the original effect size. Our analysis suggests two critical issues in understanding replication of psychological studies. First, researchers' intuitive expectations for what a replication should show do not always match with statistical estimates of replication. Second, when the results of original studies are very imprecise, they create wide prediction intervals-and a broad range of replication effects that are consistent with the original estimates. This may lead to effects that replicate successfully, in that replication results are consistent with statistical expectations, but do not provide much information about the size (or existence) of the true effect. In this light, the results of the Reproducibility Project: Psychology can be viewed as statistically consistent with what one might expect when performing a large-scale replication experiment. © The Author(s) 2016.

Rapid determination of radium-224/226 in seawater sample by alpha spectrometry.

Science.gov (United States)

Song, Lijuan; Yang, Yonggang; Luo, Maoyi; Ma, Yan; Dai, Xiongxin

2017-05-01

A new radiochemical separation method has been developed for rapid determination of alpha-emitting radium isotopes in seawater samples. This method can be applied for the measurement of 226 Ra in seawater samples when 224 Ra is used as tracer for chemical recovery correction. Likewise, 226 Ra can also be added as tracer for the determination of 224 Ra in seawater sample. In the method, radium is first pre-concentrated with hydrous titanium oxide (HTiO) and is purified by combined anion/cation exchange column chromatographic separation. The radium in the eluate is then co-precipitated with HTiO, dissolved in 9 M H 2 SO 4 , and followed through a BaSO 4 micro-precipitation step to prepare a thin-layer counting source to determine the activities of 224 Ra/ 226 Ra by alpha spectrometry. Replicate spike and blank samples were measured to evaluate the performance of the procedure. The minimum detectable activity concentration was determined to be 0.5 mBq·L -1 for 226 Ra and 0.4 mBq·L -1 for 224 Ra in 1 L of seawater sample with a counting time of 48 h. The method is a promising candidate for rapid measurement for alpha-emitting Ra isotopes in a large population of environment water samples. Copyright © 2017 Elsevier Ltd. All rights reserved.

Psychological type and attitude toward Christianity: a replication.

Science.gov (United States)

Francis, Leslie J; Robbins, Mandy; Boxer, Anna; Lewis, Christopher Alan; McGuckin, Conor; McDaid, Charles J

2003-02-01

A sample of 149 university students completed the Francis Psychological Type Scales together with the Francis Scale of Attitude Toward Christianity. The data indicated that university students classified as Feeling Types hold a more positive attitude toward Christianity than those classified as Thinking Types. These findings replicate the 1999 report of Jones and Francis.

Pressure-assisted introduction of urine samples into a short capillary for electrophoretic separation with contactless conductivity and UV spectrometry detection.

Science.gov (United States)

Makrlíková, Anna; Opekar, František; Tůma, Petr

2015-08-01

A computer-controlled hydrodynamic sample introduction method has been proposed for short-capillary electrophoresis. In the method, the BGE flushes sample from the loop of a six-way sampling valve and is carried to the injection end of the capillary. A short pressure impulse is generated in the electrolyte stream at the time when the sample zone is at the capillary, leading to injection of the sample into the capillary. Then the electrolyte flow is stopped and the separation voltage is turned on. This way of sample introduction does not involve movement of the capillary and both of its ends remain constantly in the solution during both sample injection and separation. The amount of sample introduced to the capillary is controlled by the duration of the pressure pulse. The new sample introduction method was tested in the determination of ammonia, creatinine, uric acid, and hippuric acid in human urine. The determination was performed in a capillary with an overall length of 10.5 cm, in two BGEs with compositions 50 mM MES + 5 mM NaOH (pH 5.1) and 1 M acetic acid + 1.5 mM crown ether 18-crown-6 (pH 2.4). A dual contactless conductivity/UV spectrometric detector was used for the detection. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hierarchical cluster analysis of technical replicates to identify interferents in untargeted mass spectrometry metabolomics.

Science.gov (United States)

Caesar, Lindsay K; Kvalheim, Olav M; Cech, Nadja B

2018-08-27

Mass spectral data sets often contain experimental artefacts, and data filtering prior to statistical analysis is crucial to extract reliable information. This is particularly true in untargeted metabolomics analyses, where the analyte(s) of interest are not known a priori. It is often assumed that chemical interferents (i.e. solvent contaminants such as plasticizers) are consistent across samples, and can be removed by background subtraction from blank injections. On the contrary, it is shown here that chemical contaminants may vary in abundance across each injection, potentially leading to their misidentification as relevant sample components. With this metabolomics study, we demonstrate the effectiveness of hierarchical cluster analysis (HCA) of replicate injections (technical replicates) as a methodology to identify chemical interferents and reduce their contaminating contribution to metabolomics models. Pools of metabolites with varying complexity were prepared from the botanical Angelica keiskei Koidzumi and spiked with known metabolites. Each set of pools was analyzed in triplicate and at multiple concentrations using ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS). Before filtering, HCA failed to cluster replicates in the data sets. To identify contaminant peaks, we developed a filtering process that evaluated the relative peak area variance of each variable within triplicate injections. These interferent peaks were found across all samples, but did not show consistent peak area from injection to injection, even when evaluating the same chemical sample. This filtering process identified 128 ions that appear to originate from the UPLC-MS system. Data sets collected for a high number of pools with comparatively simple chemical composition were highly influenced by these chemical interferents, as were samples that were analyzed at a low concentration. When chemical interferent masses were removed, technical replicates clustered in

DNA Replication Profiling Using Deep Sequencing.

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Saayman, Xanita; Ramos-Pérez, Cristina; Brown, Grant W

2018-01-01

Profiling of DNA replication during progression through S phase allows a quantitative snap-shot of replication origin usage and DNA replication fork progression. We present a method for using deep sequencing data to profile DNA replication in S. cerevisiae.

Purifying, Separating, and Concentrating Cells From a Sample Low in Biomass

Science.gov (United States)

Benardini, James N.; LaDuc, Myron T.; Diamond, Rochelle

2012-01-01

Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis. A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass. The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology. Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further

A confirmatory test of the underlying factor structure of scores on the collective self-esteem scale in two independent samples of Black Americans.

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Utsey, Shawn O; Constantine, Madonna G

2006-04-01

In this study, we examined the factor structure of the Collective Self-Esteem Scale (CSES; Luhtanen & Crocker, 1992) across 2 separate samples of Black Americans. The CSES was administered to a sample of Black American adolescents (n = 538) and a community sample of Black American adults (n = 313). Results of confirmatory factor analyses (CFAs), however, did not support the original 4-factor model identified by Luhtanen and Crocker (1992) as providing an adequate fit to the data for these samples. Furthermore, an exploratory CFA procedure failed to find a CSES factor structure that could be replicated across the 2 samples of Black Americans. We present and discuss implications of the findings.

Induction of UV-resistant DNA replication in Escherichia coli: Induced stable DNA replication as an SOS function

International Nuclear Information System (INIS)

Kogoma, T.; Torrey, T.A.; Connaughton, M.J.

1979-01-01

The striking similarity between the treatments that induce SOS functions and those that result in stable DNA replication (continuous DNA replication in the absence of protein synthesis) prompted us to examine the possibility of stable DNA replication being a recA + lexA + -dependent SOS function. In addition to the treatments previously reported, ultraviolet (UV) irradiation or treatment with mitomycin C was also found to induce stable DNA replication. The thermal treatment of tif-1 strains did not result in detectable levels of stable DNA replication, but nalidixic acid readily induced the activity in these strains. The induction of stable DNA replication with nalidixic acid was severely suppressed in tif-1 lex A mutant strains. The inhibitory activity of lexA3 was negated by the presence of the spr-5l mutation, an intragenic suppressor of lexA3. Induced stable DNA replication was found to be considerably more resistant to UV irradiation than normal replication both in a uvr A6 strain and a uvr + strain. The UV-resistant replication occurred mostly in the semiconservative manner. The possible roles of stable DNA replication in repair of damaged DNA are discussed. (orig.)

Chromatin replication and epigenome maintenance

DEFF Research Database (Denmark)

Alabert, Constance; Groth, Anja

2012-01-01

Stability and function of eukaryotic genomes are closely linked to chromatin structure and organization. During cell division the entire genome must be accurately replicated and the chromatin landscape reproduced on new DNA. Chromatin and nuclear structure influence where and when DNA replication...... initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA...

Critical investigation of the separation of noradrenaline and adrenaline from urine samples using Al2O3 as adsorbant

International Nuclear Information System (INIS)

Neidhart, B.; Kringe, K.-P.; Deutschmann, P.

1983-01-01

A critical investigation of the separation of free noradrenaline and adrenaline from urine samples revealed serious errors during sample pretreatment using Al 2 O 3 as adsorbent. An exact and rapid pH adjustment of the sample, using thymol-blue as indicator, proved to be the chief prerequisite for precise and accurate results. Increasing temperature and pH favour the oxidative decomposition of the catecholamines during routine analysis. This was examined, using the radiotracer method and liquid scintillation counting. (author)

On-line coupling of sample preconcentration by LVSEP with gel electrophoretic separation on T-channel chips.

Science.gov (United States)

Kitagawa, Fumihiko; Kinami, Saeko; Takegawa, Yuuki; Nukatsuka, Isoshi; Sueyoshi, Kenji; Kawai, Takayuki; Otsuka, Koji

2017-01-01

To achieve an on-line coupling of the sample preconcentration by a large-volume sample stacking with an electroosmotic flow pump (LVSEP) with microchip gel electrophoresis (MCGE), a sample solution, a background solution for LVSEP and a sieving solution for MCGE were loaded in a T-form channel and three reservoirs on PDMS microchips. By utilizing the difference in the flow resistance of the two channels, a low-viscosity sample and a viscous polymer solution were easily introduced into the LVSEP and MCGE channels, respectively. Fluorescence imaging of the sequential LVSEP-MCGE processes clearly demonstrated that a faster stacking of anionic fluorescein and successive introduction into the MCGE channel can be carried out on the T-channel chip. To evaluate the preconcentration performance, a conventional MCZE analysis of fluorescein on the cross-channel chip was compared with LVSEP-MCGE on the short T-channel chip, and as a result that the value of sensitive enhancement factor (SEF) was estimated to be 370. The repeatability of the peak height was good with the RSD value of 3.2%, indicating the robustness of the enrichment performance. In the successive LVSEP-MCGE analysis of φX174/HaeIII digest, the DNA fragments were well enriched to a sharp peak in the LVSEP channel, and they were separated in the MCGE channel, whose electropherogram was well-resembled with that in the conventional MCGE. The values of SEF for the DNA fragments were calculated to be ranging from 74 to 108. Thus, the successive LVSEP-MCGE analysis was effective for both preconcentrating and separating DNA fragments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

36 CFR 910.64 - Replication.

Science.gov (United States)

2010-07-01

... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Replication. 910.64 Section 910.64 Parks, Forests, and Public Property PENNSYLVANIA AVENUE DEVELOPMENT CORPORATION GENERAL... DEVELOPMENT AREA Glossary of Terms § 910.64 Replication. Replication means the process of using modern methods...

Replication stress-induced chromosome breakage is correlated with replication fork progression and is preceded by single-stranded DNA formation.

Science.gov (United States)

Feng, Wenyi; Di Rienzi, Sara C; Raghuraman, M K; Brewer, Bonita J

2011-10-01

Chromosome breakage as a result of replication stress has been hypothesized to be the direct consequence of defective replication fork progression, or "collapsed" replication forks. However, direct and genome-wide evidence that collapsed replication forks give rise to chromosome breakage is still lacking. Previously we showed that a yeast replication checkpoint mutant mec1-1, after transient exposure to replication impediment imposed by hydroxyurea (HU), failed to complete DNA replication, accumulated single-stranded DNA (ssDNA) at the replication forks, and fragmented its chromosomes. In this study, by following replication fork progression genome-wide via ssDNA detection and by direct mapping of chromosome breakage after HU exposure, we have tested the hypothesis that the chromosome breakage in mec1 cells occurs at collapsed replication forks. We demonstrate that sites of chromosome breakage indeed correlate with replication fork locations. Moreover, ssDNA can be detected prior to chromosome breakage, suggesting that ssDNA accumulation is the common precursor to double strand breaks at collapsed replication forks.

Automated cellular sample preparation using a Centrifuge-on-a-Chip.

Science.gov (United States)

Mach, Albert J; Kim, Jae Hyun; Arshi, Armin; Hur, Soojung Claire; Di Carlo, Dino

2011-09-07

The standard centrifuge is a laboratory instrument widely used by biologists and medical technicians for preparing cell samples. Efforts to automate the operations of concentration, cell separation, and solution exchange that a centrifuge performs in a simpler and smaller platform have had limited success. Here, we present a microfluidic chip that replicates the functions of a centrifuge without moving parts or external forces. The device operates using a purely fluid dynamic phenomenon in which cells selectively enter and are maintained in microscale vortices. Continuous and sequential operation allows enrichment of cancer cells from spiked blood samples at the mL min(-1) scale, followed by fluorescent labeling of intra- and extra-cellular antigens on the cells without the need for manual pipetting and washing steps. A versatile centrifuge-analogue may open opportunities in automated, low-cost and high-throughput sample preparation as an alternative to the standard benchtop centrifuge in standardized clinical diagnostics or resource poor settings.

Replication Research and Special Education

Science.gov (United States)

Travers, Jason C.; Cook, Bryan G.; Therrien, William J.; Coyne, Michael D.

2016-01-01

Replicating previously reported empirical research is a necessary aspect of an evidence-based field of special education, but little formal investigation into the prevalence of replication research in the special education research literature has been conducted. Various factors may explain the lack of attention to replication of special education…

The progression of replication forks at natural replication barriers in live bacteria

NARCIS (Netherlands)

Moolman, M.C.; Tiruvadi Krishnan, S; Kerssemakers, J.W.J.; de Leeuw, R.; Lorent, V.J.F.; Sherratt, David J.; Dekker, N.H.

2016-01-01

Protein-DNA complexes are one of the principal barriers the replisome encounters during replication. One such barrier is the Tus-ter complex, which is a direction dependent barrier for replication fork progression. The details concerning the dynamics of the replisome when encountering these

Flock House virus subgenomic RNA3 is replicated and its replication correlates with transactivation of RNA2

International Nuclear Information System (INIS)

Eckerle, Lance D.; Albarino, Cesar G.; Ball, L. Andrew.

2003-01-01

The nodavirus Flock House virus has a bipartite genome composed of RNAs 1 and 2, which encode the catalytic component of the RNA-dependent RNA polymerase (RdRp) and the capsid protein precursor, respectively. In addition to catalyzing replication of the viral genome, the RdRp also transcribes from RNA1 a subgenomic RNA3, which is both required for and suppressed by RNA2 replication. Here, we show that in the absence of RNA1 replication, FHV RdRp replicated positive-sense RNA3 transcripts fully and copied negative-sense RNA3 transcripts into positive strands. The two nonstructural proteins encoded by RNA3 were dispensable for replication, but sequences in the 3'-terminal 58 nucleotides were required. RNA3 variants that failed to replicate also failed to transactivate RNA2. These results imply that RNA3 is naturally produced both by transcription from RNA1 and by subsequent RNA1-independent replication and that RNA3 replication may be necessary for transactivation of RNA2

Prospective elementary teachers' conceptions of multidigit number: exemplifying a replication framework for mathematics education

Science.gov (United States)

Jacobson, Erik; Simpson, Amber

2018-04-01

Replication studies play a critical role in scientific accumulation of knowledge, yet replication studies in mathematics education are rare. In this study, the authors replicated Thanheiser's (Educational Studies in Mathematics 75:241-251, 2010) study of prospective elementary teachers' conceptions of multidigit number and examined the main claim that most elementary pre-service teachers think about digits incorrectly at least some of the time. Results indicated no statistically significant difference in the distribution of conceptions between the original and replication samples and, moreover, no statistically significant differences in the distribution of sub-conceptions among prospective teachers with the most common conception. These results suggest confidence is warranted both in the generality of the main claim and in the utility of the conceptions framework for describing prospective elementary teachers' conceptions of multidigit number. The report further contributes a framework for replication of mathematics education research adapted from the field of psychology.

Replication-independent chromatin loading of Dnmt1 during G2 and M phases

Science.gov (United States)

Easwaran, Hariharan P; Schermelleh, Lothar; Leonhardt, Heinrich; Cardoso, M Cristina

2004-01-01

The major DNA methyltransferase, Dnmt1, associates with DNA replication sites in S phase maintaining the methylation pattern in the newly synthesized strand. In view of the slow kinetics of Dnmt1 in vitro versus the fast progression of the replication fork, we have tested whether Dnmt1 associates with chromatin beyond S phase. Using time-lapse microscopy of mammalian cells expressing green-fluorescent-protein-tagged Dnmt1 and DsRed-tagged DNA Ligase I as a cell cycle progression marker, we have found that Dnmt1 associates with chromatin during G2 and M. This association is mediated by a specific targeting sequence, shows strong preference for constitutive but not facultative heterochromatin and is independent of heterochromatin-specific histone H3 Lys 9 trimethylation, SUV39H and HP1. Moreover, photobleaching analyses showed that Dnmt1 is continuously loaded onto chromatin throughout G2 and M, indicating a replication-independent role of Dnmt1 that could represent a novel and separate pathway to maintain DNA methylation. PMID:15550930

DNA Replication Control During Drosophila Development: Insights into the Onset of S Phase, Replication Initiation, and Fork Progression

Science.gov (United States)

Hua, Brian L.; Orr-Weaver, Terry L.

2017-01-01

Proper control of DNA replication is critical to ensure genomic integrity during cell proliferation. In addition, differential regulation of the DNA replication program during development can change gene copy number to influence cell size and gene expression. Drosophila melanogaster serves as a powerful organism to study the developmental control of DNA replication in various cell cycle contexts in a variety of differentiated cell and tissue types. Additionally, Drosophila has provided several developmentally regulated replication models to dissect the molecular mechanisms that underlie replication-based copy number changes in the genome, which include differential underreplication and gene amplification. Here, we review key findings and our current understanding of the developmental control of DNA replication in the contexts of the archetypal replication program as well as of underreplication and differential gene amplification. We focus on the use of these latter two replication systems to delineate many of the molecular mechanisms that underlie the developmental control of replication initiation and fork elongation. PMID:28874453

Failed Replication of Oxytocin Effects on Trust: The Envelope Task Case.

Directory of Open Access Journals (Sweden)

Anthony Lane

Full Text Available The neurohormone Oxytocin (OT has been one of the most studied peptides in behavioral sciences over the past two decades. Many studies have suggested that OT could increase trusting behaviors. A previous study, based on the "Envelope Task" paradigm, where trust is assessed by the degree of openness of an envelope containing participant's confidential information, showed that OT increases trusting behavior and reported one of the most powerful effects of OT on a behavioral variable. In this paper we present two failed replications of this effect, despite sufficient power to replicate the original large effect. The non-significant results of these two failed replications clearly exclude a large effect of OT on trust in this paradigm but are compatible with either a null effect of OT on trust, or a small effect, undetectable with small sample size (N = 95 and 61 in Study 1 and 2, respectively. Taken together, our results question the purported size of OT's effect on trust and emphasize the need for replications.

Statistical correction of the Winner's Curse explains replication variability in quantitative trait genome-wide association studies.

Directory of Open Access Journals (Sweden)

Cameron Palmer

2017-07-01

Full Text Available Genome-wide association studies (GWAS have identified hundreds of SNPs responsible for variation in human quantitative traits. However, genome-wide-significant associations often fail to replicate across independent cohorts, in apparent inconsistency with their apparent strong effects in discovery cohorts. This limited success of replication raises pervasive questions about the utility of the GWAS field. We identify all 332 studies of quantitative traits from the NHGRI-EBI GWAS Database with attempted replication. We find that the majority of studies provide insufficient data to evaluate replication rates. The remaining papers replicate significantly worse than expected (p < 10-14, even when adjusting for regression-to-the-mean of effect size between discovery- and replication-cohorts termed the Winner's Curse (p < 10-16. We show this is due in part to misreporting replication cohort-size as a maximum number, rather than per-locus one. In 39 studies accurately reporting per-locus cohort-size for attempted replication of 707 loci in samples with similar ancestry, replication rate matched expectation (predicted 458, observed 457, p = 0.94. In contrast, ancestry differences between replication and discovery (13 studies, 385 loci cause the most highly-powered decile of loci to replicate worse than expected, due to difference in linkage disequilibrium.

A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections

Science.gov (United States)

Moen, Scott T.; Hatcher, Christopher L.; Singh, Anup K.

2016-01-01

We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. This platform has great potential in both medical diagnostics and research applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing. PMID:27054764

A new method for the homogeneous precipitative separation of trace level lanthanides as oxalates: application to different types of geological samples

International Nuclear Information System (INIS)

Premadas, A.; Cyriac, Bincy; Kesavan, V.S.

2013-01-01

Oxalate precipitation of lanthanides in acidic medium is a widely used selective group separation method at percentage to trace level in different types of geological samples. Most of the procedures are based on the heterogeneous oxalate precipitation of lanthanides using calcium as carrier. In the heterogeneous precipitation, the co-precipitated impurities from the matrix elements are more, besides if the pH at the time of precipitation is not monitored carefully there is a chance of losing some of the lanthanides. In this report, we present a new homogeneous oxalate precipitation of trace level lanthanides from different types of geological samples using calcium as carrier. In the present method pH is getting adjusted (pH ∼1) on its own, after the hydrolysis of urea added to the sample solution. This acidic pH is essential for the complete precipitation of the lanthanides. Therefore, no critical parameter adjustment for the precipitation is involved in the proposed method. The oxalate precipitate obtained was in crystalline nature which facilitates the fast settlement, easy filtration; besides the co-precipitated matrix elements are very less as compared to normal heterogeneous oxalate precipitation of lanthanides. Another advantage is more quantity of the sample can be taken for the separation of lanthanides which is a limitation for other separation methods reported. Accuracy of the method was checked by analyzing nine international reference materials comprising different types of geological samples obtained from Canadian Certified Reference Project Materials such as syenite samples SY-2, SY-3 and SY-4; gabro sample MRG-1; soil samples SO-1 and SO-2; iron formation sample FeR-2; lake sediments LKSD-2 and LKSD-4. The values of the lanthanides obtained for these reference materials are comparable with recommended values, indicating that the method is accurate. The reproducibility is characterized by a relative standard deviation (RSD) of 1 to 6% (n=4). (author)

The eukaryotic bell-shaped temporal rate of DNA replication origin firing emanates from a balance between origin activation and passivation.

Science.gov (United States)

Arbona, Jean-Michel; Goldar, Arach; Hyrien, Olivier; Arneodo, Alain; Audit, Benjamin

2018-06-01

The time-dependent rate I(t) of origin firing per length of unreplicated DNA presents a universal bell shape in eukaryotes that has been interpreted as the result of a complex time-evolving interaction between origins and limiting firing factors. Here we show that a normal diffusion of replication fork components towards localized potential replication origins (p-oris) can more simply account for the I(t) universal bell shape, as a consequence of a competition between the origin firing time and the time needed to replicate DNA separating two neighboring p-oris . We predict the I(t) maximal value to be the product of the replication fork speed with the squared p-ori density. We show that this relation is robustly observed in simulations and in experimental data for several eukaryotes. Our work underlines that fork-component recycling and potential origins localization are sufficient spatial ingredients to explain the universality of DNA replication kinetics. © 2018, Arbona et al.

The progression of replication forks at natural replication barriers in live bacteria.

Science.gov (United States)

Moolman, M Charl; Tiruvadi Krishnan, Sriram; Kerssemakers, Jacob W J; de Leeuw, Roy; Lorent, Vincent; Sherratt, David J; Dekker, Nynke H

2016-07-27

Protein-DNA complexes are one of the principal barriers the replisome encounters during replication. One such barrier is the Tus-ter complex, which is a direction dependent barrier for replication fork progression. The details concerning the dynamics of the replisome when encountering these Tus-ter barriers in the cell are poorly understood. By performing quantitative fluorescence microscopy with microfuidics, we investigate the effect on the replisome when encountering these barriers in live Escherichia coli cells. We make use of an E. coli variant that includes only an ectopic origin of replication that is positioned such that one of the two replisomes encounters a Tus-ter barrier before the other replisome. This enables us to single out the effect of encountering a Tus-ter roadblock on an individual replisome. We demonstrate that the replisome remains stably bound after encountering a Tus-ter complex from the non-permissive direction. Furthermore, the replisome is only transiently blocked, and continues replication beyond the barrier. Additionally, we demonstrate that these barriers affect sister chromosome segregation by visualizing specific chromosomal loci in the presence and absence of the Tus protein. These observations demonstrate the resilience of the replication fork to natural barriers and the sensitivity of chromosome alignment to fork progression. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Replication of micro and nano surface geometries

DEFF Research Database (Denmark)

Hansen, Hans Nørgaard; Hocken, R.J.; Tosello, Guido

2011-01-01

The paper describes the state-of-the-art in replication of surface texture and topography at micro and nano scale. The description includes replication of surfaces in polymers, metals and glass. Three different main technological areas enabled by surface replication processes are presented......: manufacture of net-shape micro/nano surfaces, tooling (i.e. master making), and surface quality control (metrology, inspection). Replication processes and methods as well as the metrology of surfaces to determine the degree of replication are presented and classified. Examples from various application areas...... are given including replication for surface texture measurements, surface roughness standards, manufacture of micro and nano structured functional surfaces, replicated surfaces for optical applications (e.g. optical gratings), and process chains based on combinations of repeated surface replication steps....

Immunoassay separation technique

International Nuclear Information System (INIS)

1977-01-01

A method for effecting the immunoassay of a multiplicity of samples, each possibly containing an antigen or an antibody to be assayed, is discussed. Each sample is incubated with a solution containing a detectable antigen or antibody to form a multiplicity of mixtures, each mixture containing as components antigen-antibody, non-complexed antigen and non-complexed antibody. At least one of the components of the said mixture is separated by adsorption. There after, quantity of detectable antigen or antibody is detected in one of the non-adsorbed portions of the mixture. An improvement, compared to other techniques, is the continuous and sequential separation of at least one component, which is intended to be separated from each said multiplicity of mixtures

A New Replication Norm for Psychology

Directory of Open Access Journals (Sweden)

Etienne P LeBel

2015-10-01

Full Text Available In recent years, there has been a growing concern regarding the replicability of findings in psychology, including a mounting number of prominent findings that have failed to replicate via high-powered independent replication attempts. In the face of this replicability “crisis of confidence”, several initiatives have been implemented to increase the reliability of empirical findings. In the current article, I propose a new replication norm that aims to further boost the dependability of findings in psychology. Paralleling the extant social norm that researchers should peer review about three times as many articles that they themselves publish per year, the new replication norm states that researchers should aim to independently replicate important findings in their own research areas in proportion to the number of original studies they themselves publish per year (e.g., a 4:1 original-to-replication studies ratio. I argue this simple approach could significantly advance our science by increasing the reliability and cumulative nature of our empirical knowledge base, accelerating our theoretical understanding of psychological phenomena, instilling a focus on quality rather than quantity, and by facilitating our transformation toward a research culture where executing and reporting independent direct replications is viewed as an ordinary part of the research process. To help promote the new norm, I delineate (1 how each of the major constituencies of the research process (i.e., funders, journals, professional societies, departments, and individual researchers can incentivize replications and promote the new norm and (2 any obstacles each constituency faces in supporting the new norm.

Aberrant allele-specific replication, independent of parental origin, in blood cells of cancer patients

International Nuclear Information System (INIS)

Dotan, Zohar A; Dotan, Aviva; Ramon, Jacob; Avivi, Lydia

2008-01-01

Allelic counterparts of biallelically expressed genes display an epigenetic symmetry normally manifested by synchronous replication, different from genes subjected to monoallelic expression, which normally are characterized by an asynchronous mode of replication (well exemplified by the SNRPN imprinted locus). Malignancy was documented to be associated with gross modifications in the inherent replication-timing coordination between allelic counterparts of imprinted genes as well as of biallelically expressed loci. The cancer-related allelic replication timing aberrations are non-disease specific and appear in peripheral blood cells of cancer patients, including those with solid tumors. As such they offer potential blood markers for non-invasive cancer test. The present study was aimed to gain some insight into the mechanism leading to the replication timing alterations of genes in blood lymphocytes of cancer patients. Peripheral blood samples derived from patients with prostate cancer were chosen to represent the cancerous status, and samples taken from patients with no cancer but with benign prostate hyperplasia were used to portray the normal status. Fluorescence In Situ Hybridization (FISH) replication assay, applied to phytohemagglutinin (PHA)-stimulated blood lymphocytes, was used to evaluate the temporal order (either synchronous or asynchronous) of genes in the patients' cells. We demonstrated that: (i) the aberrant epigenetic profile, as delineated by the cancer status, is a reversible modification, evidenced by our ability to restore the normal patterns of replication in three unrelated loci (CEN15, SNRPN and RB1) by introducing an archetypical demethylating agent, 5-azacytidine; (ii) following the rehabilitating effect of demethylation, an imprinted gene (SNRPN) retains its original parental imprint; and (iii) the choice of an allele between early or late replication in the aberrant asynchronous replication, delineated by the cancer status, is not

A maize root tip system to study DNA replication programmes in somatic and endocycling nuclei during plant development.

Science.gov (United States)

Bass, Hank W; Wear, Emily E; Lee, Tae-Jin; Hoffman, Gregg G; Gumber, Hardeep K; Allen, George C; Thompson, William F; Hanley-Bowdoin, Linda

2014-06-01

The progress of nuclear DNA replication is complex in both time and space, and may reflect several levels of chromatin structure and 3-dimensional organization within the nucleus. To understand the relationship between DNA replication and developmental programmes, it is important to examine replication and nuclear substructure in different developmental contexts including natural cell-cycle progressions in situ. Plant meristems offer an ideal opportunity to analyse such processes in the context of normal growth of an organism. Our current understanding of large-scale chromosomal DNA replication has been limited by the lack of appropriate tools to visualize DNA replication with high resolution at defined points within S phase. In this perspective, we discuss a promising new system that can be used to visualize DNA replication in isolated maize (Zea mays L.) root tip nuclei after in planta pulse labelling with the thymidine analogue, 5-ethynyl-2'-deoxyuridine (EdU). Mixed populations of EdU-labelled nuclei are then separated by flow cytometry into sequential stages of S phase and examined directly using 3-dimensional deconvolution microscopy to characterize spatial patterns of plant DNA replication. Combining spatiotemporal analyses with studies of replication and epigenetic inheritance at the molecular level enables an integrated experimental approach to problems of mitotic inheritance and cellular differentiation. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication.

Science.gov (United States)

Herod, Morgan R; Ferrer-Orta, Cristina; Loundras, Eleni-Anna; Ward, Joseph C; Verdaguer, Nuria; Rowlands, David J; Stonehouse, Nicola J

2016-08-01

The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e., via expression from a separate RNA molecule), while others are required in cis (i.e., expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymatic cis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans acting. Immunofluorescence studies suggest that both cis- and trans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further. Foot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within

Centromere replication timing determines different forms of genomic instability in Saccharomyces cerevisiae checkpoint mutants during replication stress.

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Feng, Wenyi; Bachant, Jeff; Collingwood, David; Raghuraman, M K; Brewer, Bonita J

2009-12-01

Yeast replication checkpoint mutants lose viability following transient exposure to hydroxyurea, a replication-impeding drug. In an effort to understand the basis for this lethality, we discovered that different events are responsible for inviability in checkpoint-deficient cells harboring mutations in the mec1 and rad53 genes. By monitoring genomewide replication dynamics of cells exposed to hydroxyurea, we show that cells with a checkpoint deficient allele of RAD53, rad53K227A, fail to duplicate centromeres. Following removal of the drug, however, rad53K227A cells recover substantial DNA replication, including replication through centromeres. Despite this recovery, the rad53K227A mutant fails to achieve biorientation of sister centromeres during recovery from hydroxyurea, leading to secondary activation of the spindle assembly checkpoint (SAC), aneuploidy, and lethal chromosome segregation errors. We demonstrate that cell lethality from this segregation defect could be partially remedied by reinforcing bipolar attachment. In contrast, cells with the mec1-1 sml1-1 mutations suffer from severely impaired replication resumption upon removal of hydroxyurea. mec1-1 sml1-1 cells can, however, duplicate at least some of their centromeres and achieve bipolar attachment, leading to abortive segregation and fragmentation of incompletely replicated chromosomes. Our results highlight the importance of replicating yeast centromeres early and reveal different mechanisms of cell death due to differences in replication fork progression.

Hydroxyurea inhibits parvovirus B19 replication in erythroid progenitor cells.

Science.gov (United States)

Bonvicini, Francesca; Bua, Gloria; Conti, Ilaria; Manaresi, Elisabetta; Gallinella, Giorgio

2017-07-15

Parvovirus B19 (B19V) infection is restricted to erythroid progenitor cells (EPCs) of the human bone marrow, leading to transient arrest of erythropoiesis and severe complications mainly in subjects with underlying hematological disorders or with immune system deficits. Currently, there are no specific antiviral drugs for B19V treatment, but identification of compounds inhibiting B19V replication can be pursued by a drug repositioning strategy. In this frame, the present study investigates the activity of hydroxyurea (HU), the only disease-modifying therapy approved for sickle cell disease (SCD), towards B19V replication in the two relevant cellular systems, the UT7/EpoS1 cell line and EPCs. Results demonstrate that HU inhibits B19V replication with EC 50 values of 96.2µM and 147.1µM in UT7/EpoS1 and EPCs, respectively, providing experimental evidence of the antiviral activity of HU towards B19V replication, and confirming the efficacy of a drug discovery process by drug repositioning strategy. The antiviral activity occurs in vitro at concentrations lower than those affecting cellular DNA replication and viability, and at levels measured in plasma samples of SCD patients undergoing HU therapy. HU might determine a dual beneficial effect on SCD patients, not only for the treatment of the disease but also towards a virus responsible for severe complications. Copyright © 2017 Elsevier Inc. All rights reserved.

Highly Reproducible Automated Proteomics Sample Preparation Workflow for Quantitative Mass Spectrometry.

Science.gov (United States)

Fu, Qin; Kowalski, Michael P; Mastali, Mitra; Parker, Sarah J; Sobhani, Kimia; van den Broek, Irene; Hunter, Christie L; Van Eyk, Jennifer E

2018-01-05

Sample preparation for protein quantification by mass spectrometry requires multiple processing steps including denaturation, reduction, alkylation, protease digestion, and peptide cleanup. Scaling these procedures for the analysis of numerous complex biological samples can be tedious and time-consuming, as there are many liquid transfer steps and timed reactions where technical variations can be introduced and propagated. We established an automated sample preparation workflow with a total processing time for 96 samples of 5 h, including a 2 h incubation with trypsin. Peptide cleanup is accomplished by online diversion during the LC/MS/MS analysis. In a selected reaction monitoring (SRM) assay targeting 6 plasma biomarkers and spiked β-galactosidase, mean intraday and interday cyclic voltammograms (CVs) for 5 serum and 5 plasma samples over 5 days were samples repeated on 3 separate days had total CVs below 20%. Similar results were obtained when the workflow was transferred to a second site: 93% of peptides had CVs below 20%. An automated trypsin digestion workflow yields uniformly processed samples in less than 5 h. Reproducible quantification of peptides was observed across replicates, days, instruments, and laboratory sites, demonstrating the broad applicability of this approach.

The Inherent Asymmetry of DNA Replication.

Science.gov (United States)

Snedeker, Jonathan; Wooten, Matthew; Chen, Xin

2017-10-06

Semiconservative DNA replication has provided an elegant solution to the fundamental problem of how life is able to proliferate in a way that allows cells, organisms, and populations to survive and replicate many times over. Somewhat lost, however, in our admiration for this mechanism is an appreciation for the asymmetries that occur in the process of DNA replication. As we discuss in this review, these asymmetries arise as a consequence of the structure of the DNA molecule and the enzymatic mechanism of DNA synthesis. Increasing evidence suggests that asymmetries in DNA replication are able to play a central role in the processes of adaptation and evolution by shaping the mutagenic landscape of cells. Additionally, in eukaryotes, recent work has demonstrated that the inherent asymmetries in DNA replication may play an important role in the process of chromatin replication. As chromatin plays an essential role in defining cell identity, asymmetries generated during the process of DNA replication may play critical roles in cell fate decisions related to patterning and development.

Replication dynamics of the yeast genome.

Science.gov (United States)

Raghuraman, M K; Winzeler, E A; Collingwood, D; Hunt, S; Wodicka, L; Conway, A; Lockhart, D J; Davis, R W; Brewer, B J; Fangman, W L

2001-10-05

Oligonucleotide microarrays were used to map the detailed topography of chromosome replication in the budding yeast Saccharomyces cerevisiae. The times of replication of thousands of sites across the genome were determined by hybridizing replicated and unreplicated DNAs, isolated at different times in S phase, to the microarrays. Origin activations take place continuously throughout S phase but with most firings near mid-S phase. Rates of replication fork movement vary greatly from region to region in the genome. The two ends of each of the 16 chromosomes are highly correlated in their times of replication. This microarray approach is readily applicable to other organisms, including humans.

LHCb experience with LFC replication

CERN Document Server

Bonifazi, F; Perez, E D; D'Apice, A; dell'Agnello, L; Düllmann, D; Girone, M; Re, G L; Martelli, B; Peco, G; Ricci, P P; Sapunenko, V; Vagnoni, V; Vitlacil, D

2008-01-01

Database replication is a key topic in the framework of the LHC Computing Grid to allow processing of data in a distributed environment. In particular, the LHCb computing model relies on the LHC File Catalog, i.e. a database which stores information about files spread across the GRID, their logical names and the physical locations of all the replicas. The LHCb computing model requires the LFC to be replicated at Tier-1s. The LCG 3D project deals with the database replication issue and provides a replication service based on Oracle Streams technology. This paper describes the deployment of the LHC File Catalog replication to the INFN National Center for Telematics and Informatics (CNAF) and to other LHCb Tier-1 sites. We performed stress tests designed to evaluate any delay in the propagation of the streams and the scalability of the system. The tests show the robustness of the replica implementation with performance going much beyond the LHCb requirements.

LHCb experience with LFC replication

International Nuclear Information System (INIS)

Bonifazi, F; Carbone, A; D'Apice, A; Dell'Agnello, L; Re, G L; Martelli, B; Ricci, P P; Sapunenko, V; Vitlacil, D; Perez, E D; Duellmann, D; Girone, M; Peco, G; Vagnoni, V

2008-01-01

Database replication is a key topic in the framework of the LHC Computing Grid to allow processing of data in a distributed environment. In particular, the LHCb computing model relies on the LHC File Catalog, i.e. a database which stores information about files spread across the GRID, their logical names and the physical locations of all the replicas. The LHCb computing model requires the LFC to be replicated at Tier-1s. The LCG 3D project deals with the database replication issue and provides a replication service based on Oracle Streams technology. This paper describes the deployment of the LHC File Catalog replication to the INFN National Center for Telematics and Informatics (CNAF) and to other LHCb Tier-1 sites. We performed stress tests designed to evaluate any delay in the propagation of the streams and the scalability of the system. The tests show the robustness of the replica implementation with performance going much beyond the LHCb requirements

Determination of 63Ni and 55Fe in nuclear waste samples using radiochemical separation and liquid scintillation counting

DEFF Research Database (Denmark)

Hou, Xiaolin; Frøsig Østergaard, L.; Nielsen, S.P.

2005-01-01

An analytical method for the determination of Ni-63 and Fe-55 in nuclear waste samples such as graphite, heavy concrete, aluminium and lead was developed. Different decomposition methods (i.e. ashing, acid digestion and alkali fusion) were investigated for the decomposition of the samples...... by extraction chromatography. The purified Ni-63 and Fe-55 was then measured by liquid scintillation counting. The chemical yields of the separation procedures for Fe-55 and Ni-63 are above 90% and the decontamination factors for all interfering radionuclides are more than 10(5). The detection limits...

Validation of limited sampling models (LSM) for estimating AUC in therapeutic drug monitoring - is a separate validation group required?

NARCIS (Netherlands)

Proost, J. H.

Objective: Limited sampling models (LSM) for estimating AUC in therapeutic drug monitoring are usually validated in a separate group of patients, according to published guidelines. The aim of this study is to evaluate the validation of LSM by comparing independent validation with cross-validation

A study on the separation and extraction of polycyclic aromatic hydrocarbons in water sample by gas chromatography/mass spectrometry

International Nuclear Information System (INIS)

Lee, Won; Hong, Jee Eun; Park, Song Ja; Pyo, Hee Soo; Kim, In Hwan

1998-01-01

The separation and sample extraction methods of 19 polycyclic aromatic hydrocarbons (PAH S ) in water samples were investigated by gas chromatography/mass spectrometry (GC/MS) and some ex-traction methods involved liquid-liquid extraction, disk extraction and solid-phase extraction methods. The separation of 19 PAH s was possible by partial variation of oven temperature of GC/MS in temperature range 80∼310.deg.C. Extraction procedures of PAH s in water samples were somewhat modified and com-pared as extraction recoveries and the simplicity of methods. Extraction recoveries of PAH s were 71.3∼109.5% by liquid-liquid extraction method. By using disk extraction, good extraction recoveries (80.7∼94.9%) were obtained in case of C 1 8 disk extraction method by filtration. And extraction recoveries of PAH s by C 1 8 solid-phase were in the range of 51.8∼77.9%. Method detection limits (S/N=5) of 19 PAH s were in the range of 0.25∼6.25 ppb by liquid-liquid extraction and solid-phase extraction and 0.05∼1.25 ppb by disk extraction methods

Separation and screening of short-chain chlorinated paraffins in environmental samples using comprehensive two-dimensional gas chromatography with micro electron capture detection.

Science.gov (United States)

Xia, Dan; Gao, Lirong; Zhu, Shuai; Zheng, Minghui

2014-11-01

Short-chain chlorinated paraffins (SCCPs) are highly complex technical mixtures with thousands of isomers and numerous homologs. They are classified as priority candidate persistent organic pollutants under the Stockholm Convention for their persistence, bioaccumulation, and toxicity. Analyzing SCCPs is challenging because of the complexity of the mixtures. Chromatograms of SCCPs acquired using one-dimensional (1D) gas chromatography (GC) contain a large characteristic "peak" with a broad and unresolved profile. Comprehensive two-dimensional GC (GC×GC) shows excellent potential for separating complex mixtures. In this study, GC×GC coupled with micro electron capture detection (μECD) was used to separate and screen SCCPs. The chromatographic parameters, including the GC column types, oven temperature program, and modulation period, were systematically optimized. The SCCP congeners were separated into groups using a DM-1 column connected to a BPX-50 column. The SCCP congeners in technical mixtures were separated according to the number of chlorine substituents for a given carbon chain length and according to the number of carbon atoms plus chlorine atoms for different carbon chain lengths. A fish tissue sample was analyzed to illustrate the feasibility of the GC×GC-μECD method in analyzing biological samples. Over 1,500 compounds were identified in the fish extract, significantly more than were identified using 1D GC. The detection limits for five selected SCCP congeners were between 1 and 5 pg/L using the GC×GC method, and these were significantly lower than those achieved using 1D GC. This method is a good choice for analysis of SCCPs in environmental samples, exhibiting good separation and good sensitivity.

High-Resolution Replication Profiles Define the Stochastic Nature of Genome Replication Initiation and Termination

Directory of Open Access Journals (Sweden)

Michelle Hawkins

2013-11-01

Full Text Available Eukaryotic genome replication is stochastic, and each cell uses a different cohort of replication origins. We demonstrate that interpreting high-resolution Saccharomyces cerevisiae genome replication data with a mathematical model allows quantification of the stochastic nature of genome replication, including the efficiency of each origin and the distribution of termination events. Single-cell measurements support the inferred values for stochastic origin activation time. A strain, in which three origins were inactivated, confirmed that the distribution of termination events is primarily dictated by the stochastic activation time of origins. Cell-to-cell variability in origin activity ensures that termination events are widely distributed across virtually the whole genome. We propose that the heterogeneity in origin usage contributes to genome stability by limiting potentially deleterious events from accumulating at particular loci.

Characterization of the replication timing program of 6 human model cell lines

Directory of Open Access Journals (Sweden)

Djihad Hadjadj

2016-09-01

Full Text Available During the S-phase, the DNA replication process is finely orchestrated and regulated by two programs: the spatial program that determines where replication will start in the genome (Cadoret et al. (2008 Oct 14, Cayrou et al. (2011 Sep, Picard et al. (2014 May 1 [1–3], and the temporal program that determines when during the S phase different parts of the genome are replicated and when origins are activated. The temporal program is so well conserved for each cell type from independent individuals [4] that it is possible to identify a cell type from an unknown sample just by determining its replication timing program. Moreover, replicative domains are strongly correlated with the partition of the genome into topological domains (determined by the Hi-C method, Lieberman-Aiden et al. (2009 Oct 9, Pope et al. (2014 Nov 20 [5,6]. On the one hand, replicative areas are well defined and participate in shaping the spatial organization of the genome for a given cell type. On the other hand, studies on the timing program during cell differentiation showed a certain plasticity of this program according to the stage of cell differentiation Hiratani et al. (2008 Oct 7, 2010 Feb [7,8]. Domains where a replication timing change was observed went through a nuclear re-localization. Thus the temporal program of replication can be considered as an epigenetic mark Hiratani and Gilbert (2009 Feb 16 [9]. We present the genomic data of replication timing in 6 human model cell lines: U2OS (GSM2111308, RKO (GSM2111309, HEK 293T (GSM2111310, HeLa (GSM2111311, MRC5-SV (GSM2111312 and K562 (GSM2111313. A short comparative analysis was performed that allowed us to define regions common to the 6 cell lines. These replication timing data can be taken into account when performing studies that use these model cell lines.

DNA replication and post-replication repair in U.V.-sensitive mouse neuroblastoma cells

International Nuclear Information System (INIS)

Lavin, M.F.; McCombe, P.; Kidson, C.

1976-01-01

Mouse neuroblastoma cells differentiated when grown in the absence of serum; differentiation was reversed on the addition of serum. Differentiated cells were more sensitive to U.V.-radiation than proliferating cells. Whereas addition of serum to differentiated neuroblastoma cells normally resulted in immediate, synchronous entry into S phase, irradiation just before the addition of serum resulted in a long delay in the onset of DNA replication. During this lag period, incorporated 3 H-thymidine appeared in the light density region of CsCl gradients, reflecting either repair synthesis or abortive replication. Post-replication repair (gap-filling) was found to be present in proliferating cells and at certain times in differentiated cells. It is suggested that the sensitivity of differentiated neuroblastoma cells to U.V.-radiation may have been due to ineffective post-replication repair or to deficiencies in more than one repair mechanism, with reduction in repair capacity beyond a critical threshold. (author)

Role of the hydrophilic channels of simian virus 40 T-antigen helicase in DNA replication.

Science.gov (United States)

Wang, Weiping; Manna, David; Simmons, Daniel T

2007-05-01

The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higher-than-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.

DATABASE REPLICATION IN HETEROGENOUS PLATFORM

OpenAIRE

Hendro Nindito; Evaristus Didik Madyatmadja; Albert Verasius Dian Sano

2014-01-01

The application of diverse database technologies in enterprises today is increasingly a common practice. To provide high availability and survavibality of real-time information, a database replication technology that has capability to replicate databases under heterogenous platforms is required. The purpose of this research is to find the technology with such capability. In this research, the data source is stored in MSSQL database server running on Windows. The data will be replicated to MyS...

Comparison of the radiochemical separation procedures od plutonium applied for its determination in the environmental samples using alpha spectrometry

International Nuclear Information System (INIS)

Komosa, A.; Michalik, S.

2006-01-01

Alpha spectrometry of the plutonium isotopes can be performed only after the perfect plutonium separation from other components of the matrix. So, till now numerous procedures have been elaborated and tested. The communication presents comparison of the plutonium content determination in soil, bones, eggshells and in the reference materials obtained by alpha spectrometry combined with two different separation procedures. The samples were mineralized in the concentrated HCl or HF prior to plutonium electrodeposition or coprecipitation with NdF 3 . Some other details were also tested in various variants. Quality of the spectra is discussed in terms of all these pre-treatment methods

Overcoming natural replication barriers: differential helicase requirements.

Science.gov (United States)

Anand, Ranjith P; Shah, Kartik A; Niu, Hengyao; Sung, Patrick; Mirkin, Sergei M; Freudenreich, Catherine H

2012-02-01

DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.

Exploiting replicative stress to treat cancer

DEFF Research Database (Denmark)

Dobbelstein, Matthias; Sørensen, Claus Storgaard

2015-01-01

DNA replication in cancer cells is accompanied by stalling and collapse of the replication fork and signalling in response to DNA damage and/or premature mitosis; these processes are collectively known as 'replicative stress'. Progress is being made to increase our understanding of the mechanisms...

Chromatin maturation depends on continued DNA-replication

International Nuclear Information System (INIS)

Schlaeger, E.J.; Puelm, W.; Knippers, R.

1983-01-01

The structure of [ 3 H]thymidine pulse-labeled chromatin in lymphocytes differs from that of non-replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin. These structural features of replicating chromatin rapidly disappear when the [ 3 H]thymidine pulse is followed by a chase in the presence of an excess of non-radioactive thymidine. However, when the rate of DNA replication is reduced, as in cycloheximide-treated lymphocytes, chromatin maturation is retarded. No chromatin maturation is observed when nuclei from pulse-labeled lymphocytes are incubated in vitro in the absence of DNA precursors. In contrast, when these nuclei are incubated under conditions known to be optimal for DNA replication, the structure of replicating chromatin is efficiently converted to that of 'mature', non-replicating chromatin. The authors conclude that the properties of nascent DNA and/or the distance from the replication fork are important factors in chromatin maturation. (Auth.)

Genome-scale cluster analysis of replicated microarrays using shrinkage correlation coefficient.

Science.gov (United States)

Yao, Jianchao; Chang, Chunqi; Salmi, Mari L; Hung, Yeung Sam; Loraine, Ann; Roux, Stanley J

2008-06-18

Currently, clustering with some form of correlation coefficient as the gene similarity metric has become a popular method for profiling genomic data. The Pearson correlation coefficient and the standard deviation (SD)-weighted correlation coefficient are the two most widely-used correlations as the similarity metrics in clustering microarray data. However, these two correlations are not optimal for analyzing replicated microarray data generated by most laboratories. An effective correlation coefficient is needed to provide statistically sufficient analysis of replicated microarray data. In this study, we describe a novel correlation coefficient, shrinkage correlation coefficient (SCC), that fully exploits the similarity between the replicated microarray experimental samples. The methodology considers both the number of replicates and the variance within each experimental group in clustering expression data, and provides a robust statistical estimation of the error of replicated microarray data. The value of SCC is revealed by its comparison with two other correlation coefficients that are currently the most widely-used (Pearson correlation coefficient and SD-weighted correlation coefficient) using statistical measures on both synthetic expression data as well as real gene expression data from Saccharomyces cerevisiae. Two leading clustering methods, hierarchical and k-means clustering were applied for the comparison. The comparison indicated that using SCC achieves better clustering performance. Applying SCC-based hierarchical clustering to the replicated microarray data obtained from germinating spores of the fern Ceratopteris richardii, we discovered two clusters of genes with shared expression patterns during spore germination. Functional analysis suggested that some of the genetic mechanisms that control germination in such diverse plant lineages as mosses and angiosperms are also conserved among ferns. This study shows that SCC is an alternative to the Pearson

The Escherichia coli Tus-Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

DEFF Research Database (Denmark)

Larsen, Nicolai B; Sass, Ehud; Suski, Catherine

2014-01-01

Replication fork (RF) pausing occurs at both 'programmed' sites and non-physiological barriers (for example, DNA adducts). Programmed RF pausing is required for site-specific DNA replication termination in Escherichia coli, and this process requires the binding of the polar terminator protein, Tus...... as a versatile, site-specific, heterologous DNA replication-perturbing system, with a variety of potential applications....

Separation and enrichment of gold(III) from environmental samples prior to its flame atomic absorption spectrometric determination

International Nuclear Information System (INIS)

Senturk, Hasan Basri; Gundogdu, Ali; Bulut, Volkan Numan; Duran, Celal; Soylak, Mustafa; Elci, Latif; Tufekci, Mehmet

2007-01-01

A simple and accurate method was developed for separation and enrichment of trace levels of gold in environmental samples. The method is based on the adsorption of Au(III)-diethyldithiocarbamate complex on Amberlite XAD-2000 resin prior to the analysis of gold by flame atomic absorption spectrometry after elution with 1 mol L -1 HNO 3 in acetone. Some parameters including nitric acid concentration, eluent type, matrix ions, sample volume, sample flow rate and adsorption capacity were investigated on the recovery of gold(III). The recovery values for gold(III) and detection limit of gold were greater than 95% and 16.6 μg L -1 , respectively. The preconcentration factor was 200. The relative standard deviation of the method was -1 . The validation of the presented procedure was checked by the analysis of CRM-SA-C Sandy Soil certified reference material. The presented procedure was applied to the determination of gold in some environmental samples

Personality and attitude toward Christianity among churchgoers: a replication.

Science.gov (United States)

Williams, Emyr; Francis, Leslie J

2006-08-01

A sample of 158 churchgoers attending eight Anglican churches in the United Kingdom completed the abbreviated Revised Eysenck Personality Questionnaire together with the Francis Scale of Attitude toward Christianity to replicate a 1996 study by Carter, Kay, and Francis. Data confirm that scores on Attitude toward Christianity were significantly negatively related to Psychoticism, but to neither Extraversion nor Neuroticism scores.

Roles of three amino acids of capsid proteins in mink enteritis parvovirus replication.

Science.gov (United States)

Mao, Yaping; Su, Jun; Wang, Jigui; Zhang, Xiaomei; Hou, Qiang; Bian, Dawei; Liu, Weiquan

2016-08-15

Virulent mink enteritis parvovirus (MEV) strain MEV-LHV replicated to higher titers in feline F81 cells than attenuated strain MEV-L. Phylogenetic and sequence analyses of the VP2 gene of MEV-LHV, MEV-L and other strains in GenBank revealed two evolutionary branches separating virulent and attenuated strains. Three residues, 101, 232 and 411, differed between virulent and attenuated strains but were conserved within the two branches. Site-directed mutagenesis of the VP2 gene of infectious plasmids of attenuated strain MEV-L respectively replacing residues 101 Ile and 411 Ala with Thr and Glu of virulent strains (MEV-L I101T and MEV-L A411E) increased replication efficiency but still to lower levels than MEV-LHV. However, viruses with mutation of residue 232 (MEV-L I232V and MEV-L I101T/I232V/A411E) decreased viral transcription and replication levels. The three VP2 residues 101, 232 and 411, located on or near the capsid surface, played different roles in the infection processes of MEV. Copyright © 2016 Elsevier B.V. All rights reserved.

Treatment with a non-toxic, self-replicating anti-prion delays or prevents prion disease in vivo.

Science.gov (United States)

Diaz-Espinoza, R; Morales, R; Concha-Marambio, L; Moreno-Gonzalez, I; Moda, F; Soto, C

2018-03-01

Transmissible spongiform encephalopathies (TSEs) are fatal neurological disorders caused by prions, which are composed of a misfolded protein (PrP Sc ) that self-propagates in the brain of infected individuals by converting the normal prion protein (PrP C ) into the pathological isoform. Here, we report a novel experimental strategy for preventing prion disease based on producing a self-replicating, but innocuous PrP Sc -like form, termed anti-prion, which can compete with the replication of pathogenic prions. Our results show that a prophylactic inoculation of prion-infected animals with an anti-prion delays the onset of the disease and in some animals completely prevents the development of clinical symptoms and brain damage. The data indicate that a single injection of the anti-prion eliminated ~99% of the infectivity associated to pathogenic prions. Furthermore, this treatment caused significant changes in the profile of regional PrP Sc deposition in the brains of animals that were treated, but still succumbed to the disease. Our findings provide new insights for a mechanistic understanding of prion replication and support the concept that prion replication can be separated from toxicity, providing a novel target for therapeutic intervention.

Enzymatic recognition of DNA replication origins

International Nuclear Information System (INIS)

Stayton, M.M.; Bertsch, L.; Biswas, S.

1983-01-01

In this paper we discuss the process of recognition of the complementary-strand origin with emphasis on RNA polymerase action in priming M13 DNA replication, the role of primase in G4 DNA replication, and the function of protein n, a priming protein, during primosome assembly. These phage systems do not require several of the bacterial DNA replication enzymes, particularly those involved in the regulation of chromosome copy number of the initiatiion of replication of duplex DNA. 51 references, 13 figures, 1 table

Surface Microstructure Replication in Injection Moulding

DEFF Research Database (Denmark)

Hansen, Hans Nørgaard; Arlø, Uffe Rolf

2005-01-01

topography is transcribed onto the plastic part through complex mechanisms. This replication however, is not perfect, and the replication quality depends on the plastic material properties, the topography itself, and the process conditions. This paper describes and discusses an investigation of injection...... moulding of surface microstructures. Emphasis is put on the ability to replicate surface microstructures under normal injection moulding conditions, notably with low cost materials at low mould temperatures. The replication of surface microstructures in injection moulding has been explored...... for Polypropylene at low mould temperatures. The process conditions were varied over the recommended process window for the material. The geometry of the obtained structures was analyzed. Evidence suggests that step height replication quality depends linearly on structure width in a certain range. Further...

Inconsistent Effects of Parietal α-tACS on Pseudoneglect across Two Experiments: A Failed Internal Replication

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Domenica Veniero

2017-06-01

Full Text Available Transcranial electrical stimulation (tES is being investigated as an experimental and clinical interventional technique in human participants. While promising, important limitations have been identified, including weak effect sizes and high inter- and intra-individual variability of outcomes. Here, we compared two “inhibitory” tES-techniques with supposedly different mechanisms of action as to their effects on performance in a visuospatial attention task, and report on a direct replication attempt. In two experiments, 2 × 20 healthy participants underwent tES in three separate sessions testing different protocols (10 min stimulation each with a montage targeting right parietal cortex (right parietal–left frontal, electrode-sizes: 3cm × 3cm–7 cm × 5 cm, while performing a perceptual line bisection (landmark task. The tES-protocols were compared as to their ability to modulate pseudoneglect (thought to be under right hemispheric control. In experiment 1, sham-tES was compared to transcranial alternating current stimulation at alpha frequency (10 Hz; α-tACS (expected to entrain “inhibitory” alpha oscillations and to cathodal transcranial direct current stimulation (c-tDCS (shown to suppress neuronal spiking activity. In experiment 2, we attempted to replicate the findings of experiment 1, and establish frequency-specificity by adding a 45 Hz-tACS condition to α-tACS and sham. In experiment 1, right parietal α-tACS led to the expected changes in spatial attention bias, namely a rightward shift in subjective midpoint estimation (relative to sham. However, this was not confirmed in experiment 2 and in the complete sample. Right parietal c-tDCS and 45 Hz-tACS had no effect. These results highlight the importance of replication studies, adequate statistical power and optimizing tES-interventions for establishing the robustness and reliability of electrical stimulation effects, and best practice.

Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

OpenAIRE

Vassin, Vitaly M.; Wold, Marc S.; Borowiec, James A.

2004-01-01

Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2D) or alanine (RPA2A). Although RPA2D was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2D mutant was selectively unable to associate with re...

Speeding Up Non-Parametric Bootstrap Computations for Statistics Based on Sample Moments in Small/Moderate Sample Size Applications.

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Elias Chaibub Neto

Full Text Available In this paper we propose a vectorized implementation of the non-parametric bootstrap for statistics based on sample moments. Basically, we adopt the multinomial sampling formulation of the non-parametric bootstrap, and compute bootstrap replications of sample moment statistics by simply weighting the observed data according to multinomial counts instead of evaluating the statistic on a resampled version of the observed data. Using this formulation we can generate a matrix of bootstrap weights and compute the entire vector of bootstrap replications with a few matrix multiplications. Vectorization is particularly important for matrix-oriented programming languages such as R, where matrix/vector calculations tend to be faster than scalar operations implemented in a loop. We illustrate the application of the vectorized implementation in real and simulated data sets, when bootstrapping Pearson's sample correlation coefficient, and compared its performance against two state-of-the-art R implementations of the non-parametric bootstrap, as well as a straightforward one based on a for loop. Our investigations spanned varying sample sizes and number of bootstrap replications. The vectorized bootstrap compared favorably against the state-of-the-art implementations in all cases tested, and was remarkably/considerably faster for small/moderate sample sizes. The same results were observed in the comparison with the straightforward implementation, except for large sample sizes, where the vectorized bootstrap was slightly slower than the straightforward implementation due to increased time expenditures in the generation of weight matrices via multinomial sampling.

Variance Swap Replication: Discrete or Continuous?

Directory of Open Access Journals (Sweden)

Fabien Le Floc’h

2018-02-01

Full Text Available The popular replication formula to price variance swaps assumes continuity of traded option strikes. In practice, however, there is only a discrete set of option strikes traded on the market. We present here different discrete replication strategies and explain why the continuous replication price is more relevant.

Dpb11/TopBP1 plays distinct roles in DNA replication, checkpoint response and homologous recombination

DEFF Research Database (Denmark)

Germann, Susanne Manuela; Østergaard, Vibe Hallundbæk; Haas, Caroline

2011-01-01

DPB11/TopBP1 is an essential evolutionarily conserved gene involved in initiation of DNA replication and checkpoint signaling. Here, we show that Saccharomyces cerevisiae Dpb11 forms nuclear foci that localize to sites of DNA damage in G1, S and G2 phase, a recruitment that is conserved for its...... and Tel1, and of the checkpoint mediator Rad9. In a site-directed mutagenesis screen, we identify a separation-of-function mutant, dpb11-PF, that is sensitive to DSB-inducing agents yet remains proficient for DNA replication and the S-phase checkpoint at the permissive temperature. The dpb11-PF mutant...... homologue TopBP1 in Gallus gallus. Damage-induced Dpb11 foci are distinct from Sld3 replication initiation foci. Further, Dpb11 foci are dependent on the checkpoint proteins Mec3 (9-1-1 complex) and Rad24, and require the C-terminal domain of Dpb11. Dpb11 foci are independent of the checkpoint kinases Mec1...

Cryogenic separation of an oxygen-argon mixture in natural air samples for the determination of isotope and molecular ratios.

Science.gov (United States)

Keedakkadan, Habeeb Rahman; Abe, Osamu

2015-04-30

The separation and purification of oxygen-argon mixtures are critical in the high-precision analysis of Δ(17) O and δ(O2 /Ar) for geochemical applications. At present, chromatographic methods are used for the separation and purification of oxygen-argon mixtures or pure oxygen, but these methods require the use of high-purity helium as a carrier gas. Considerable interest has been expressed in the development of a helium-free cryogenic separation of oxygen-argon mixtures in natural air samples. The precise and simplified cryogenic separation of oxygen-argon mixtures from natural air samples presented here was made possible using a single 5A (30/60 mesh) molecular sieve column. The method involves the trapping of eluted gases using molecular sieves at liquid nitrogen temperature, which is associated with isotopic fractionation. We tested the proposed method for the determination of isotopic fractionations during the gas exchange between water and atmospheric air at equilibrium. The dependency of fractionation was studied at different water temperatures and for different methods of equilibration (bubbling and stirring). Isotopic and molecular fractionations during gas desorption from molecular sieves were studied for different amounts and types of molecular sieves. Repeated measurements of atmospheric air yielded a reproducibility (±SD) of 0.021 ‰, 0.044 ‰, 15 per meg and 1.9 ‰ for δ(17) O, δ(18) O, Δ(17) O and δ(O2 /Ar) values, respectively. We applied the method to determine equilibrium isotope fractionation during gas exchange between air and water. Consistent δ(18) O and Δ(17) O results were obtained with the latest two studies, whereas there was a significant difference in δ(18) O values between seawater and deionized water. We have revised a helium-free, cryogenic separation of oxygen-argon mixtures in natural air samples for isotopic and molecular ratio analysis. The use of a single 13X (1/8" pellet) molecular sieve yielded the smallest isotopic

On the cross-cultural replicability of the resilient, undercontrolled, and overcontrolled personality types.

Science.gov (United States)

Alessandri, Guido; Vecchione, Michele; Donnellan, Brent M; Eisenberg, Nancy; Caprara, Gian Vittorio; Cieciuch, Jan

2014-08-01

Personality types reflect typical configurations of personality attributes within individuals. Over the last 20 years, researchers have identified a set of three replicable personality types: resilient (R), undercontrolled (U), and overcontrolled (O) types. In this study, we examined the cross-cultural replicability of the RUO types in Italy, Poland, Spain, and the United States. Personality types were identified using cluster analyses of Big Five profiles in large samples of college students from Italy (n = 322), the United States (n = 499), Spain (n = 420), and Poland (n = 235). Prior to clustering the profiles, the measurement invariance of the Big Five measure across samples was tested. We found evidence for the RUO types in all four samples. The three-cluster solution showed a better fit over alternative solutions and had a relatively high degree of cross-cultural generalizability. The RUO types are evident in samples from four countries with distinct linguistic and cultural traditions. Results were discussed in light of the importance of considering how traits are organized within individuals for advancing contemporary personality psychology. © 2013 Wiley Periodicals, Inc.

Grey Matter Abnormalities in Social Anxiety Disorder: Primary, Replication, and Specificity Studies

Science.gov (United States)

Talati, Ardesheer; Pantazatos, Spiro P.; Schneier, Franklin R.; Weissman, Myrna M; Hirsch, Joy

2012-01-01

Background Despite increasing evidence that neuroanatomical abnormalities underlie pathological anxiety, social anxiety disorder (SAD), although among the most common of anxiety disorders, has received little attention. Using Magnetic Resonance Imaging, we (1) examined grey matter (GM) differences between generalized SAD and healthy control groups; (2) retested the findings in an independent clinical sample; and (3) tested for specificity by contrasting the SAD group to a separate group of panic disorder (PD) subjects. Methods The primary SAD group (N=16) was required to meet DSM-IV criteria for SAD, with onset by age 30; controls (N=20) had no lifetime history of anxiety. The replication sample included 17 generalized SAD and 17 control subjects. The PD comparison group (N=16) was required to have no lifetime SAD. Images were acquired on a 1.5Tesla GE Signa MRI scanner using a 3D T1-weighted spoiled gradient recalled pulse sequence. Morphological differences were determined using voxel based morphometry, in SPM8. Results After adjusting for age, gender, and total intracranial volume, SAD (as compared to control) subjects had greater GM in the left parahippocampal and middle occipital, and bilateral supramarginal and angular cortices, and left cerebellum; and lower GM in bilateral temporal poles and left lateral orbitofrontal cortex. Cerebellar, parahippocampal, and temporal pole differences were observed in both samples, survived whole brain corrections, and were not observed in the PD group, pointing to relative specificity to SAD. Conclusions These findings parallel the functional literature on SAD, and suggest structural abnormalities underlying the functional disturbances. PMID:22748614

Chemical process to separate iron oxides particles in pottery sample for EPR dating

Science.gov (United States)

Watanabe, S.; Farias, T. M. B.; Gennari, R. F.; Ferraz, G. M.; Kunzli, R.; Chubaci, J. F. D.

2008-12-01

Ancient potteries usually are made of the local clay material, which contains relatively high concentration of iron. The powdered samples are usually quite black, due to magnetite, and, although they can be used for thermoluminescene (TL) dating, it is easiest to obtain better TL reading when clearest natural or pre-treated sample is used. For electron paramagnetic resonance (EPR) measurements, the huge signal due to iron spin-spin interaction, promotes an intense interference overlapping any other signal in this range. Sample dating is obtained by dividing the radiation dose, determined by the concentration of paramagnetic species generated by irradiation, by the natural dose so as a consequence, EPR dating cannot be used, since iron signal do not depend on radiation dose. In some cases, the density separation method using hydrated solution of sodium polytungstate [Na 6(H 2W 12O 40)·H 2O] becomes useful. However, the sodium polytungstate is very expensive in Brazil; hence an alternative method for eliminating this interference is proposed. A chemical process to eliminate about 90% of magnetite was developed. A sample of powdered ancient pottery was treated in a mixture (3:1:1) of HCl, HNO 3 and H 2O 2 for 4 h. After that, it was washed several times in distilled water to remove all acid matrixes. The original black sample becomes somewhat clearer. The resulting material was analyzed by plasma mass spectrometry (ICP-MS), with the result that the iron content is reduced by a factor of about 9. In EPR measurements a non-treated natural ceramic sample shows a broad spin-spin interaction signal, the chemically treated sample presents a narrow signal in g = 2.00 region, possibly due to a radical of (SiO 3) 3-, mixed with signal of remaining iron [M. Ikeya, New Applications of Electron Spin Resonance, World Scientific, Singapore, 1993, p. 285]. This signal increases in intensity under γ-irradiation. However, still due to iron influence, the additive method yielded too

Surface microstructure replication in injection molding

DEFF Research Database (Denmark)

Theilade, Uffe Arlø; Hansen, Hans Nørgaard

2006-01-01

topography is transcribed onto the plastic part through complex mechanisms. This replication, however, is not perfect, and the replication quality depends on the plastic material properties, the topography itself, and the process conditions. This paper describes and discusses an investigation of injection...... molding of surface microstructures. The fundamental problem of surface microstructure replication has been studied. The research is based on specific microstructures as found in lab-on-a-chip products and on rough surfaces generated from EDM (electro discharge machining) mold cavities. Emphasis is put...... on the ability to replicate surface microstructures under normal injection-molding conditions, i.e., with commodity materials within typical process windows. It was found that within typical process windows the replication quality depends significantly on several process parameters, and especially the mold...

Activation of human herpesvirus replication by apoptosis.

Science.gov (United States)

Prasad, Alka; Remick, Jill; Zeichner, Steven L

2013-10-01

A central feature of herpesvirus biology is the ability of herpesviruses to remain latent within host cells. Classically, exposure to inducing agents, like activating cytokines or phorbol esters that stimulate host cell signal transduction events, and epigenetic agents (e.g., butyrate) was thought to end latency. We recently showed that Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus-8 [HHV-8]) has another, alternative emergency escape replication pathway that is triggered when KSHV's host cell undergoes apoptosis, characterized by the lack of a requirement for the replication and transcription activator (RTA) protein, accelerated late gene kinetics, and production of virus with decreased infectivity. Caspase-3 is necessary and sufficient to initiate the alternative replication program. HSV-1 was also recently shown to initiate replication in response to host cell apoptosis. These observations suggested that an alternative apoptosis-triggered replication program might be a general feature of herpesvirus biology and that apoptosis-initiated herpesvirus replication may have clinical implications, particularly for herpesviruses that almost universally infect humans. To explore whether an alternative apoptosis-initiated replication program is a common feature of herpesvirus biology, we studied cell lines latently infected with Epstein-Barr virus/HHV-4, HHV-6A, HHV-6B, HHV-7, and KSHV. We found that apoptosis triggers replication for each HHV studied, with caspase-3 being necessary and sufficient for HHV replication. An alternative apoptosis-initiated replication program appears to be a common feature of HHV biology. We also found that commonly used cytotoxic chemotherapeutic agents activate HHV replication, which suggests that treatments that promote apoptosis may lead to activation of latent herpesviruses, with potential clinical significance.

Enhanced replication of herpes simplex virus type 1 in human cells

International Nuclear Information System (INIS)

Miller, C.S.; Smith, K.O.

1991-01-01

The effects of DNA-damaging agents on the replication of herpes simplex virus type 1 (HSV-1) were assessed in vitro. Monolayers of human lung fibroblast cell lines were exposed to DNA-damaging agents (methyl methanesulfonate [MMS], methyl methanethiosulfonate [MMTS], ultraviolet light [UV], or gamma radiation [GR]) at specific intervals, before or after inoculation with low levels of HSV-1. The ability of cell monolayers to support HSV-1 replication was measured by direct plaque assay and was compared with that of untreated control samples. In this system, monolayers of different cell lines infected with identical HSV-1 strains demonstrated dissimilar levels of recovery of the infectious virus. Exposure of DNA-repair-competent cell cultures to DNA-damaging agents produced time-dependent enhanced virus replication. Treatment with agent before virus inoculation significantly (p less than 0.025) increased the number of plaques by 10 to 68%, compared with untreated control cultures, while treatment with agent after virus adsorption significantly increased (p less than 0.025) the number of plaques by 7 to 15%. In a parallel series of experiments, cells deficient in DNA repair (xeroderma pigmentosum) failed to support enhanced virus replication. These results suggest that after exposure to DNA-damaging agents, fibroblasts competent in DNA repair amplify the replication of HSV-1, and that DNA-repair mechanisms that act on a variety of chromosomal lesions may be involved in the repair and biological activation of HSV-1 genomes

Evolution of Replication Machines

Science.gov (United States)

Yao, Nina Y.; O'Donnell, Mike E.

2016-01-01

The machines that decode and regulate genetic information require the translation, transcription and replication pathways essential to all living cells. Thus, it might be expected that all cells share the same basic machinery for these pathways that were inherited from the primordial ancestor cell from which they evolved. A clear example of this is found in the translation machinery that converts RNA sequence to protein. The translation process requires numerous structural and catalytic RNAs and proteins, the central factors of which are homologous in all three domains of life, bacteria, archaea and eukarya. Likewise, the central actor in transcription, RNA polymerase, shows homology among the catalytic subunits in bacteria, archaea and eukarya. In contrast, while some “gears” of the genome replication machinery are homologous in all domains of life, most components of the replication machine appear to be unrelated between bacteria and those of archaea and eukarya. This review will compare and contrast the central proteins of the “replisome” machines that duplicate DNA in bacteria, archaea and eukarya, with an eye to understanding the issues surrounding the evolution of the DNA replication apparatus. PMID:27160337

DNA replication origins—where do we begin?

Science.gov (United States)

Prioleau, Marie-Noëlle; MacAlpine, David M.

2016-01-01

For more than three decades, investigators have sought to identify the precise locations where DNA replication initiates in mammalian genomes. The development of molecular and biochemical approaches to identify start sites of DNA replication (origins) based on the presence of defining and characteristic replication intermediates at specific loci led to the identification of only a handful of mammalian replication origins. The limited number of identified origins prevented a comprehensive and exhaustive search for conserved genomic features that were capable of specifying origins of DNA replication. More recently, the adaptation of origin-mapping assays to genome-wide approaches has led to the identification of tens of thousands of replication origins throughout mammalian genomes, providing an unprecedented opportunity to identify both genetic and epigenetic features that define and regulate their distribution and utilization. Here we summarize recent advances in our understanding of how primary sequence, chromatin environment, and nuclear architecture contribute to the dynamic selection and activation of replication origins across diverse cell types and developmental stages. PMID:27542827

Determination of Ultralow Level 129I/127I in Natural Samples by Separation of Microgram Carrier Free Iodine and Accelerator Mass Spectrometry Detection

DEFF Research Database (Denmark)

Hou, Xiaolin; Zhou, Weijian; Chen, Ning

2010-01-01

of 129I/127I, and a detection limit of this method for 129I is calculated to be 105 atoms. This will allow us to accurately determine 129I in prenuclear geological samples of low iodine concentration with 129I/127I of 10−12, such as loess, soil, coral, rock, sediment, and groundwater. Some samples......Separation of carrier free iodine from low iodine level samples and accurate measurement of ultralow level 129I in micrograms of iodine target are essential but a bottleneck in geological dating of terrestrial system and tracer research using naturally produced 129I. In this work, we present...... a carrier free method using coprecipitation of AgI with AgCl for preparing micrograms of iodine target, associated with combustion using a tube furnace for separating iodine from solid samples and anion exchange chromatography for preconcentrating iodine from a large volume of water. An accelerator mass...

A novel technique with enhanced detection and quantitation of HPV-16 E1- and E2-mediated DNA replication

International Nuclear Information System (INIS)

Taylor, Ewan R.; Morgan, Iain M.

2003-01-01

Transient DNA replication assays to detect papillomavirus E1/E2-mediated DNA replication have depended upon Southern blotting. This technique is hazardous (radioactive), labour intensive, semiquantitative, and physically limited in the number of samples that can be processed at any one time. We have overcome these problems by developing a real-time PCR protocol for the detection of E1/E2-mediated transient DNA replication. The results demonstrate detection of replication at levels not seen using Southern blotting demonstrating enhanced sensitivity. This technique is also, by definition, highly quantitative. Therefore, the real-time PCR technique is the optimal method for the detection of E1/E2-mediated DNA replication

Separation of Platinum from Palladium and Iridium in Iron Meteorites and Accurate High-Precision Determination of Platinum Isotopes by Multi-Collector ICP-MS.

Science.gov (United States)

Hunt, Alison C; Ek, Mattias; Schönbächler, Maria

2017-12-01

This study presents a new measurement procedure for the isolation of Pt from iron meteorite samples. The method also allows for the separation of Pd from the same sample aliquot. The separation entails a two-stage anion-exchange procedure. In the first stage, Pt and Pd are separated from each other and from major matrix constituents including Fe and Ni. In the second stage, Ir is reduced with ascorbic acid and eluted from the column before Pt collection. Platinum yields for the total procedure were typically 50-70%. After purification, high-precision Pt isotope determinations were performed by multi-collector ICP-MS. The precision of the new method was assessed using the IIAB iron meteorite North Chile. Replicate analyses of multiple digestions of this material yielded an intermediate precision for the measurement results of 0.73 for ε 192 Pt, 0.15 for ε 194 Pt and 0.09 for ε 196 Pt (2 standard deviations). The NIST SRM 3140 Pt solution reference material was passed through the measurement procedure and yielded an isotopic composition that is identical to the unprocessed Pt reference material. This indicates that the new technique is unbiased within the limit of the estimated uncertainties. Data for three iron meteorites support that Pt isotope variations in these samples are due to exposure to galactic cosmic rays in space.

Chromosomal DNA replication of Vicia faba cells

International Nuclear Information System (INIS)

Ikushima, Takaji

1976-01-01

The chromosomal DNA replication of higher plant cells has been investigated by DNA fiber autoradiography. The nuclear DNA fibers of Vicia root meristematic cells are organized into many tandem arrays of replication units or replicons which exist as clusters with respect to replication. DNA is replicated bidirectionally from the initiation points at the average rate of 0.15 μm/min at 20 0 C, and the average interinitiation interval is about 16 μm. The manner of chromosomal DNA replication in this higher plant is similar to that found in other eukaryotic cells at a subchromosomal level. (auth.)

Surface micro topography replication in injection moulding

DEFF Research Database (Denmark)

Arlø, Uffe Rolf

Thermoplastic injection moulding is a widely used industrial process that involves surface generation by replication. The surface topography of injection moulded plastic parts can be important for aesthetical or technical reasons. With the emergence of microengineering and nanotechnology additional...... importance of surface topography follows. In general the replication is not perfect and the topography of the plastic part differs from the inverse topography of the mould cavity. It is desirable to be able to control the degree of replication perfection or replication quality. This requires an understanding...... of the physical mechanisms of replication. Such understanding can lead to improved process design and facilitate in-line process quality control with respect to surface properties. The purpose of the project is to identify critical factors that affect topography replication quality and to obtain an understanding...

DNA replication in ultraviolet light irradiated Chinese hamster cells: the nature of replicon inhibition and post-replication repair

International Nuclear Information System (INIS)

Doniger, J.

1978-01-01

DNA replication in ultraviolet light irradiated Chinese hamster cells was studied using techniques of DNA fiber autoradiography and alkaline sucrose sedimentation. Bidirectionally growing replicons were observed in the autoradiograms independent of the irradiation conditions. After a dose of 5 J/m 2 at 254 nm the rate of fork progression was the same as in unirradiated cells, while the rate of replication was reduced by 50%. After a dose of 10J/m 2 the rate of fork progression was reduced 40%, while the replication rate was only 25% of normal. Therefore, at low doses of ultraviolet light irradiation, the inhibition of DNA replication is due to reduction in the number of functioning replicons, while at higher doses the rate of fork progression is also slowed. Those replicons which no longer function after irradiation are blocked in fork movement rather than replicon initiation. After irradiation, pulse label was first incorporated into short nascent strands, the average size of which was approximately equal to the distance between pyrimidine dimers. Under conditions where post-replication repair occurs these short strands were eventually joined into larger pieces. Finally, the data show that slowing post-replication repair with caffeine does not slow fork movement. The results presented here support the post-replication repair model of 'gapped synthesis' and rule out a major role for 'replicative bypass'. (author)

Separation and enrichment of gold(III) from environmental samples prior to its flame atomic absorption spectrometric determination

Energy Technology Data Exchange (ETDEWEB)

Senturk, Hasan Basri; Gundogdu, Ali [Department of Chemistry, Faculty of Arts and Sciences, Karadeniz Technical University, 61080 Trabzon (Turkey); Bulut, Volkan Numan [Department of Chemistry, Faculty of Arts and Sciences, Karadeniz Technical University, 28049 Giresun (Turkey); Duran, Celal [Department of Chemistry, Faculty of Arts and Sciences, Karadeniz Technical University, 61080 Trabzon (Turkey); Soylak, Mustafa [Department of Chemistry, Faculty of Arts and Sciences, Erciyes University, 38039 Kayseri (Turkey)], E-mail: soylak@erciyes.edu.tr; Elci, Latif [Department of Chemistry, Faculty of Arts and Sciences, Pamukkale University, 20020 Denizli (Turkey); Tufekci, Mehmet [Department of Chemistry, Faculty of Arts and Sciences, Karadeniz Technical University, 61080 Trabzon (Turkey)

2007-10-22

A simple and accurate method was developed for separation and enrichment of trace levels of gold in environmental samples. The method is based on the adsorption of Au(III)-diethyldithiocarbamate complex on Amberlite XAD-2000 resin prior to the analysis of gold by flame atomic absorption spectrometry after elution with 1 mol L{sup -1} HNO{sub 3} in acetone. Some parameters including nitric acid concentration, eluent type, matrix ions, sample volume, sample flow rate and adsorption capacity were investigated on the recovery of gold(III). The recovery values for gold(III) and detection limit of gold were greater than 95% and 16.6 {mu}g L{sup -1}, respectively. The preconcentration factor was 200. The relative standard deviation of the method was <6%. The adsorption capacity of the resin was 12.3 mg g{sup -1}. The validation of the presented procedure was checked by the analysis of CRM-SA-C Sandy Soil certified reference material. The presented procedure was applied to the determination of gold in some environmental samples.

Nonequilibrium Entropic Bounds for Darwinian Replicators

Directory of Open Access Journals (Sweden)

Jordi Piñero

2018-01-01

Full Text Available Life evolved on our planet by means of a combination of Darwinian selection and innovations leading to higher levels of complexity. The emergence and selection of replicating entities is a central problem in prebiotic evolution. Theoretical models have shown how populations of different types of replicating entities exclude or coexist with other classes of replicators. Models are typically kinetic, based on standard replicator equations. On the other hand, the presence of thermodynamical constraints for these systems remain an open question. This is largely due to the lack of a general theory of statistical methods for systems far from equilibrium. Nonetheless, a first approach to this problem has been put forward in a series of novel developements falling under the rubric of the extended second law of thermodynamics. The work presented here is twofold: firstly, we review this theoretical framework and provide a brief description of the three fundamental replicator types in prebiotic evolution: parabolic, malthusian and hyperbolic. Secondly, we employ these previously mentioned techinques to explore how replicators are constrained by thermodynamics. Finally, we comment and discuss where further research should be focused on.

Automated radioanalytical system incorporating microwave-assisted sample preparation, chemical separation, and online radiometric detection for the monitoring of total 99Tc in nuclear waste processing streams.

Science.gov (United States)

Egorov, Oleg B; O'Hara, Matthew J; Grate, Jay W

2012-04-03

An automated fluidic instrument is described that rapidly determines the total (99)Tc content of aged nuclear waste samples, where the matrix is chemically and radiologically complex and the existing speciation of the (99)Tc is variable. The monitor links microwave-assisted sample preparation with an automated anion exchange column separation and detection using a flow-through solid scintillator detector. The sample preparation steps acidify the sample, decompose organics, and convert all Tc species to the pertechnetate anion. The column-based anion exchange procedure separates the pertechnetate from the complex sample matrix, so that radiometric detection can provide accurate measurement of (99)Tc. We developed a preprogrammed spike addition procedure to automatically determine matrix-matched calibration. The overall measurement efficiency that is determined simultaneously provides a self-diagnostic parameter for the radiochemical separation and overall instrument function. Continuous, automated operation was demonstrated over the course of 54 h, which resulted in the analysis of 215 samples plus 54 hly spike-addition samples, with consistent overall measurement efficiency for the operation of the monitor. A sample can be processed and measured automatically in just 12.5 min with a detection limit of 23.5 Bq/mL of (99)Tc in low activity waste (0.495 mL sample volume), with better than 10% RSD precision at concentrations above the quantification limit. This rapid automated analysis method was developed to support nuclear waste processing operations planned for the Hanford nuclear site.

Rescue from replication stress during mitosis.

Science.gov (United States)

Fragkos, Michalis; Naim, Valeria

2017-04-03

Genomic instability is a hallmark of cancer and a common feature of human disorders, characterized by growth defects, neurodegeneration, cancer predisposition, and aging. Recent evidence has shown that DNA replication stress is a major driver of genomic instability and tumorigenesis. Cells can undergo mitosis with under-replicated DNA or unresolved DNA structures, and specific pathways are dedicated to resolving these structures during mitosis, suggesting that mitotic rescue from replication stress (MRRS) is a key process influencing genome stability and cellular homeostasis. Deregulation of MRRS following oncogene activation or loss-of-function of caretaker genes may be the cause of chromosomal aberrations that promote cancer initiation and progression. In this review, we discuss the causes and consequences of replication stress, focusing on its persistence in mitosis as well as the mechanisms and factors involved in its resolution, and the potential impact of incomplete replication or aberrant MRRS on tumorigenesis, aging and disease.

Personality and Academic Motivation: Replication, Extension, and Replication

Science.gov (United States)

Jones, Martin H.; McMichael, Stephanie N.

2015-01-01

Previous work examines the relationships between personality traits and intrinsic/extrinsic motivation. We replicate and extend previous work to examine how personality may relate to achievement goals, efficacious beliefs, and mindset about intelligence. Approximately 200 undergraduates responded to the survey with a 150 participants replicating…

Adoption of the B2SAFE EUDAT replication service by the EPOS community

Science.gov (United States)

Cacciari, Claudio; Fares, Massimo; Fiameni, Giuseppe; Michelini, Alberto; Danecek, Peter; Wittenburg, Peter

2014-05-01

B2SAFE is the EUDAT service for moving and replicating data between sites and storage systems for different purposes. The goal of B2SAFE is to keep the data from a repository safe by replicating it across different geographical and administrative zones according to a set of well-defined policies. It is also a way to store large volumes of data permanently at those sites which are providing powerful on-demand data analysis facilities. In particular, B2SAFE operates on the domain of registered data where data objects are referable via persistent identifiers (PIDs). B2SAFE is more than just copying data because the PIDs must be carefully managed when data objects are moved or replicated. The EUDAT B2SAFE Service offers functionality to replicate datasets across different data centres in a safe and efficient way while maintaining all information required to easily find and query information about the replica locations. The information about the replica locations and other important information is stored in PID records, each managed in separate administrative domains. The B2SAFE Service is implemented as an iRODS module providing a set of iRODS rules or policies to interface with the EPIC handle API and uses the iRODS middleware to replicate datasets from a source data (or community) centre to a destination data centre. The definition of the dataset(s) to replicate is flexible and up to the communities using the B2SAFE service. While the B2SAFE is internally using the EPIC handle API, communities have the choice to use any PID system they prefer to assign PIDs to their digital objects. A reference to one or more EUDAT B2SAFE PIDs is returned by the B2SAFE service when a dataset is replicated. The presentation will introduce the problem space of B2SAFE, presents the achievements that have been made during the last year for enabling communities to make use of the B2SAFE service, demonstrates a EPOS use cases, outlines the commonalities and differences between the policies

DNA replication origins-where do we begin?

Science.gov (United States)

Prioleau, Marie-Noëlle; MacAlpine, David M

2016-08-01

For more than three decades, investigators have sought to identify the precise locations where DNA replication initiates in mammalian genomes. The development of molecular and biochemical approaches to identify start sites of DNA replication (origins) based on the presence of defining and characteristic replication intermediates at specific loci led to the identification of only a handful of mammalian replication origins. The limited number of identified origins prevented a comprehensive and exhaustive search for conserved genomic features that were capable of specifying origins of DNA replication. More recently, the adaptation of origin-mapping assays to genome-wide approaches has led to the identification of tens of thousands of replication origins throughout mammalian genomes, providing an unprecedented opportunity to identify both genetic and epigenetic features that define and regulate their distribution and utilization. Here we summarize recent advances in our understanding of how primary sequence, chromatin environment, and nuclear architecture contribute to the dynamic selection and activation of replication origins across diverse cell types and developmental stages. © 2016 Prioleau and MacAlpine; Published by Cold Spring Harbor Laboratory Press.

Pattern replication by confined dewetting

NARCIS (Netherlands)

Harkema, S.; Schäffer, E.; Morariu, M.D.; Steiner, U

2003-01-01

The dewetting of a polymer film in a confined geometry was employed in a pattern-replication process. The instability of dewetting films is pinned by a structured confining surface, thereby replicating its topographic pattern. Depending on the surface energy of the confining surface, two different

Selective separation of iron from uranium in quantitative determination of traces of uranium by alpha spectrometry in soil/sediment sample.

Science.gov (United States)

Singhal, R K; Narayanan, Usha; Karpe, Rupali; Kumar, Ajay; Ranade, A; Ramachandran, V

2009-04-01

During this work, controlled redox potential methodology was adopted for the complete separation of traces of uranium from the host matrix of mixed hydroxide of Iron. Precipitates of Fe(+2) and Fe(+3) along with other transuranic elements were obtained from acid leached solution of soil by raising the pH to 9 with 14N ammonia solution. The concentration of the uranium observed in the soil samples was 200-600 ppb, whereas in sediment samples, the concentration range was 61-400 ppb.

Parametrised Constants and Replication for Spatial Mobility

DEFF Research Database (Denmark)

Hüttel, Hans; Haagensen, Bjørn

2009-01-01

Parametrised replication and replication are common ways of expressing infinite computation in process calculi. While parametrised constants can be encoded using replication in the π-calculus, this changes in the presence of spatial mobility as found in e.g. the distributed π- calculus...... of the distributed π-calculus with parametrised constants and replication are incomparable. On the other hand, we shall see that there exists a simple encoding of recursion in mobile ambients....

Adenovirus sequences required for replication in vivo.

OpenAIRE

Wang, K; Pearson, G D

1985-01-01

We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occup...

Extraction chromatographic separation of Sr, Pu and Am in environmental samples

Energy Technology Data Exchange (ETDEWEB)

Sidhu, Rajdeep

2004-04-01

An accurate determination of radionuclides from various sources in the environment is essential for assessment of the potential hazards and suitable countermeasures both in case of an authorised release, accidents and routine surveillance. Due to the short range of alpha and beta radiation, the accurate determination of pure alpha and beta emitters must always include radiochemical separations to separate the analytes from the matrix and from other interfering stable and radioactive nuclides. Hence, the procedures used for their determination are usually tedious and involve several preconcentration and separation steps. This work deals with the determination of {sup 90}Sr, {sup 241}Am, {sup 238}Pu, {sup 239,240}Pu and {sup 244}Cm, whic some of the most important artificial radionuclides. Due to either absence or low yield of gamma radiation, a secure determination of low concentrations of all these nuclides requires a dedicated chemistry. Selective extraction chromatographic resins (TRU- and Sr-Resin) have been utilised to develop new procedures for the analyses of Pu, Am and Cm isotopes in seawater and both these and {sup 90}Sr in soil, sediment, urine and low-level liquid radioactive effluents. The proposed method for the analyses of Pu and Am in seawater offers a quick and secure mode for the determination of these radionuclides in marine waters. Combined pre-concentration of actinides and strontium (oxalate or phosphate precipitation) followed by dual column separation on TRU- and Sr-Resin significantly reduces the through put time and costs compared to traditional ion exchange and precipitation methods. The greatest gain in productivity and environmental friendliness is achieved in Sr separations using Sr-Resin instead of precipitations involving fuming nitric acid and oxalate, hydroxide, chromate and carbonate precipitations. (author)

Evolution of microstructure and elastic wave velocities in dehydrated gypsum samples

Science.gov (United States)

Milsch, Harald; Priegnitz, Mike

2012-12-01

We report on changes in P and S-wave velocities and rock microstructure induced by devolatilization reactions using gypsum as a reference analog material. Cylindrical samples of natural alabaster were dehydrated in air, at ambient pressure, and temperatures between 378 and 423 K. Dehydration did not proceed homogeneously but via a reaction front moving sample inwards separating an outer highly porous rim from the remaining gypsum which, above approximately 393 (±5) K, concurrently decomposed into hemihydrate. Overall porosity was observed to continuously increase with reaction progress from approximately 2% for fully hydrated samples to 30% for completely dehydrated ones. Concurrently, P and S-wave velocities linearly decreased with porosity from 5.2 and 2.7 km/s to 1.0 and 0.7 km/s, respectively. It is concluded that a linearized empirical Raymer-type model extended by a critical porosity term and based on the respective time dependent mineral and pore volumes reasonably replicates the P and S-wave data in relation to reaction progress and porosity.

Separation and quantitation of radionuclides in Hanford environmental and waste tank samples using IC-ICP/MS techniques

International Nuclear Information System (INIS)

Farmer, O.T. III; Smith, M.R.; Wyse, E.J.; Barinage, C.J.; Koppenaal, D.W.

1996-01-01

The use of shielded ICP/MS instrumentation to characterize radioactive material has seen marked growth over the past few years. With a lower limit detection now in the fg/mL range for newer units, ICP/MS has become the method of choice for many studies requiring rapid, isotopic analysis of both stable elements and radionuclides with half-lives greater than 102-103 years. However, despite its sensitivity and versatility, ICP/MS has heretofore had some notable limitations in certain radiological applications. For example, Hanford waste tank samples contain a variety of nuclides with altered isotopic abundances, which complicate identification of these nuclides. Even if a nuclide can be identified, conventional quantitation using vendor-supplied software assumes natural, or knowledge of, isotopic abundance. Difficult sample matrices like those obtained from the Hanford tanks can further complicate interpretation and quantitation. On-line ion chromatography (IC) has been recently employed to mitigate some these problems. By sequentially separating elements, isobars can be resolved, thereby permitting the unequivocal determination of isotopic abundances. But even though the technique resolves isobaric interferences and is effective in reducing matrix problems, IC does not easily lend itself to quantitation. Internal standards, which are crucial for quantitation during a prolonged ICP/MS analysis with varying eluant matrices, are separated or affected as any indigenous element is using standard IC methodology

Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks.

Science.gov (United States)

Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia; Evangelou, Konstantinos; Da-Ré, Caterina; Huber, Florian; Padayachy, Laura; Tardy, Sebastien; Nicati, Noemie L; Barriot, Samia; Ochs, Fena; Lukas, Claudia; Lukas, Jiri; Gorgoulis, Vassilis G; Scapozza, Leonardo; Halazonetis, Thanos D

2016-12-15

Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Suppression of Poxvirus Replication by Resveratrol.

Science.gov (United States)

Cao, Shuai; Realegeno, Susan; Pant, Anil; Satheshkumar, Panayampalli S; Yang, Zhilong

2017-01-01

Poxviruses continue to cause serious diseases even after eradication of the historically deadly infectious human disease, smallpox. Poxviruses are currently being developed as vaccine vectors and cancer therapeutic agents. Resveratrol is a natural polyphenol stilbenoid found in plants that has been shown to inhibit or enhance replication of a number of viruses, but the effect of resveratrol on poxvirus replication is unknown. In the present study, we found that resveratrol dramatically suppressed the replication of vaccinia virus (VACV), the prototypic member of poxviruses, in various cell types. Resveratrol also significantly reduced the replication of monkeypox virus, a zoonotic virus that is endemic in Western and Central Africa and causes human mortality. The inhibitory effect of resveratrol on poxviruses is independent of VACV N1 protein, a potential resveratrol binding target. Further experiments demonstrated that resveratrol had little effect on VACV early gene expression, while it suppressed VACV DNA synthesis, and subsequently post-replicative gene expression.

Suppression of Poxvirus Replication by Resveratrol

Directory of Open Access Journals (Sweden)

Shuai Cao

2017-11-01

Full Text Available Poxviruses continue to cause serious diseases even after eradication of the historically deadly infectious human disease, smallpox. Poxviruses are currently being developed as vaccine vectors and cancer therapeutic agents. Resveratrol is a natural polyphenol stilbenoid found in plants that has been shown to inhibit or enhance replication of a number of viruses, but the effect of resveratrol on poxvirus replication is unknown. In the present study, we found that resveratrol dramatically suppressed the replication of vaccinia virus (VACV, the prototypic member of poxviruses, in various cell types. Resveratrol also significantly reduced the replication of monkeypox virus, a zoonotic virus that is endemic in Western and Central Africa and causes human mortality. The inhibitory effect of resveratrol on poxviruses is independent of VACV N1 protein, a potential resveratrol binding target. Further experiments demonstrated that resveratrol had little effect on VACV early gene expression, while it suppressed VACV DNA synthesis, and subsequently post-replicative gene expression.

Reduced genetic distance and high replication levels increase the RNA recombination rate of hepatitis delta virus.

Science.gov (United States)

Lin, Chia-Chi; Yang, Zhi-Wei; Iang, Shan-Bei; Chao, Mei

2015-01-02

Hepatitis delta virus (HDV) replication is carried out by host RNA polymerases. Since homologous inter-genotypic RNA recombination is known to occur in HDV, possibly via a replication-dependent process, we hypothesized that the degree of sequence homology and the replication level should be related to the recombination frequency in cells co-expressing two HDV sequences. To confirm this, we separately co-transfected cells with three different pairs of HDV genomic RNAs and analyzed the obtained recombinants by RT-PCR followed by restriction fragment length polymorphism and sequencing analyses. The sequence divergence between the clones ranged from 24% to less than 0.1%, and the difference in replication levels was as high as 100-fold. As expected, significant differences were observed in the recombination frequencies, which ranged from 0.5% to 47.5%. Furthermore, varying the relative amounts of parental RNA altered the dominant recombinant species produced, suggesting that template switching occurs frequently during the synthesis of genomic HDV RNA. Taken together, these data suggest that during the host RNA polymerase-driven RNA recombination of HDV, both inter- and intra-genotypic recombination events are important in shaping the genetic diversity of HDV. Copyright © 2014 Elsevier B.V. All rights reserved.

Teacher Perceptions of School Consultant Social Influence Strategies: Replication and Expansion

Science.gov (United States)

Owens, Julie Sarno; Schwartz, Madeleine E.; Erchul, William P.; Himawan, Lina K.; Evans, Steven W.; Coles, Erika K.; Schulte, Ann C.

2017-01-01

The goals were to (a) replicate the findings of previous research with regard to the Consultee/Teacher Version of the Interpersonal Power Inventory (IPI), and (b) advance the literature by examining IPI scores about a current consultation relationship. Sample 1 included 99 elementary school teachers (44.4% Hispanic) who completed the IPI. Results…

Crinivirus replication and host interactions

Directory of Open Access Journals (Sweden)

Zsofia A Kiss

2013-05-01

Full Text Available Criniviruses comprise one of the genera within the family Closteroviridae. Members in this family are restricted to the phloem and rely on whitefly vectors of the genera Bemisia and/or Trialeurodes for plant-to-plant transmission. All criniviruses have bipartite, positive-sense ssRNA genomes, although there is an unconfirmed report of one having a tripartite genome. Lettuce infectious yellows virus (LIYV is the type species of the genus, the best studied so far of the criniviruses and the first for which a reverse genetics system was available. LIYV RNA 1 encodes for proteins predicted to be involved in replication, and alone is competent for replication in protoplasts. Replication results in accumulation of cytoplasmic vesiculated membranous structures which are characteristic of most studied members of the Closteroviridae. These membranous structures, often referred to as BYV-type vesicles, are likely sites of RNA replication. LIYV RNA 2 is replicated in trans when co-infecting cells with RNA 1, but is temporally delayed relative to RNA1. Efficient RNA 2 replication also is dependent on the RNA 1-encoded RNA binding protein, P34. No LIYV RNA 2-encoded proteins have been shown to affect RNA replication, but at least four, CP, CPm, Hsp70h, and p59 are virion structural components and CPm is a determinant of whitefly transmissibility. Roles of other LIYV RNA 2-encoded proteins are largely as yet unknown, but P26 is a non-virion protein that accumulates in cells as characteristic plasmalemma deposits which in plants are localized within phloem parenchyma and companion cells over plasmodesmata connections to sieve elements. The two remaining crinivirus-conserved RNA 2-encoded proteins are P5 and P9. P5 is 39 amino acid protein and is encoded at the 5’ end of RNA 2 as ORF1 and is part of the hallmark closterovirus gene array. The orthologous gene in BYV has been shown to play a role in cell-to-cell movement and indicated to be localized to the

Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides

International Nuclear Information System (INIS)

Takegami, T.; Semler, B.L.; Anderson, C.W.; Wimmer, E.

1983-01-01

The poliovirus specific polypeptide P3-9 is of special interest for studies of viral RNA replication because it contains a hydrophobic region and, separated by only seven amino acids from that region, the amino acid sequence of the genome-linked protein VPg. Membraneous complexes of poliovirus-infected HeLa cells that contain poliovirus RNA replicating proteins have been analyzed for the presence of P3-9 by immunoprecipitation. Incubation of a membrane fraction rich in P3-9 with proteinase leaves the C-terminal 69 amino acids of P3-9 intact, an observation suggesting that this portion is protected by its association with the cellular membrane. These studies have also revealed two hitherto undescribed viral polypeptides consisting of amino acid sequences of the P2 andf P3 regions of the polyprotein. Sequence analysis by stepwise Edman degradation show that these proteins are 3b/9 (M/sub r/77,000) and X/9 (M/sub r/50,000). 3b/9 and X/9 are membrane bound and are turned over rapidly and may be direct precursors to proteins P2-X and P3-9 of the RNA replication complex. P2-X, a polypeptide void of hydrophobic amino acid sequences but also found associated with membranes, is rapidly degraded when the membraneous complex is treated with trypsin. It is speculated that P2-X is associated with membranes by its affinity to the N-terminus of P3-9

Epoxy replication for Wolter x-ray microscope fabrication

International Nuclear Information System (INIS)

Priedhorsky, W.

1981-01-01

An epoxy replica of a test piece designed to simulate a Wolter x-ray microscope geometry showed no loss of x-ray reflectivity or resolution, compared to the original. The test piece was a diamond-turned cone with 1.5 0 half angle. A flat was fly-cut on one side, then super- and conventionally polished. The replica was separated at the 1.5 0 -draft angle, simulating a shallow angle Wolter microscope geometry. A test with 8.34 A x rays at 0.9 0 grazing angle showed a reflectivity of 67% for the replica flat surface, and 70% for the original. No spread of the reflected beam was observed with a 20-arc second wide test beam. This test verifies the epoxy replication technique for production of Wolter x-ray microscopes

The role of inducer cells in mediating in vitro suppression of feline immunodeficiency virus replication

International Nuclear Information System (INIS)

Phadke, Anagha P.; Choi, In-Soo; Li Zhongxia; Weaver, Eric; Collisson, Ellen W.

2004-01-01

CD8 + T-cell-mediated suppression of feline immunodeficiency virus (FIV) replication has been described by several groups, although the mechanisms of activation and conditions for viral suppression vary with the methodologies. We have previously reported that CD8 + T-cell-mediated suppression of FIV replication required inducer cell stimulation of the effector cells. The focus of the present study was to examine the essential role of inducer cells required for the induction of this soluble anti-FIV activity. Both FIV-PPR-infected T cells and feline skin fibroblasts (FSF) infected with an alphavirus vector expressing FIV capsid or the irrelevant antigen lacZ, stimulated autologous or heterologous effector cells to produce supernatants that suppressed FIV replication. Thus, induction of this suppression of FIV replication did not strictly require autologous inducer cells and did not require the presence of FIV antigen. Anti-viral activity correlated with the presence of CD8 + T cells. Suppression was maximal when the inducer cells and the effector cells were in contact with each other, because separation of the inducer and effector cells by a 0.45-μm membrane reduced FIV suppression by approximately 50%. These findings emphasize the importance for membrane antigen interactions and cytokines in the optimal induction of effector cell synthesis of the soluble anti-FIV activity

Online combination of reversed-phase/reversed-phase and porous graphitic carbon liquid chromatography for multicomponent separation of proteomics and glycoproteomics samples.

Science.gov (United States)

Lam, Maggie P Y; Lau, Edward; Siu, S O; Ng, Dominic C M; Kong, Ricky P W; Chiu, Philip C N; Yeung, William S B; Lo, Clive; Chu, Ivan K

2011-11-01

In this paper, we describe an online combination of reversed-phase/reversed-phase (RP-RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP-RP portion of this system provides comprehensive 2-D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP-RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11-protein mixture, we found that the system could efficiently separate native peptides and released N-glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25 μg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP-RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A-extracted glycoproteome from human serum; in total, 134 potentially N-glycosylated serum proteins, 151 possible N-glycosylation sites, and more than 40 possible N-glycan structures recognized by concanavalin A were simultaneously detected. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

How many bootstrap replicates are necessary?

Science.gov (United States)

Pattengale, Nicholas D; Alipour, Masoud; Bininda-Emonds, Olaf R P; Moret, Bernard M E; Stamatakis, Alexandros

2010-03-01

Phylogenetic bootstrapping (BS) is a standard technique for inferring confidence values on phylogenetic trees that is based on reconstructing many trees from minor variations of the input data, trees called replicates. BS is used with all phylogenetic reconstruction approaches, but we focus here on one of the most popular, maximum likelihood (ML). Because ML inference is so computationally demanding, it has proved too expensive to date to assess the impact of the number of replicates used in BS on the relative accuracy of the support values. For the same reason, a rather small number (typically 100) of BS replicates are computed in real-world studies. Stamatakis et al. recently introduced a BS algorithm that is 1 to 2 orders of magnitude faster than previous techniques, while yielding qualitatively comparable support values, making an experimental study possible. In this article, we propose stopping criteria--that is, thresholds computed at runtime to determine when enough replicates have been generated--and we report on the first large-scale experimental study to assess the effect of the number of replicates on the quality of support values, including the performance of our proposed criteria. We run our tests on 17 diverse real-world DNA--single-gene as well as multi-gene--datasets, which include 125-2,554 taxa. We find that our stopping criteria typically stop computations after 100-500 replicates (although the most conservative criterion may continue for several thousand replicates) while producing support values that correlate at better than 99.5% with the reference values on the best ML trees. Significantly, we also find that the stopping criteria can recommend very different numbers of replicates for different datasets of comparable sizes. Our results are thus twofold: (i) they give the first experimental assessment of the effect of the number of BS replicates on the quality of support values returned through BS, and (ii) they validate our proposals for

Optical method for distance and displacement measurements of the probe-sample separation in a scanning near-field optical microscope

International Nuclear Information System (INIS)

Santamaria, L.; Siller, H. R.; Garcia-Ortiz, C. E.; Cortes, R.; Coello, V.

2016-01-01

In this work, we present an alternative optical method to determine the probe-sample separation distance in a scanning near-field optical microscope. The experimental method is based in a Lloyd’s mirror interferometer and offers a measurement precision deviation of ∼100 nm using digital image processing and numerical analysis. The technique can also be strategically combined with the characterization of piezoelectric actuators and stability evaluation of the optical system. It also opens the possibility for the development of an automatic approximation control system valid for probe-sample distances from 5 to 500 μm.

Optical method for distance and displacement measurements of the probe-sample separation in a scanning near-field optical microscope

Energy Technology Data Exchange (ETDEWEB)

Santamaria, L.; Siller, H. R. [Tecnológico de Monterrey, Eugenio Garza Sada 2501 Sur, Monterrey, N.L., 64849 (Mexico); Garcia-Ortiz, C. E., E-mail: cegarcia@cicese.mx [CONACYT Research Fellow – CICESE, Unidad Monterrey, Alianza Centro 504, Apodaca, NL, 66629 (Mexico); Cortes, R.; Coello, V. [CICESE, Unidad Monterrey, PIIT, Alianza Centro 504, Apodaca, NL, 66629 (Mexico)

2016-04-15

In this work, we present an alternative optical method to determine the probe-sample separation distance in a scanning near-field optical microscope. The experimental method is based in a Lloyd’s mirror interferometer and offers a measurement precision deviation of ∼100 nm using digital image processing and numerical analysis. The technique can also be strategically combined with the characterization of piezoelectric actuators and stability evaluation of the optical system. It also opens the possibility for the development of an automatic approximation control system valid for probe-sample distances from 5 to 500 μm.

Non‐Canonical Replication Initiation: You’re Fired!

Directory of Open Access Journals (Sweden)

Bazilė Ravoitytė

2017-01-01

Full Text Available The division of prokaryotic and eukaryotic cells produces two cells that inherit a perfect copy of the genetic material originally derived from the mother cell. The initiation of canonical DNA replication must be coordinated to the cell cycle to ensure the accuracy of genome duplication. Controlled replication initiation depends on a complex interplay of cis‐acting DNA sequences, the so‐called origins of replication (ori, with trans‐acting factors involved in the onset of DNA synthesis. The interplay of cis‐acting elements and trans‐acting factors ensures that cells initiate replication at sequence‐specific sites only once, and in a timely order, to avoid chromosomal endoreplication. However, chromosome breakage and excessive RNA:DNA hybrid formation can cause breakinduced (BIR or transcription‐initiated replication (TIR, respectively. These non‐canonical replication events are expected to affect eukaryotic genome function and maintenance, and could be important for genome evolution and disease development. In this review, we describe the difference between canonical and non‐canonical DNA replication, and focus on mechanistic differences and common features between BIR and TIR. Finally, we discuss open issues on the factors and molecular mechanisms involved in TIR.

Factors influencing microinjection molding replication quality

Science.gov (United States)

Vera, Julie; Brulez, Anne-Catherine; Contraires, Elise; Larochette, Mathieu; Trannoy-Orban, Nathalie; Pignon, Maxime; Mauclair, Cyril; Valette, Stéphane; Benayoun, Stéphane

2018-01-01

In recent years, there has been increased interest in producing and providing high-precision plastic parts that can be manufactured by microinjection molding: gears, pumps, optical grating elements, and so on. For all of these applications, the replication quality is essential. This study has two goals: (1) fabrication of high-precision parts using the conventional injection molding machine; (2) identification of robust parameters that ensure production quality. Thus, different technological solutions have been used: cavity vacuuming and the use of a mold coated with DLC or CrN deposits. AFM and SEM analyses were carried out to characterize the replication profile. The replication quality was studied in terms of the process parameters, coated and uncoated molds and crystallinity of the polymer. Specific studies were processed to quantify the replicability of injection molded parts (ABS, PC and PP). Analysis of the Taguchi experimental designs permits prioritization of the impact of each parameter on the replication quality. A discussion taking into account these new parameters and the thermal and spreading properties on the coatings is proposed. It appeared that, in general, increasing the mold temperature improves the molten polymer fill in submicron features except for the steel insert (for which the presence of a vacuum is the most important factor). Moreover, the DLC coating was the best coating to increase the quality of the replication. This result could be explained by the lower thermal diffusivity of this coating. We noted that the viscosity of the polymers is not a primordial factor of the replication quality.

Realistic Vascular Replicator for TAVR Procedures.

Science.gov (United States)

Rotman, Oren M; Kovarovic, Brandon; Sadasivan, Chander; Gruberg, Luis; Lieber, Baruch B; Bluestein, Danny

2018-04-13

Transcatheter aortic valve replacement (TAVR) is an over-the-wire procedure for treatment of severe aortic stenosis (AS). TAVR valves are conventionally tested using simplified left heart simulators (LHS). While those provide baseline performance reliably, their aortic root geometries are far from the anatomical in situ configuration, often overestimating the valves' performance. We report on a novel benchtop patient-specific arterial replicator designed for testing TAVR and training interventional cardiologists in the procedure. The Replicator is an accurate model of the human upper body vasculature for training physicians in percutaneous interventions. It comprises of fully-automated Windkessel mechanism to recreate physiological flow conditions. Calcified aortic valve models were fabricated and incorporated into the Replicator, then tested for performing TAVR procedure by an experienced cardiologist using the Inovare valve. EOA, pressures, and angiograms were monitored pre- and post-TAVR. A St. Jude mechanical valve was tested as a reference that is less affected by the AS anatomy. Results in the Replicator of both valves were compared to the performance in a commercial ISO-compliant LHS. The AS anatomy in the Replicator resulted in a significant decrease of the TAVR valve performance relative to the simplified LHS, with EOA and transvalvular pressures comparable to clinical data. Minor change was seen in the mechanical valve performance. The Replicator showed to be an effective platform for TAVR testing. Unlike a simplified geometric anatomy LHS, it conservatively provides clinically-relevant outcomes and complement it. The Replicator can be most valuable for testing new valves under challenging patient anatomies, physicians training, and procedural planning.

Preparation of calcium-separated isotope targets using small samples

International Nuclear Information System (INIS)

Thomas, G.E.

1975-01-01

Targets are routinely evaporated using a few milligram quantities of separated isotopes of calcium with reducing agents. The source to target distance is 3.0 cm with the substrate, if necessary, as thin as 15 μg/cm 2 carbon or 100 μg/cm 2 of gold. A tantalum closed boat, heat shield, and special collimator system are used

Risk Factors as Major Determinants of Resilience: A Replication Study.

Science.gov (United States)

Eshel, Yohanan; Kimhi, Shaul; Lahad, Mooli; Leykin, Dmitry; Goroshit, Marina

2018-03-16

The present study was conducted in the context of current concerns about replication in psychological research. It claims that risk factors should be regarded as an integral part of the definition of individual resilience, which should be defined in terms of the balance between individual strength or protective factors, and individual vulnerability or risk factors (IND-SVR). Five independent samples, including 3457 Israeli participants, were employed to determine the effects of resilience promoting and resilience suppressing variables on the IND-SVR index of resilience, and on its two components: recovery from adversity, and distress symptoms. Five path analyses were employed for determining the role of distress symptoms as a measure of psychological resilience, as compared to other indices of this resilience. Results indicated the major role of risk factors (distress symptoms) as an integral component of resilience. This role was generally replicated in the five investigated samples. Risk factors are legitimate, valid, and useful parts of the definition of psychological resilience. Resilience research has shifted away from studying individual risk factors to investigating the process through which individuals overcome the hardships they experience. The present data seem to suggest that this shift should be reexamined.

One-year prospective replication study of an untreated sample of community dysthymia subjects.

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McCullough, J P; McCune, K J; Kaye, A L; Braith, J A; Friend, R; Roberts, W C; Belyea-Caldwell, S; Norris, S L; Hampton, C

1994-07-01

This study replicates an earlier naturalistic-prospective investigation of nontreatment, community DSM-III-R dysthymia subjects. Major goals were to determine spontaneous remission rates and monitor the stability of psychosocial functioning levels over time. Twenty-four dysthymia subjects were followed for 1 year. Three remissions (13%) were diagnosed at the final interview. At a 4-year diagnostic follow-up contact with the remitters only, one remitter had relapsed and two remained in remission. Subjects were monitored for depressive symptom intensity, personality functioning, general medical distress, cognitive functioning, coping stylistics, interpersonal functioning, quality of their social support resources, and general family functioning. Stable levels of psychosocial functioning were maintained across all measures over the 1-year period. Current psychometric findings confirm the conclusions of the earlier nontreatment prospective study that dysthymia is a chronic mood disorder with stable psychosocial features and is unlikely to remit spontaneously over time.

Replication Validity of Initial Association Studies: A Comparison between Psychiatry, Neurology and Four Somatic Diseases

Science.gov (United States)

Dumas-Mallet, Estelle; Button, Katherine; Boraud, Thomas; Munafo, Marcus; Gonon, François

2016-01-01

Context There are growing concerns about effect size inflation and replication validity of association studies, but few observational investigations have explored the extent of these problems. Objective Using meta-analyses to measure the reliability of initial studies and explore whether this varies across biomedical domains and study types (cognitive/behavioral, brain imaging, genetic and “others”). Methods We analyzed 663 meta-analyses describing associations between markers or risk factors and 12 pathologies within three biomedical domains (psychiatry, neurology and four somatic diseases). We collected the effect size, sample size, publication year and Impact Factor of initial studies, largest studies (i.e., with the largest sample size) and the corresponding meta-analyses. Initial studies were considered as replicated if they were in nominal agreement with meta-analyses and if their effect size inflation was below 100%. Results Nominal agreement between initial studies and meta-analyses regarding the presence of a significant effect was not better than chance in psychiatry, whereas it was somewhat better in neurology and somatic diseases. Whereas effect sizes reported by largest studies and meta-analyses were similar, most of those reported by initial studies were inflated. Among the 256 initial studies reporting a significant effect (p<0.05) and paired with significant meta-analyses, 97 effect sizes were inflated by more than 100%. Nominal agreement and effect size inflation varied with the biomedical domain and study type. Indeed, the replication rate of initial studies reporting a significant effect ranged from 6.3% for genetic studies in psychiatry to 86.4% for cognitive/behavioral studies. Comparison between eight subgroups shows that replication rate decreases with sample size and “true” effect size. We observed no evidence of association between replication rate and publication year or Impact Factor. Conclusion The differences in reliability

Replicating chromatin: a tale of histones

DEFF Research Database (Denmark)

Groth, Anja

2009-01-01

Chromatin serves structural and functional roles crucial for genome stability and correct gene expression. This organization must be reproduced on daughter strands during replication to maintain proper overlay of epigenetic fabric onto genetic sequence. Nucleosomes constitute the structural...... framework of chromatin and carry information to specify higher-order organization and gene expression. When replication forks traverse the chromosomes, nucleosomes are transiently disrupted, allowing the replication machinery to gain access to DNA. Histone recycling, together with new deposition, ensures...

Varicella-zoster virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription.

Science.gov (United States)

Khalil, Mohamed I; Sommer, Marvin H; Hay, John; Ruyechan, William T; Arvin, Ann M

2015-07-01

The VZV genome has two origins of DNA replication (oriS), each of which consists of an AT-rich sequence and three origin binding protein (OBP) sites called Box A, C and B. In these experiments, the mutation in the core sequence CGC of the Box A and C not only inhibited DNA replication but also inhibited both ORF62 and ORF63 expression in reporter gene assays. In contrast the Box B mutation did not influence DNA replication or flanking gene transcription. These results suggest that efficient DNA replication enhances ORF62 and ORF63 transcription. Recombinant viruses carrying these mutations in both sites and one with a deletion of the whole oriS were constructed. Surprisingly, the recombinant virus lacking both copies of oriS retained the capacity to replicate in melanoma and HELF cells suggesting that VZV has another origin of DNA replication. Copyright © 2015 Elsevier Inc. All rights reserved.

Modes of DNA repair and replication

International Nuclear Information System (INIS)

Hanawalt, P.; Kondo, S.

1979-01-01

Modes of DNA repair and replication require close coordination as well as some overlap of enzyme functions. Some classes of recovery deficient mutants may have defects in replication rather than repair modes. Lesions such as the pyrimidine dimers produced by ultraviolet light irradiation are the blocks to normal DNA replication in vivo and in vitro. The DNA synthesis by the DNA polymerase 1 of E. coli is blocked at one nucleotide away from the dimerized pyrimidines in template strands. Thus, some DNA polymerases seem to be unable to incorporate nucleotides opposite to the non-pairing lesions in template DNA strands. The lesions in template DNA strands may block the sequential addition of nucleotides in the synthesis of daughter strands. Normal replication utilizes a constitutive ''error-free'' mode that copies DNA templates with high fidelity, but which may be totally blocked at a lesion that obscures the appropriate base pairing specificity. It might be expected that modified replication system exhibits generally high error frequency. The error rate of DNA polymerases may be controlled by the degree of phosphorylation of the enzyme. Inducible SOS system is controlled by recA genes that also control the pathways for recombination. It is possible that SOS system involves some process other than the modification of a blocked replication apparatus to permit error-prone transdimer synthesis. (Yamashita, S.)

Charter School Replication. Policy Guide

Science.gov (United States)

Rhim, Lauren Morando

2009-01-01

"Replication" is the practice of a single charter school board or management organization opening several more schools that are each based on the same school model. The most rapid strategy to increase the number of new high-quality charter schools available to children is to encourage the replication of existing quality schools. This policy guide…

Replication of kinetoplast minicircle DNA

International Nuclear Information System (INIS)

Sheline, C.T.

1989-01-01

These studies describe the isolation and characterization of early minicircle replication intermediates from Crithidia fasciculata, and Leishmania tarentolae, the mitochondrial localization of a type II topoisomerase (TIImt) in C. fasciculata, and the implication of the aforementioned TIImt in minicircle replication in L. tarentolae. Early minicircle replication intermediates from C. fasciculata were identified and characterized using isolated kinetoplasts to incorporate radiolabeled nucleotides into its DNA. The pulse-label in an apparent theta-type intermediate chase into two daughter molecules. A uniquely gapped, ribonucleotide primed, knotted molecule represents the leading strand in the model proposed, and a highly gapped molecule represents the lagging strand. This theta intermediate is repaired in vitro to a doubly nicked catenated dimer which was shown to result from the replication of a single parental molecule. Very similar intermediates were found in the heterogeneous population of minicircles of L. tarentolae. The sites of the Leishmania specific discontinuities were mapped and shown to lie within the universally conserved sequence blocks in identical positions as compared to C. fasciculata and Trypanosoma equiperdum

Manual of Cupule Replication Technology

Directory of Open Access Journals (Sweden)

Giriraj Kumar

2015-09-01

Full Text Available Throughout the world, iconic rock art is preceded by non-iconic rock art. Cupules (manmade, roughly semi-hemispherical depressions on rocks form the major bulk of the early non-iconic rock art globally. The antiquity of cupules extends back to the Lower Paleolithic in Asia and Africa, hundreds of thousand years ago. When one observes these cupules, the inquisitive mind poses so many questions with regard to understanding their technology, reasons for selecting the site, which rocks were used to make the hammer stones used, the skill and cognitive abilities employed to create the different types of cupules, the objective of their creation, their age, and so on. Replication of the cupules can provide satisfactory answers to some of these questions. Comparison of the hammer stones and cupules produced by the replication process with those obtained from excavation can provide support to observations. This paper presents a manual of cupule replication technology based on our experience of cupule replication on hard quartzite rock near Daraki-Chattan in the Chambal Basin, India.

Targeting DNA Replication Stress for Cancer Therapy

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Jun Zhang

2016-08-01

Full Text Available The human cellular genome is under constant stress from extrinsic and intrinsic factors, which can lead to DNA damage and defective replication. In normal cells, DNA damage response (DDR mediated by various checkpoints will either activate the DNA repair system or induce cellular apoptosis/senescence, therefore maintaining overall genomic integrity. Cancer cells, however, due to constitutive growth signaling and defective DDR, may exhibit “replication stress” —a phenomenon unique to cancer cells that is described as the perturbation of error-free DNA replication and slow-down of DNA synthesis. Although replication stress has been proven to induce genomic instability and tumorigenesis, recent studies have counterintuitively shown that enhancing replicative stress through further loosening of the remaining checkpoints in cancer cells to induce their catastrophic failure of proliferation may provide an alternative therapeutic approach. In this review, we discuss the rationale to enhance replicative stress in cancer cells, past approaches using traditional radiation and chemotherapy, and emerging approaches targeting the signaling cascades induced by DNA damage. We also summarize current clinical trials exploring these strategies and propose future research directions including the use of combination therapies, and the identification of potential new targets and biomarkers to track and predict treatment responses to targeting DNA replication stress.

Recycling of WEEE by magnetic density separation

NARCIS (Netherlands)

Hu, B.; Giacometti, L.; Di Maio, F.; Rem, P.C.

2011-01-01

The paper introduces a new recycling method of WEEE: Magnetic Density Separation. By using this technology, both grade and recovery rate of recycled products are over 90%. Good separations are not only observed in relatively big WEEE samples, but also in samples with smaller sizes or electrical

Global profiling of DNA replication timing and efficiency reveals that efficient replication/firing occurs late during S-phase in S. pombe.

Directory of Open Access Journals (Sweden)

Majid Eshaghi

Full Text Available BACKGROUND: During S. pombe S-phase, initiation of DNA replication occurs at multiple sites (origins that are enriched with AT-rich sequences, at various times. Current studies of genome-wide DNA replication profiles have focused on the DNA replication timing and origin location. However, the replication and/or firing efficiency of the individual origins on the genomic scale remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using the genome-wide ORF-specific DNA microarray analysis, we show that in S. pombe, individual origins fire with varying efficiencies and at different times during S-phase. The increase in DNA copy number plotted as a function of time is approximated to the near-sigmoidal model, when considering the replication start and end timings at individual loci in cells released from HU-arrest. Replication efficiencies differ from origin to origin, depending on the origin's firing efficiency. We have found that DNA replication is inefficient early in S-phase, due to inefficient firing at origins. Efficient replication occurs later, attributed to efficient but late-firing origins. Furthermore, profiles of replication timing in cds1Delta cells are abnormal, due to the failure in resuming replication at the collapsed forks. The majority of the inefficient origins, but not the efficient ones, are found to fire in cds1Delta cells after HU removal, owing to the firing at the remaining unused (inefficient origins during HU treatment. CONCLUSIONS/SIGNIFICANCE: Taken together, our results indicate that efficient DNA replication/firing occurs late in S-phase progression in cells after HU removal, due to efficient late-firing origins. Additionally, checkpoint kinase Cds1p is required for maintaining the efficient replication/firing late in S-phase. We further propose that efficient late-firing origins are essential for ensuring completion of DNA duplication by the end of S-phase.

Replicated Data Management for Mobile Computing

CERN Document Server

Douglas, Terry

2008-01-01

Managing data in a mobile computing environment invariably involves caching or replication. In many cases, a mobile device has access only to data that is stored locally, and much of that data arrives via replication from other devices, PCs, and services. Given portable devices with limited resources, weak or intermittent connectivity, and security vulnerabilities, data replication serves to increase availability, reduce communication costs, foster sharing, and enhance survivability of critical information. Mobile systems have employed a variety of distributed architectures from client-server

pUL34 binding near the human cytomegalovirus origin of lytic replication enhances DNA replication and viral growth.

Science.gov (United States)

Slayton, Mark; Hossain, Tanvir; Biegalke, Bonita J

2018-05-01

The human cytomegalovirus (HCMV) UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are required for viral replication. Interactions of pUL34 with DNA binding sites represses transcription of two viral immune evasion genes, US3 and US9. 12 additional predicted pUL34-binding sites are present in the HCMV genome (strain AD169) with three binding sites concentrated near the HCMV origin of lytic replication (oriLyt). We used ChIP-seq analysis of pUL34-DNA interactions to confirm that pUL34 binds to the oriLyt region during infection. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated oriLyt-dependent DNA replication. Mutagenesis of these sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with the viral proteins IE2, UL44, and UL84, that are essential for viral DNA replication, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade. Copyright © 2018 Elsevier Inc. All rights reserved.

Replication of urban innovations - prioritization of strategies for the replication of Dhaka's community-based decentralized composting model.

Science.gov (United States)

Yedla, Sudhakar

2012-01-01

Dhaka's community-based decentralized composting (DCDC) is a successful demonstration of solid waste management by adopting low-cost technology, local resources community participation and partnerships among the various actors involved. This paper attempts to understand the model, necessary conditions, strategies and their priorities to replicate DCDC in the other developing cities of Asia. Thirteen strategies required for its replication are identified and assessed based on various criteria, namely transferability, longevity, economic viability, adaptation and also overall replication. Priority setting by multi-criteria analysis by applying analytic hierarchy process revealed that immediate transferability without long-term and economic viability consideration is not advisable as this would result in unsustainable replication of DCDC. Based on the analysis, measures to ensure the product quality control; partnership among stakeholders (public-private-community); strategies to achieve better involvement of the private sector in solid waste management (entrepreneurship in approach); simple and low-cost technology; and strategies to provide an effective interface among the complementing sectors are identified as important strategies for its replication.

Replication of bacteriophage lambda DNA

International Nuclear Information System (INIS)

Tsurimoto, T.; Matsubara, K.

1983-01-01

In this paper results of studies on the mechanism of bacteriophage lambda replication using molecular biological and biochemical approaches are reported. The purification of the initiator proteins, O and P, and the role of the O and P proteins in the initiation of lambda DNA replication through interactions with specific DNA sequences are described. 47 references, 15 figures

Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

Science.gov (United States)

Kuriya, Kenji; Higashiyama, Eriko; Avşar-Ban, Eriko; Tamaru, Yutaka; Ogata, Shin; Takebayashi, Shin-ichiro; Ogata, Masato; Okumura, Katsuzumi

2015-12-01

Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

The effects of ozone and water exchange rates on water quality and rainbow trout Oncorhynchus mykiss performance in replicated water recirculating systems

Science.gov (United States)

Rainbow trout Oncorhynchus mykiss performance and water quality were evaluated and compared within six replicated 9.5 cubic meter water recirculating aquaculture systems (WRAS) operated with and without ozone at various water exchange rates. Three separate studies were conducted: 1) low water exchan...

Insulated hsp70B' promoter: stringent heat-inducible activity in replication-deficient, but not replication-competent adenoviruses.

Science.gov (United States)

Rohmer, Stanimira; Mainka, Astrid; Knippertz, Ilka; Hesse, Andrea; Nettelbeck, Dirk M

2008-04-01

Key to the realization of gene therapy is the development of efficient and targeted gene transfer vectors. Therapeutic gene transfer by replication-deficient or more recently by conditionally replication-competent/oncolytic adenoviruses has shown much promise. For specific applications, however, it will be advantageous to provide vectors that allow for external control of gene expression. The efficient cellular heat shock system in combination with available technology for focused and controlled hyperthermia suggests heat-regulated transcription control as a promising tool for this purpose. We investigated the feasibility of a short fragment of the human hsp70B' promoter, with and without upstream insulator elements, for the regulation of transgene expression by replication-deficient or oncolytic adenoviruses. Two novel adenoviral vectors with an insulated hsp70B' promoter were developed and showed stringent heat-inducible gene expression with induction ratios up to 8000-fold. In contrast, regulation of gene expression from the hsp70B' promoter without insulation was suboptimal. In replication-competent/oncolytic adenoviruses regulation of the hsp70B' promoter was lost specifically during late replication in permissive cells and could not be restored by the insulators. We developed novel adenovirus gene transfer vectors that feature improved and stringent regulation of transgene expression from the hsp70B' promoter using promoter insulation. These vectors have potential for gene therapy applications that benefit from external modulation of therapeutic gene expression or for combination therapy with hyperthermia. Furthermore, our study reveals that vector replication can deregulate inserted cellular promoters, an observation which is of relevance for the development of replication-competent/oncolytic gene transfer vectors. (c) 2008 John Wiley & Sons, Ltd.

Development of a cloud-point extraction method for copper and nickel determination in food samples

International Nuclear Information System (INIS)

Azevedo Lemos, Valfredo; Selis Santos, Moacy; Teixeira David, Graciete; Vasconcelos Maciel, Mardson; Almeida Bezerra, Marcos de

2008-01-01

A new, simple and versatile cloud-point extraction (CPE) methodology has been developed for the separation and preconcentration of copper and nickel. The metals in the initial aqueous solution were complexed with 2-(2'-benzothiazolylazo)-5-(N,N-diethyl)aminophenol (BDAP) and Triton X-114 was added as surfactant. Dilution of the surfactant-rich phase with acidified methanol was performed after phase separation, and the copper and nickel contents were measured by flame atomic absorption spectrometry. The variables affecting the cloud-point extraction were optimized using a Box-Behnken design. Under the optimum experimental conditions, enrichment factors of 29 and 25 were achieved for copper and nickel, respectively. The accuracy of the method was evaluated and confirmed by analysis of the followings certified reference materials: Apple Leaves, Spinach Leaves and Tomato Leaves. The limits of detection expressed to solid sample analysis were 0.1 μg g -1 (Cu) and 0.4 μg g -1 (Ni). The precision for 10 replicate measurements of 75 μg L -1 Cu or Ni was 6.4 and 1.0, respectively. The method has been successfully applied to the analysis of food samples

Replication assessment of surface texture at sub-micrometre scale

DEFF Research Database (Denmark)

Quagliotti, Danilo; Tosello, Guido; Hansen, Hans Nørgaard

2017-01-01

[2]. A replication process requires reproducing a master geometry by conveying it to a substrate material. It is typically induced by means of different energy sources (usually heat and force) and a direct physical contact between the master and the substrate. Furthermore, concepts of advanced......, because of the replication nature of molding processes, the required specifications for the manufacture of micro molded components must be ensured by means of a metrological approach to surface replication and dimensional control of both master geometry and replicated substrate [3]-[4]. Therefore...... replication was assessed by the replication fidelity, i.e., comparing the produced parts with the tool used to replicate the geometry. Furthermore, the uncertainty of the replication fidelity was achieved by propagating the uncertainties evaluated for both masters and replicas. Finally, despite the specimens...

Mechanisms and regulation of DNA replication initiation in eukaryotes.

Science.gov (United States)

Parker, Matthew W; Botchan, Michael R; Berger, James M

2017-04-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.

Commercial Building Partnerships Replication and Diffusion

Energy Technology Data Exchange (ETDEWEB)

Antonopoulos, Chrissi A.; Dillon, Heather E.; Baechler, Michael C.

2013-09-16

This study presents findings from survey and interview data investigating replication efforts of Commercial Building Partnership (CBP) partners that worked directly with the Pacific Northwest National Laboratory (PNNL). PNNL partnered directly with 12 organizations on new and retrofit construction projects, which represented approximately 28 percent of the entire U.S. Department of Energy (DOE) CBP program. Through a feedback survey mechanism, along with personal interviews, PNNL gathered quantitative and qualitative data relating to replication efforts by each organization. These data were analyzed to provide insight into two primary research areas: 1) CBP partners’ replication efforts of technologies and approaches used in the CBP project to the rest of the organization’s building portfolio (including replication verification), and, 2) the market potential for technology diffusion into the total U.S. commercial building stock, as a direct result of the CBP program. The first area of this research focused specifically on replication efforts underway or planned by each CBP program participant. Factors that impact replication include motivation, organizational structure and objectives firms have for implementation of energy efficient technologies. Comparing these factors between different CBP partners revealed patterns in motivation for constructing energy efficient buildings, along with better insight into market trends for green building practices. The second area of this research develops a diffusion of innovations model to analyze potential broad market impacts of the CBP program on the commercial building industry in the United States.

Fast automatic quantitative cell replication with fluorescent live cell imaging

Directory of Open Access Journals (Sweden)

Wang Ching-Wei

2012-01-01

Full Text Available Abstract Background live cell imaging is a useful tool to monitor cellular activities in living systems. It is often necessary in cancer research or experimental research to quantify the dividing capabilities of cells or the cell proliferation level when investigating manipulations of the cells or their environment. Manual quantification of fluorescence microscopic image is difficult because human is neither sensitive to fine differences in color intensity nor effective to count and average fluorescence level among cells. However, auto-quantification is not a straightforward problem to solve. As the sampling location of the microscopy changes, the amount of cells in individual microscopic images varies, which makes simple measurement methods such as the sum of stain intensity values or the total number of positive stain within each image inapplicable. Thus, automated quantification with robust cell segmentation techniques is required. Results An automated quantification system with robust cell segmentation technique are presented. The experimental results in application to monitor cellular replication activities show that the quantitative score is promising to represent the cell replication level, and scores for images from different cell replication groups are demonstrated to be statistically significantly different using ANOVA, LSD and Tukey HSD tests (p-value Conclusion A robust automated quantification method of live cell imaging is built to measure the cell replication level, providing a robust quantitative analysis system in fluorescent live cell imaging. In addition, the presented unsupervised entropy based cell segmentation for live cell images is demonstrated to be also applicable for nuclear segmentation of IHC tissue images.

Extremal dynamics in random replicator ecosystems

Energy Technology Data Exchange (ETDEWEB)

Kärenlampi, Petri P., E-mail: petri.karenlampi@uef.fi

2015-10-02

The seminal numerical experiment by Bak and Sneppen (BS) is repeated, along with computations with replicator models, including a greater amount of features. Both types of models do self-organize, and do obey power-law scaling for the size distribution of activity cycles. However species extinction within the replicator models interferes with the BS self-organized critical (SOC) activity. Speciation–extinction dynamics ruins any stationary state which might contain a steady size distribution of activity cycles. The BS-type activity appears as a dissimilar phenomenon in comparison to speciation–extinction dynamics in the replicator system. No criticality is found from the speciation–extinction dynamics. Neither are speciations and extinctions in real biological macroevolution known to contain any diverging distributions, or self-organization towards any critical state. Consequently, biological macroevolution probably is not a self-organized critical phenomenon. - Highlights: • Extremal Dynamics organizes random replicator ecosystems to two phases in fitness space. • Replicator systems show power-law scaling of activity. • Species extinction interferes with Bak–Sneppen type mutation activity. • Speciation–extinction dynamics does not show any critical phase transition. • Biological macroevolution probably is not a self-organized critical phenomenon.

Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells.

Science.gov (United States)

Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J; Méndez, Ernesto; Arias, Carlos F

2015-10-01

Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients

Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells

Science.gov (United States)

Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J.; Méndez, Ernesto

2015-01-01

ABSTRACT Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. IMPORTANCE Astroviruses are common etiological agents of acute gastroenteritis in children and

An amphipathic alpha-helix controls multiple roles of brome mosaic virus protein 1a in RNA replication complex assembly and function.

Directory of Open Access Journals (Sweden)

Ling Liu

2009-03-01

Full Text Available Brome mosaic virus (BMV protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2a(Pol and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic alpha-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a-ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a-membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2a(Pol accumulation. The second class of helix A mutations not only maintains efficient 1a-membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2a(Pol accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.

Sex Differences in Preferences for Humor: A Replication, Modification, and Extension

Directory of Open Access Journals (Sweden)

Liana S. E. Hone

2015-01-01

Full Text Available Evolutionary-minded scientists have proposed that humor is a sexually selected trait in men that signals mate quality. Indeed, women tend to prefer men who make them laugh and men tend to prefer women who laugh at their jokes. However, it is unclear how robust this pattern is. Here we report a replication of one of the first studies (Bressler, Martin, and Balshine, 2006 to examine the sex differences in preferences for humor receptivity versus humor production. We replicate Bressler et al.'s (2006 findings that men prefer women who are receptive to their humor whereas women prefer men who produce humor. These findings held even after we modified Bressler et al.'s questionnaire for better conceptual validity. Furthermore, using a separate measure designed to assess trade-offs, we found that men viewed humor receptivity as a necessity and humor production as a luxury when they were asked to create an ideal long-term partner. For women, it was just the opposite. These results bolster the claim that sexual selection has shaped sex differences regarding preferences for a prospective mate's sense of humor and that what one means by “sense of humor” can vary.

Sex differences in preferences for humor: a replication, modification, and extension.

Science.gov (United States)

Hone, Liana S E; Hurwitz, William; Lieberman, Debra

2015-02-10

Evolutionary-minded scientists have proposed that humor is a sexually selected trait in men that signals mate quality. Indeed, women tend to prefer men who make them laugh and men tend to prefer women who laugh at their jokes. However, it is unclear how robust this pattern is. Here we report a replication of one of the first studies (Bressler, Martin, and Balshine, 2006) to examine the sex differences in preferences for humor receptivity versus humor production. We replicate Bressler et al.'s (2006) findings that men prefer women who are receptive to their humor whereas women prefer men who produce humor. These findings held even after we modified Bressler et al.'s questionnaire for better conceptual validity. Furthermore, using a separate measure designed to assess trade-offs, we found that men viewed humor receptivity as a necessity and humor production as a luxury when they were asked to create an ideal long-term partner. For women, it was just the opposite. These results bolster the claim that sexual selection has shaped sex differences regarding preferences for a prospective mate's sense of humor and that what one means by "sense of humor" can vary.

A Replication of the Technical Adequacy of the Student Subjective Wellbeing Questionnaire

Science.gov (United States)

Renshaw, Tyler L.

2015-01-01

The present study reports on a replication of the technical adequacy of the Student Subjective Wellbeing Questionnaire (SSWQ), which is a 16-item self-report instrument for assessing youth's academic efficacy, educational purpose, joy of learning, and school connectedness, with a sample of adolescents in Grades 6 to 7 (N = 438). Findings confirmed…

The Genomic Replication of the Crenarchaeal Virus SIRV2

DEFF Research Database (Denmark)

Martinez Alvarez, Laura

reinitiation events may partially explain the branched topology of the viral replication intermediates. We also analyzed the intracellular location of viral replication, showing the formation of viral peripheral replication centers in SIRV2-infected cells, where viral DNA synthesis and replication...

Separation in 5 Msun Binaries

Science.gov (United States)

Evans, Nancy R.; Bond, H. E.; Schaefer, G.; Mason, B. D.; Karovska, M.; Tingle, E.

2013-01-01

Cepheids (5 Msun stars) provide an excellent sample for determining the binary properties of fairly massive stars. International Ultraviolet Explorer (IUE) observations of Cepheids brighter than 8th magnitude resulted in a list of ALL companions more massive than 2.0 Msun uniformly sensitive to all separations. Hubble Space Telescope Wide Field Camera 3 (WFC3) has resolved three of these binaries (Eta Aql, S Nor, and V659 Cen). Combining these separations with orbital data in the literature, we derive an unbiased distribution of binary separations for a sample of 18 Cepheids, and also a distribution of mass ratios. The distribution of orbital periods shows that the 5 Msun binaries prefer shorter periods than 1 Msun stars, reflecting differences in star formation processes.

Quantification of fatty acids as methyl esters and phospholipids in cheese samples after separation of triacylglycerides and phospholipids

International Nuclear Information System (INIS)

Hauff, Simone; Vetter, Walter

2009-01-01

Determination of the individual fatty acid composition of neutral- and phospholipids as well as the phospholipid content of dairy food and other foodstuffs are important tasks in life sciences. For these purposes, a method was developed for the separation of lipids (standards of triolein and diacylphosphatidylcholines as well as three cheese samples) by solid-phase extraction using a self-packed column filled with partly deactivated silica. Non-halogenated solvents were used for the elution of the lipid classes. Cyclohexane/ethyl acetate (1:1, v/v) served for the elution of neutral lipids, while polar lipids were eluted with three solvents (ethyl acetate/methanol, methanol, and methanol/water) into one fraction. The separated lipid fractions were transesterified and the individual fatty acids were quantified by using gas chromatography coupled to electron ionization mass spectrometry (GC/EI-MS) in the selected ion monitoring (SIM) mode. The recovery rate for standard phosphatidylcholines was ∼90% and cross-contamination from neutral lipids was negligible. The method was applied to cheese samples. Quantitative amounts of individual fatty acids in the phospholipid fraction were eq ) were found to be representative for the average contribution of fatty acids to all classes of phospholipids in dairy products. Using this approach, the phospholipid content of lipids from mozzarella, camembert, and goat cream cheese was 0.60%, 1.42% and 0.79%, respectively

Rapid and accurate species tree estimation for phylogeographic investigations using replicated subsampling.

Science.gov (United States)

Hird, Sarah; Kubatko, Laura; Carstens, Bryan

2010-11-01

We describe a method for estimating species trees that relies on replicated subsampling of large data matrices. One application of this method is phylogeographic research, which has long depended on large datasets that sample intensively from the geographic range of the focal species; these datasets allow systematicists to identify cryptic diversity and understand how contemporary and historical landscape forces influence genetic diversity. However, analyzing any large dataset can be computationally difficult, particularly when newly developed methods for species tree estimation are used. Here we explore the use of replicated subsampling, a potential solution to the problem posed by large datasets, with both a simulation study and an empirical analysis. In the simulations, we sample different numbers of alleles and loci, estimate species trees using STEM, and compare the estimated to the actual species tree. Our results indicate that subsampling three alleles per species for eight loci nearly always results in an accurate species tree topology, even in cases where the species tree was characterized by extremely rapid divergence. Even more modest subsampling effort, for example one allele per species and two loci, was more likely than not (>50%) to identify the correct species tree topology, indicating that in nearly all cases, computing the majority-rule consensus tree from replicated subsampling provides a good estimate of topology. These results were supported by estimating the correct species tree topology and reasonable branch lengths for an empirical 10-locus great ape dataset. Copyright © 2010 Elsevier Inc. All rights reserved.

Sample-interpolation timing: an optimized technique for the digital measurement of time of flight for γ rays and neutrons at relatively low sampling rates

International Nuclear Information System (INIS)

Aspinall, M D; Joyce, M J; Mackin, R O; Jarrah, Z; Boston, A J; Nolan, P J; Peyton, A J; Hawkes, N P

2009-01-01

A unique, digital time pick-off method, known as sample-interpolation timing (SIT) is described. This method demonstrates the possibility of improved timing resolution for the digital measurement of time of flight compared with digital replica-analogue time pick-off methods for signals sampled at relatively low rates. Three analogue timing methods have been replicated in the digital domain (leading-edge, crossover and constant-fraction timing) for pulse data sampled at 8 GSa s −1 . Events arising from the 7 Li(p, n) 7 Be reaction have been detected with an EJ-301 organic liquid scintillator and recorded with a fast digital sampling oscilloscope. Sample-interpolation timing was developed solely for the digital domain and thus performs more efficiently on digital signals compared with analogue time pick-off methods replicated digitally, especially for fast signals that are sampled at rates that current affordable and portable devices can achieve. Sample interpolation can be applied to any analogue timing method replicated digitally and thus also has the potential to exploit the generic capabilities of analogue techniques with the benefits of operating in the digital domain. A threshold in sampling rate with respect to the signal pulse width is observed beyond which further improvements in timing resolution are not attained. This advance is relevant to many applications in which time-of-flight measurement is essential

The Role of the Transcriptional Response to DNA Replication Stress.

Science.gov (United States)

Herlihy, Anna E; de Bruin, Robertus A M

2017-03-02

During DNA replication many factors can result in DNA replication stress. The DNA replication stress checkpoint prevents the accumulation of replication stress-induced DNA damage and the potential ensuing genome instability. A critical role for post-translational modifications, such as phosphorylation, in the replication stress checkpoint response has been well established. However, recent work has revealed an important role for transcription in the cellular response to DNA replication stress. In this review, we will provide an overview of current knowledge of the cellular response to DNA replication stress with a specific focus on the DNA replication stress checkpoint transcriptional response and its role in the prevention of replication stress-induced DNA damage.

The Role of the Transcriptional Response to DNA Replication Stress

Science.gov (United States)

Herlihy, Anna E.; de Bruin, Robertus A.M.

2017-01-01

During DNA replication many factors can result in DNA replication stress. The DNA replication stress checkpoint prevents the accumulation of replication stress-induced DNA damage and the potential ensuing genome instability. A critical role for post-translational modifications, such as phosphorylation, in the replication stress checkpoint response has been well established. However, recent work has revealed an important role for transcription in the cellular response to DNA replication stress. In this review, we will provide an overview of current knowledge of the cellular response to DNA replication stress with a specific focus on the DNA replication stress checkpoint transcriptional response and its role in the prevention of replication stress-induced DNA damage. PMID:28257104

Does it actually feel right? A replication attempt of the rounded price effect.

Science.gov (United States)

Harms, Christopher; Genau, Hanna A; Meschede, Carolin; Beauducel, André

2018-04-01

How does the roundedness of prices affect product evaluations? The 'rounded price effect' postulates that depending on the context, rounded or non-rounded prices increase the purchase likelihood of consumers. The study presented here is a replication attempt of these findings and the proposed mediation of the effect through a sense of 'feeling right' when evaluating the product. p -Curve analysis and the R-Index are used to assess the robustness of the originally reported statistics since original data were not available. A pre-registered replication of study 5 from the original article was conducted in a sample of N =588 participants. For both the original product and one alternative product neither an interaction between price roundedness and context, nor a mediation through 'a sense of feeling right' was found. Our results suggest that the effect is either smaller than originally reported or contingent on other, not investigated factors. Further studies might investigate contingencies in larger samples.

DNA Copy-Number Control through Inhibition of Replication Fork Progression

Directory of Open Access Journals (Sweden)

Jared T. Nordman

2014-11-01

Full Text Available Proper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell-cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein SUUR through an unknown mechanism. We demonstrate that SUUR is recruited to active replication forks and mediates the repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass spectrometry identification of SUUR-associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. Our results reveal that control of eukaryotic DNA copy number can occur through the inhibition of replication fork progression.

Initiation of Replication in Escherichia coli

DEFF Research Database (Denmark)

Frimodt-Møller, Jakob

The circular chromosome of Escherichia coli is replicated by two replisomes assembled at the unique origin and moving in the opposite direction until they meet in the less well defined terminus. The key protein in initiation of replication, DnaA, facilitates the unwinding of double-stranded DNA...... to single-stranded DNA in oriC. Although DnaA is able to bind both ADP and ATP, DnaA is only active in initiation when bound to ATP. Although initiation of replication, and the regulation of this, is thoroughly investigated it is still not fully understood. The overall aim of the thesis was to investigate...... the regulation of initiation, the effect on the cell when regulation fails, and if regulation was interlinked to chromosomal organization. This thesis uncovers that there exists a subtle balance between chromosome replication and reactive oxygen species (ROS) inflicted DNA damage. Thus, failure in regulation...

LHCb Data Replication During SC3

CERN Multimedia

Smith, A

2006-01-01

LHCb's participation in LCG's Service Challenge 3 involves testing the bulk data transfer infrastructure developed to allow high bandwidth distribution of data across the grid in accordance with the computing model. To enable reliable bulk replication of data, LHCb's DIRAC system has been integrated with gLite's File Transfer Service middleware component to make use of dedicated network links between LHCb computing centres. DIRAC's Data Management tools previously allowed the replication, registration and deletion of files on the grid. For SC3 supplementary functionality has been added to allow bulk replication of data (using FTS) and efficient mass registration to the LFC replica catalog.Provisional performance results have shown that the system developed can meet the expected data replication rate required by the computing model in 2007. This paper details the experience and results of integration and utilisation of DIRAC with the SC3 transfer machinery.

Buffer layers grown by replicating the texture of an original template tape

International Nuclear Information System (INIS)

Lim, Sunme; Yoo, Jaeun; Park, Chan; Youm, Dojun

2007-01-01

We propose a fabrication method of the buffer layers, whose biaxial textures are replicated from an original template tape. The purpose of this method is economical texturing process for coated conductors. At first we prepared a biaxially textured metal tape (TM-tape). Then a sacrifice layer (SA), a buffer layer (BU) and a thick metallic layer (SM) were sequentially deposited on the TM-tape. SA-layer and BU-layer were deposited epitaxially to copy the texture of the TM-tape. SA-layer was dissoluble in water. SM-layer with the textured BU-layer was separated and could be used for a supporting tape for the further growth of a superconducting layer. In this way, it is possible to reuse the original textured TM-tape many times. In this paper, we report the results of our experiments, in which we used a biaxially Ni tape, BaO film, STO film, and a thick Ag film for TM-tape, SA-layer, BU-layer, and SM-layer, respectively. The Ag/STO layers were successfully separated form the Ni tape by dissolving the BaO layer in water. The texture quality of the STO layer was well secured after the separation

The conserved structures of the 5' nontranslated region of Citrus tristeza virus are involved in replication and virion assembly

International Nuclear Information System (INIS)

Gowda, Siddarame; Satyanarayana, Tatineni; Ayllon, Maria A.; Moreno, Pedro; Flores, Ricardo; Dawson, William O.

2003-01-01

The genomic RNA of different isolates of Citrus tristeza virus (CTV) reveals an unusual pattern of sequence diversity: the 3' halves are highly conserved (homology >90%), while the 5' halves show much more dissimilarity, with the 5' nontranslated region (NTR) containing the highest diversity (homology as low as 42%). Yet, positive-sense sequences of the 5' NTR were predicted to fold into nearly identical structures consisting of two stem-loops (SL1 and SL2) separated by a short spacer region. The predicted most stable secondary structures of the negative-sense sequences were more variable. We introduced mutations into the 5' NTR of a CTV replicon to alter the sequence and/or the predicted secondary structures with or without additional compensatory changes designed to restore predicted secondary structures, and examined their effect on replication in transfected protoplasts. The results suggested that the predicted secondary structures of the 5' NTR were more important for replication than the primary structure. Most mutations that were predicted to disrupt the secondary structures fail to replicate, while compensatory mutations were allowed replication to resume. The 5' NTR mutations that were tolerated by the CTV replicon were examined in the full-length virus for effects on replication and production of the multiple subgenomic RNAs. Additionally, the ability of these mutants to produce virions was monitored by electron microscopy and by passaging the progeny nucleocapsids to another batch of protoplasts. Some of the mutants with compensatory sequence alterations predicted to rebuild similar secondary structures allowed replication at near wild-type levels but failed to passage, suggesting that the 5' NTR contains sequences required for both replication and virion assembly

Ultrastructural Characterization of Zika Virus Replication Factories

Directory of Open Access Journals (Sweden)

Mirko Cortese

2017-02-01

Full Text Available Summary: A global concern has emerged with the pandemic spread of Zika virus (ZIKV infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus replicating in virus-induced membranous replication factories (RFs. Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles. Analyses of human hepatic cells and neural progenitor cells infected with ZIKV revealed endoplasmic reticulum (ER membrane invaginations containing pore-like openings toward the cytosol, reminiscent to RFs in Dengue virus-infected cells. Both the MR766 African strain and the H/PF/2013 Asian strain, the latter linked to neurological diseases, induce RFs of similar architecture. Importantly, ZIKV infection causes a drastic reorganization of microtubules and intermediate filaments forming cage-like structures surrounding the viral RF. Consistently, ZIKV replication is suppressed by cytoskeleton-targeting drugs. Thus, ZIKV RFs are tightly linked to rearrangements of the host cell cytoskeleton. : Cortese et al. show that ZIKV infection in both human hepatoma and neuronal progenitor cells induces drastic structural modification of the cellular architecture. Microtubules and intermediate filaments surround the viral replication factory composed of vesicles corresponding to ER membrane invagination toward the ER lumen. Importantly, alteration of microtubule flexibility impairs ZIKV replication. Keywords: Zika virus, flavivirus, human neural progenitor cells, replication factories, replication organelles, microtubules, intermediate filaments, electron microscopy, electron tomography, live-cell imaging

Tombusviruses upregulate phospholipid biosynthesis via interaction between p33 replication protein and yeast lipid sensor proteins during virus replication in yeast

International Nuclear Information System (INIS)

Barajas, Daniel; Xu, Kai; Sharma, Monika; Wu, Cheng-Yu; Nagy, Peter D.

2014-01-01

Positive-stranded RNA viruses induce new membranous structures and promote membrane proliferation in infected cells to facilitate viral replication. In this paper, the authors show that a plant-infecting tombusvirus upregulates transcription of phospholipid biosynthesis genes, such as INO1, OPI3 and CHO1, and increases phospholipid levels in yeast model host. This is accomplished by the viral p33 replication protein, which interacts with Opi1p FFAT domain protein and Scs2p VAP protein. Opi1p and Scs2p are phospholipid sensor proteins and they repress the expression of phospholipid genes. Accordingly, deletion of OPI1 transcription repressor in yeast has a stimulatory effect on TBSV RNA accumulation and enhanced tombusvirus replicase activity in an in vitro assay. Altogether, the presented data convincingly demonstrate that de novo lipid biosynthesis is required for optimal TBSV replication. Overall, this work reveals that a (+)RNA virus reprograms the phospholipid biosynthesis pathway in a unique way to facilitate its replication in yeast cells. - Highlights: • Tombusvirus p33 replication protein interacts with FFAT-domain host protein. • Tombusvirus replication leads to upregulation of phospholipids. • Tombusvirus replication depends on de novo lipid synthesis. • Deletion of FFAT-domain host protein enhances TBSV replication. • TBSV rewires host phospholipid synthesis

Regulation of beta cell replication

DEFF Research Database (Denmark)

Lee, Ying C; Nielsen, Jens Høiriis

2008-01-01

Beta cell mass, at any given time, is governed by cell differentiation, neogenesis, increased or decreased cell size (cell hypertrophy or atrophy), cell death (apoptosis), and beta cell proliferation. Nutrients, hormones and growth factors coupled with their signalling intermediates have been...... suggested to play a role in beta cell mass regulation. In addition, genetic mouse model studies have indicated that cyclins and cyclin-dependent kinases that determine cell cycle progression are involved in beta cell replication, and more recently, menin in association with cyclin-dependent kinase...... inhibitors has been demonstrated to be important in beta cell growth. In this review, we consider and highlight some aspects of cell cycle regulation in relation to beta cell replication. The role of cell cycle regulation in beta cell replication is mostly from studies in rodent models, but whether...

Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation

Energy Technology Data Exchange (ETDEWEB)

Eyre, Nicholas S., E-mail: nicholas.eyre@adelaide.edu.au [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia); Hampton-Smith, Rachel J.; Aloia, Amanda L. [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia); Eddes, James S. [Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide (Australia); Simpson, Kaylene J. [Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne (Australia); The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville (Australia); Hoffmann, Peter [Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide (Australia); Institute for Photonics and Advanced Sensing (IPAS), University of Adelaide, Adelaide (Australia); Beard, Michael R. [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia)

2016-04-15

Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using ‘APEX2’-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using a customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα. - Highlights: • NS5A residues Ser-222, Ser-235 and Thr-348 are phosphorylated during HCV infection. • Phosphorylation of Ser-235 is essential to HCV RNA replication. • Mutation of Ser-235 alters replication compartment localization and morphology. • Phosphatidylinositol-4 kinase III alpha is important for Ser-235 phosphorylation.

Uncoupling of Sister Replisomes during Eukaryotic DNA Replication

NARCIS (Netherlands)

Yardimci, Hasan; Loveland, Anna B.; Habuchi, Satoshi; van Oijen, Antoine M.; Walter, Johannes C.

2010-01-01

The duplication of eukaryotic genomes involves the replication of DNA from multiple origins of replication. In S phase, two sister replisomes assemble at each active origin, and they replicate DNA in opposite directions. Little is known about the functional relationship between sister replisomes.

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

NARCIS (Netherlands)

Tanner, Nathan A.; Loparo, Joseph J.; Oijen, Antoine M. van

2009-01-01

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides. The

Dynamic behavior of DNA replication domains

NARCIS (Netherlands)

Manders, E. M.; Stap, J.; Strackee, J.; van Driel, R.; Aten, J. A.

1996-01-01

Like many nuclear processes, DNA replication takes place in distinct domains that are scattered throughout the S-phase nucleus. Recently we have developed a fluorescent double-labeling procedure that allows us to visualize nascent DNA simultaneously with "newborn" DNA that had replicated earlier in

The evolutionary ecology of molecular replicators.

Science.gov (United States)

Nee, Sean

2016-08-01

By reasonable criteria, life on the Earth consists mainly of molecular replicators. These include viruses, transposons, transpovirons, coviruses and many more, with continuous new discoveries like Sputnik Virophage. Their study is inherently multidisciplinary, spanning microbiology, genetics, immunology and evolutionary theory, and the current view is that taking a unified approach has great power and promise. We support this with a new, unified, model of their evolutionary ecology, using contemporary evolutionary theory coupling the Price equation with game theory, studying the consequences of the molecular replicators' promiscuous use of each others' gene products for their natural history and evolutionary ecology. Even at this simple expository level, we can make a firm prediction of a new class of replicators exploiting viruses such as lentiviruses like SIVs, a family which includes HIV: these have been explicitly stated in the primary literature to be non-existent. Closely connected to this departure is the view that multicellular organism immunology is more about the management of chronic infections rather than the elimination of acute ones and new understandings emerging are changing our view of the kind of theatre we ourselves provide for the evolutionary play of molecular replicators. This study adds molecular replicators to bacteria in the emerging field of sociomicrobiology.

RNA-seq: technical variability and sampling

Science.gov (United States)

2011-01-01

Background RNA-seq is revolutionizing the way we study transcriptomes. mRNA can be surveyed without prior knowledge of gene transcripts. Alternative splicing of transcript isoforms and the identification of previously unknown exons are being reported. Initial reports of differences in exon usage, and splicing between samples as well as quantitative differences among samples are beginning to surface. Biological variation has been reported to be larger than technical variation. In addition, technical variation has been reported to be in line with expectations due to random sampling. However, strategies for dealing with technical variation will differ depending on the magnitude. The size of technical variance, and the role of sampling are examined in this manuscript. Results In this study three independent Solexa/Illumina experiments containing technical replicates are analyzed. When coverage is low, large disagreements between technical replicates are apparent. Exon detection between technical replicates is highly variable when the coverage is less than 5 reads per nucleotide and estimates of gene expression are more likely to disagree when coverage is low. Although large disagreements in the estimates of expression are observed at all levels of coverage. Conclusions Technical variability is too high to ignore. Technical variability results in inconsistent detection of exons at low levels of coverage. Further, the estimate of the relative abundance of a transcript can substantially disagree, even when coverage levels are high. This may be due to the low sampling fraction and if so, it will persist as an issue needing to be addressed in experimental design even as the next wave of technology produces larger numbers of reads. We provide practical recommendations for dealing with the technical variability, without dramatic cost increases. PMID:21645359

Contextual mediation of perceptions during hauntings and poltergeist-like experiences: a replication and extension.

Science.gov (United States)

Harte, T M

2000-10-01

This study is a replication of the experiment by Lange, Houran, Harte, and Havens (1996 on contextual variables, in which hallucinations appear to be affected by the environmental context. These contextual variables are influential in the reporting of haunting and poltergeist-like episodes. This study extended the previous study by adding new factors of time of day, climactic conditions, and emotional feelings. These were analyzed for a different sample, looking for further congruency between experiential content and the context. The sample (N=8431 were reports found on the Internet and in one book. The Lange, et al. study was replicated in that contextual variables were identified in 99.2% of the reports, the content of the reports was judged to be consistent with the nature of the contextual variables in 58.8% of the reports, and contextual variables were related to the percipients' state of arousal and the modalities of experience.

A Fuzzy Modeling Approach for Replicated Response Measures Based on Fuzzification of Replications with Descriptive Statistics and Golden Ratio

Directory of Open Access Journals (Sweden)

Özlem TÜRKŞEN

2018-03-01

Full Text Available Some of the experimental designs can be composed of replicated response measures in which the replications cannot be identified exactly and may have uncertainty different than randomness. Then, the classical regression analysis may not be proper to model the designed data because of the violation of probabilistic modeling assumptions. In this case, fuzzy regression analysis can be used as a modeling tool. In this study, the replicated response values are newly formed to fuzzy numbers by using descriptive statistics of replications and golden ratio. The main aim of the study is obtaining the most suitable fuzzy model for replicated response measures through fuzzification of the replicated values by taking into account the data structure of the replications in statistical framework. Here, the response and unknown model coefficients are considered as triangular type-1 fuzzy numbers (TT1FNs whereas the inputs are crisp. Predicted fuzzy models are obtained according to the proposed fuzzification rules by using Fuzzy Least Squares (FLS approach. The performances of the predicted fuzzy models are compared by using Root Mean Squared Error (RMSE criteria. A data set from the literature, called wheel cover component data set, is used to illustrate the performance of the proposed approach and the obtained results are discussed. The calculation results show that the combined formulation of the descriptive statistics and the golden ratio is the most preferable fuzzification rule according to the well-known decision making method, called TOPSIS, for the data set.

Separation and sampling technique of light element isotopes by chemical exchange process

International Nuclear Information System (INIS)

Kato, Shunsaku; Oi, Kenta; Takagi, Norio; Hirotsu, Takafumi; Kano, Hirofumi; Sonoda, Akinari; Makita, Yoji

2000-01-01

Lithium and boron isotope separation technique were studied. Granulation of lithium isotope separation agent was carried out by cure covering in solution. Separation of lithium isotope was stepped up by ammonium carbonate used as elusion agent. Styrene and ester resin derived three kinds of agents such as 2-amino-1, 3-propanediol (1, 3-PD), 2-amino-2-methyl-1, 3-propanediol (Me-1,3-PD) and tris(2-hydroxyethyl)amine (Tris) were used as absorbent.The ester resin with Tris showed larger amount of adsorption (1.4 mmol/g) than other resins. However, all resins with agent indicated more large adsorption volume of boron than the objective value (0.5 mmol/g). Large isotope shift was shown by the unsymmetrical vibration mode of lithium ion on the basis of quantum chemical calculation of isotope effect on dehydration of hydrated lithium ion. (S.Y.)

Replication Improves Sorting-Task Results Analyzed by DISTATIS in a Consumer Study of American Bourbon and Rye Whiskeys.

Science.gov (United States)

Lahne, Jacob; Collins, Thomas S; Heymann, Hildegarde

2016-05-01

In consumer food-sensory studies, sorting and closely related methods (for example, projective mapping) have often been applied to large product sets which are complex and fatiguing for panelists. Analysis of sorting by Multi-Dimensional Scaling (MDS) is common, but this method discards relevant individual decisions; analysis by DISTATIS, which accounts for individual differences, is gaining acceptance. This research posits that replication can improve DISTATIS analysis by stabilizing consumer sensory maps, which are often extremely unstable. As a case study a fatiguing product set was sorted: 10 American whiskeys-5 bourbons and 5 ryes-were sorted into groups by 21 consumers over 2 replications. These products were chosen because American whiskeys are some of the most important distilled beverages in today's market; in particular, "bourbon" (mashbill more than 50% corn) and "rye" (more than 50% rye) whiskeys are important and assumed to be products with distinct sensory attributes. However, there is almost no scientific information about their sensory properties. Data were analyzed using standard and aggregated DISTATIS and MDS. No significant relationship between mashbill and consumer categorization in whiskeys was found; instead, there was evidence of producer and aging effects. aggregated DISTATIS was found to provide more stable results than without replication, and DISTATIS results provided a number of benefits over MDS, including bootstrapped confidence intervals for product separation. In addition, this is the first published evidence that mashbill does not determine sensory properties of American whiskey: bourbons and ryes, while legally distinct, were not separated by consumers. © 2016 Institute of Food Technologists®

Rapid transient production in plants by replicating and non-replicating vectors yields high quality functional anti-HIV antibody.

Directory of Open Access Journals (Sweden)

Frank Sainsbury

2010-11-01

Full Text Available The capacity of plants and plant cells to produce large amounts of recombinant protein has been well established. Due to advantages in terms of speed and yield, attention has recently turned towards the use of transient expression systems, including viral vectors, to produce proteins of pharmaceutical interest in plants. However, the effects of such high level expression from viral vectors and concomitant effects on host cells may affect the quality of the recombinant product.To assess the quality of antibodies transiently expressed to high levels in plants, we have expressed and characterised the human anti-HIV monoclonal antibody, 2G12, using both replicating and non-replicating systems based on deleted versions of Cowpea mosaic virus (CPMV RNA-2. The highest yield (approximately 100 mg/kg wet weight leaf tissue of affinity purified 2G12 was obtained when the non-replicating CPMV-HT system was used and the antibody was retained in the endoplasmic reticulum (ER. Glycan analysis by mass-spectrometry showed that the glycosylation pattern was determined exclusively by whether the antibody was retained in the ER and did not depend on whether a replicating or non-replicating system was used. Characterisation of the binding and neutralisation properties of all the purified 2G12 variants from plants showed that these were generally similar to those of the Chinese hamster ovary (CHO cell-produced 2G12.Overall, the results demonstrate that replicating and non-replicating CPMV-based vectors are able to direct the production of a recombinant IgG similar in activity to the CHO-produced control. Thus, a complex recombinant protein was produced with no apparent effect on its biochemical properties using either high-level expression or viral replication. The speed with which a recombinant pharmaceutical with excellent biochemical characteristics can be produced transiently in plants makes CPMV-based expression vectors an attractive option for

Dynamics of picornavirus RNA replication within infected cells

DEFF Research Database (Denmark)

Belsham, Graham; Normann, Preben

2008-01-01

Replication of many picornaviruses is inhibited by low concentrations of guanidine. Guanidine-resistant mutants are readily isolated and the mutations map to the coding region for the 2C protein. Using in vitro replication assays it has been determined previously that guanidine blocks the initiat......Replication of many picornaviruses is inhibited by low concentrations of guanidine. Guanidine-resistant mutants are readily isolated and the mutations map to the coding region for the 2C protein. Using in vitro replication assays it has been determined previously that guanidine blocks...... the initiation of negative-strand synthesis. We have now examined the dynamics of RNA replication, measured by quantitative RT-PCR, within cells infected with either swine vesicular disease virus (an enterovirus) or foot-and-mouth disease virus as regulated by the presence or absence of guanidine. Following...... the removal of guanidine from the infected cells, RNA replication occurs after a significant lag phase. This restoration of RNA synthesis requires de novo protein synthesis. Viral RNA can be maintained for at least 72 h within cells in the absence of apparent replication but guanidine-resistant virus can...

Pyrimidine dimers block simian virus 40 replication forks

International Nuclear Information System (INIS)

Berger, C.A.; Edenberg, H.J.

1986-01-01

UV light produces lesions, predominantly pyrimidine dimers, which inhibit DNA replication in mammalian cells. The mechanism of inhibition is controversial: is synthesis of a daughter strand halted at a lesion while the replication fork moves on and reinitiates downstream, or is fork progression itself blocked for some time at the site of a lesion? We directly addressed this question by using electron microscopy to examine the distances of replication forks from the origin in unirradiated and UV-irradiated simian virus 40 chromosomes. If UV lesions block replication fork progression, the forks should be asymmetrically located in a large fraction of the irradiated molecules; if replication forks move rapidly past lesions, the forks should be symmetrically located. A large fraction of the simian virus 40 replication forks in irradiated molecules were asymmetrically located, demonstrating that UV lesions present at the frequency of pyrimidine dimers block replication forks. As a mechanism for this fork blockage, we propose that polymerization of the leading strand makes a significant contribution to the energetics of fork movement, so any lesion in the template for the leading strand which blocks polymerization should also block fork movement

Initiation preference at a yeast origin of replication.

Science.gov (United States)

Brewer, B J; Fangman, W L

1994-04-12

Replication origins in the yeast Saccharomyces cerevisiae are identified as autonomous replication sequence (ARS) elements. To examine the effect of origin density on replication initiation, we have analyzed the replication of a plasmid that contains two copies of the same origin, ARS1. The activation of origins and the direction that replication forks move through flanking sequences can be physically determined by analyzing replication intermediates on two-dimensional agarose gels. We find that only one of the two identical ARSs on the plasmid initiates replication on any given plasmid molecule; that is, this close spacing of ARSs results in an apparent interference between the potential origins. Moreover, in the particular plasmid that we constructed, one of the two identical copies of ARS1 is used four times more frequently than the other one. These results show that the plasmid context is critical for determining the preferred origin. This origin preference is also exhibited when the tandem copies of ARS1 are introduced into a yeast chromosome. The sequences responsible for establishing the origin preference have been identified by deletion analysis and are found to reside in a portion of the yeast URA3 gene.

Mapping replication origins in yeast chromosomes.

Science.gov (United States)

Brewer, B J; Fangman, W L

1991-07-01

The replicon hypothesis, first proposed in 1963 by Jacob and Brenner, states that DNA replication is controlled at sites called origins. Replication origins have been well studied in prokaryotes. However, the study of eukaryotic chromosomal origins has lagged behind, because until recently there has been no method for reliably determining the identity and location of origins from eukaryotic chromosomes. Here, we review a technique we developed with the yeast Saccharomyces cerevisiae that allows both the mapping of replication origins and an assessment of their activity. Two-dimensional agarose gel electrophoresis and Southern hybridization with total genomic DNA are used to determine whether a particular restriction fragment acquires the branched structure diagnostic of replication initiation. The technique has been used to localize origins in yeast chromosomes and assess their initiation efficiency. In some cases, origin activation is dependent upon the surrounding context. The technique is also being applied to a variety of eukaryotic organisms.

Assembly of Slx4 signaling complexes behind DNA replication forks.

Science.gov (United States)

Balint, Attila; Kim, TaeHyung; Gallo, David; Cussiol, Jose Renato; Bastos de Oliveira, Francisco M; Yimit, Askar; Ou, Jiongwen; Nakato, Ryuichiro; Gurevich, Alexey; Shirahige, Katsuhiko; Smolka, Marcus B; Zhang, Zhaolei; Brown, Grant W

2015-08-13

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress. © 2015 The Authors.

The Multi-Template Molecularly Imprinted Polymer Based on SBA-15 for Selective Separation and Determination of Panax notoginseng Saponins Simultaneously in Biological Samples

Directory of Open Access Journals (Sweden)

Chenghong Sun

2017-11-01

Full Text Available The feasible, reliable and selective multi-template molecularly imprinted polymers (MT-MIPs based on SBA-15 (SBA-15@MT-MIPs for the selective separation and determination of the trace level of ginsenoside Rb1 (Rb1, ginsenoside Rg1 (Rg1 and notoginsenoside R1 (R1 simultaneously from biological samples were developed. The polymers were constructed by SBA-15 as support, Rb1, Rg1, R1 as multi-template, acrylamide (AM as functional monomer and ethylene glycol dimethacrylate (EGDMA as cross-linker. The new synthetic SBA-15@MT-MIPs were satisfactorily applied to solid-phase extraction (SPE coupled with high performance liquid chromatography (HPLC for the separation and determination of trace Rb1, Rg1 and R1 in plasma samples. Under the optimized conditions, the limits of detection (LODs and quantitation (LOQs of the proposed method for Rb1, Rg1 and R1 were in the range of 0.63–0.75 ng·mL−1 and 2.1–2.5 ng·mL−1, respectively. The recoveries of R1, Rb1 and Rg1 were obtained between 93.4% and 104.3% with relative standard deviations (RSDs in the range of 3.3–4.2%. All results show that the obtained SBA-15@MT-MIPs could be a promising prospect for the practical application in the selective separation and enrichment of trace Panax notoginseng saponins (PNS in the biological samples.

Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis

Energy Technology Data Exchange (ETDEWEB)

Kai, Mihoko; Wang, Teresa S.-F

2003-11-27

Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Pol{kappa}). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks. Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development.

Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis

International Nuclear Information System (INIS)

Kai, Mihoko; Wang, Teresa S.-F.

2003-01-01

Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Polκ). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks. Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development

Right and Wrong and Cultural Diversity: Replication of the 2002 NAS/Zogby Poll on Business Ethics

Science.gov (United States)

Ludlum, Marty; Mascaloinov, Sergei

2004-01-01

In April 2002, a NAS/Zogby poll found that only a quarter of sampled students perceived uniform standards of "right and wrong" and that most students felt that ethical behavior depends on cultural diversity. In this effort to replicate those findings in a larger sample of American college students, the authors obtained results that…

Hyperthermia stimulates HIV-1 replication.

Directory of Open Access Journals (Sweden)

Ferdinand Roesch

Full Text Available HIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42-45°C and Heat Shock Proteins (HSPs modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38-40°C on HIV-1 infection has not been extensively investigated. Here, we show that culturing primary CD4+ T lymphocytes and cell lines at a fever-like temperature (39.5°C increased the efficiency of HIV-1 replication by 2 to 7 fold. Hyperthermia did not facilitate viral entry nor reverse transcription, but increased Tat transactivation of the LTR viral promoter. Hyperthermia also boosted HIV-1 reactivation in a model of latently-infected cells. By imaging HIV-1 transcription, we further show that Hsp90 co-localized with actively transcribing provirus, and this phenomenon was enhanced at 39.5°C. The Hsp90 inhibitor 17-AAG abrogated the increase of HIV-1 replication in hyperthermic cells. Altogether, our results indicate that fever may directly stimulate HIV-1 replication, in a process involving Hsp90 and facilitation of Tat-mediated LTR activity.

T135I substitution in the nonstructural protein 2C enhances foot-and-mouth disease virus replication.

Science.gov (United States)

Yuan, Tiangang; Wang, Haiwei; Li, Chen; Yang, Decheng; Zhou, Guohui; Yu, Li

2017-12-01

The foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays an important role in viral replication, virulence, and host range. It has been shown that deletions of 10 or 19-20 amino acids in the C-terminal half of 3A attenuate serotype O and C FMDVs, which replicate poorly in bovine cells but normally in porcine-derived cells, and the C-terminal half of 3A is not essential for serotype Asia1 FMDV replication in BHK-21 cells. In this study, we constructed a 3A deletion FMDV mutant based on a serotype O FMDV, the wild-type virus O/YS/CHA/05, with a 60-amino acid deletion in the 3A protein sequence, between residues 84 and 143. The rescued virus O/YS/CHA/05-Δ3A exhibited slower growth kinetics and formed smaller plaques compared to O/YS/CHA/05 in both BHK-21 and IBRS-2 cells, indicating that the 60-amino acid deletion in the 3A protein impaired FMDV replication. After 14 passages in BHK-21 cells, the replication capacity of the passaged virus O/YS/CHA/05-Δ3A-P14 returned to a level similar to the wild-type virus, suggesting that amino acid substitutions responsible for the enhanced replication capacity occurred in the genome of O/YS/CHA/05-Δ3A-P14. By sequence analysis, two amino acid substitutions, P153L in VP1 and T135I in 2C, were found in the O/YS/CHA/05-Δ3A-P14 genome compared to the O/YS/CHA/05-Δ3A genome. Subsequently, the amino acid substitutions VP1 P153L and 2C T135I were separately introduced into O/YS/CHA/05-Δ3A to rescue mutant viruses for examining their growth kinetics. Results showed that the 2C T135I instead of the VP1 P153L enhanced the virus replication capacity. The 2C T135I substitution also improved the replication of the wild-type virus, indicating that the effect of 2C T135I substitution on FMDV replication is not associated with the 3A deletion. Furthermore, our results showed that the T135I substitution in the nonstructural protein 2C enhanced O/YS/CHA/05 replication through promoting viral RNA synthesis.

Replication and Robustness in Developmental Research

Science.gov (United States)

Duncan, Greg J.; Engel, Mimi; Claessens, Amy; Dowsett, Chantelle J.

2014-01-01

Replications and robustness checks are key elements of the scientific method and a staple in many disciplines. However, leading journals in developmental psychology rarely include explicit replications of prior research conducted by different investigators, and few require authors to establish in their articles or online appendices that their key…

Systematic Sampling and Cluster Sampling of Packet Delays

OpenAIRE

Lindh, Thomas

2006-01-01

Based on experiences of a traffic flow performance meter this papersuggests and evaluates cluster sampling and systematic sampling as methods toestimate average packet delays. Systematic sampling facilitates for exampletime analysis, frequency analysis and jitter measurements. Cluster samplingwith repeated trains of periodically spaced sampling units separated by randomstarting periods, and systematic sampling are evaluated with respect to accuracyand precision. Packet delay traces have been ...

Biomarkers of replicative senescence revisited

DEFF Research Database (Denmark)

Nehlin, Jan

2016-01-01

Biomarkers of replicative senescence can be defined as those ultrastructural and physiological variations as well as molecules whose changes in expression, activity or function correlate with aging, as a result of the gradual exhaustion of replicative potential and a state of permanent cell cycle...... arrest. The biomarkers that characterize the path to an irreversible state of cell cycle arrest due to proliferative exhaustion may also be shared by other forms of senescence-inducing mechanisms. Validation of senescence markers is crucial in circumstances where quiescence or temporary growth arrest may...... be triggered or is thought to be induced. Pre-senescence biomarkers are also important to consider as their presence indicate that induction of aging processes is taking place. The bona fide pathway leading to replicative senescence that has been extensively characterized is a consequence of gradual reduction...

Quantification of fatty acids as methyl esters and phospholipids in cheese samples after separation of triacylglycerides and phospholipids.

Science.gov (United States)

Hauff, Simone; Vetter, Walter

2009-03-23

Determination of the individual fatty acid composition of neutral- and phospholipids as well as the phospholipid content of dairy food and other foodstuffs are important tasks in life sciences. For these purposes, a method was developed for the separation of lipids (standards of triolein and diacylphosphatidylcholines as well as three cheese samples) by solid-phase extraction using a self-packed column filled with partly deactivated silica. Non-halogenated solvents were used for the elution of the lipid classes. Cyclohexane/ethyl acetate (1:1, v/v) served for the elution of neutral lipids, while polar lipids were eluted with three solvents (ethyl acetate/methanol, methanol, and methanol/water) into one fraction. The separated lipid fractions were transesterified and the individual fatty acids were quantified by using gas chromatography coupled to electron ionization mass spectrometry (GC/EI-MS) in the selected ion monitoring (SIM) mode. The recovery rate for standard phosphatidylcholines was approximately 90% and cross-contamination from neutral lipids was negligible. The method was applied to cheese samples. Quantitative amounts of individual fatty acids in the phospholipid fraction were camembert, cheese. Differences in the fatty acid pattern of neutral and polar lipids were detected. The quantity of the fatty acids determined in the phospholipid fraction was divided by the factor 0.7 in order to convert the fatty acid content into the phospholipid content of the cheese samples. This factor is based on the contribution of 16:0 to dipalmitoylphosphatidylcholine (DPPC). The resulting DPPC equivalents (DPPC(eq)) were found to be representative for the average contribution of fatty acids to all classes of phospholipids in dairy products. Using this approach, the phospholipid content of lipids from mozzarella, camembert, and goat cream cheese was 0.60%, 1.42% and 0.79%, respectively.

Intrapulmonary Human Cytomegalovirus Replication in Lung Transplant Recipients Is Associated With a Rise of CCL-18 and CCL-20 Chemokine Levels.

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Weseslindtner, Lukas; Görzer, Irene; Roedl, Kevin; Küng, Erik; Jaksch, Peter; Klepetko, Walter; Puchhammer-Stöckl, Elisabeth

2017-01-01

In lung transplant recipients (LTRs), human cytomegalovirus (HCMV) DNA detection in the bronchoalveolar lavage fluid (BALF) indicates HCMV replication in the pulmonary compartment. Such local HCMV replication episodes may remain asymptomatic or may lead to symptomatic HCMV disease. Here, we investigated LTRs with intrapulmonary HCMV replication for the chemokines CCL-18 and CCL-20. In particular, we analyzed whether these chemokines rise in the allograft and/or the blood and are associated with HCMV disease. CCL-18 and CCL-20 levels were quantitated by ELISA in BALF and serum samples from 60 LTRs. During the posttransplant follow-up, these LTRs displayed HCMV DNA detection in the BALF by PCR, whereas other infectious agents were undetectable. Furthermore, we investigated samples from 10 controls who did not display any HCMV replication episode during the follow-up. HCMV replication in the allograft was associated with a significant increase of CCL-18 and CCL-20 BALF levels (P Wilcoxon signed-rank test) and a significant rise of CCL-20 (P Wilcoxon signed-rank test) but not of CCL-18 in the blood. In controls, no such chemokine increase was observed. Furthermore, CCL-18 BALF levels were significantly higher in 8 LTRs who additionally developed HCMV disease, as compared with the other 52 patients in whom HCMV replication remained asymptomatic (P test). HCMV replication in the allograft causes an intrapulmonary increase of CCL-18 and CCL-20 and a systemic rise of CCL-20 serum levels. Strong intrapulmonary CCL-18 responses are associated with symptomatic HCMV disease, proposing that CCL-18 BALF levels could serve as a marker.

Enzymes involved in organellar DNA replication in photosynthetic eukaryotes.

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Moriyama, Takashi; Sato, Naoki

2014-01-01

Plastids and mitochondria possess their own genomes. Although the replication mechanisms of these organellar genomes remain unclear in photosynthetic eukaryotes, several organelle-localized enzymes related to genome replication, including DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, primer removal enzyme, and several DNA recombination-related enzymes, have been identified. In the reference Eudicot plant Arabidopsis thaliana, the replication-related enzymes of plastids and mitochondria are similar because many of them are dual targeted to both organelles, whereas in the red alga Cyanidioschyzon merolae, plastids and mitochondria contain different replication machinery components. The enzymes involved in organellar genome replication in green plants and red algae were derived from different origins, including proteobacterial, cyanobacterial, and eukaryotic lineages. In the present review, we summarize the available data for enzymes related to organellar genome replication in green plants and red algae. In addition, based on the type and distribution of replication enzymes in photosynthetic eukaryotes, we discuss the transitional history of replication enzymes in the organelles of plants.

Viral hijacking of a replicative helicase loader and its implications for helicase loading control and phage replication

Energy Technology Data Exchange (ETDEWEB)

Hood, Iris V.; Berger, James M.

2016-05-31

Replisome assembly requires the loading of replicative hexameric helicases onto origins by AAA+ ATPases. How loader activity is appropriately controlled remains unclear. Here, we use structural and biochemical analyses to establish how an antimicrobial phage protein interferes with the function of theStaphylococcus aureusreplicative helicase loader, DnaI. The viral protein binds to the loader’s AAA+ ATPase domain, allowing binding of the host replicative helicase but impeding loader self-assembly and ATPase activity. Close inspection of the complex highlights an unexpected locus for the binding of an interdomain linker element in DnaI/DnaC-family proteins. We find that the inhibitor protein is genetically coupled to a phage-encoded homolog of the bacterial helicase loader, which we show binds to the host helicase but not to the inhibitor itself. These findings establish a new approach by which viruses can hijack host replication processes and explain how loader activity is internally regulated to prevent aberrant auto-association.

The transcription elongation factor Bur1-Bur2 interacts with replication protein A and maintains genome stability during replication stress

DEFF Research Database (Denmark)

Clausing, Emanuel; Mayer, Andreas; Chanarat, Sittinan

2010-01-01

Multiple DNA-associated processes such as DNA repair, replication, and recombination are crucial for the maintenance of genome integrity. Here, we show a novel interaction between the transcription elongation factor Bur1-Bur2 and replication protein A (RPA), the eukaryotic single-stranded DNA......-binding protein with functions in DNA repair, recombination, and replication. Bur1 interacted via its C-terminal domain with RPA, and bur1-¿C mutants showed a deregulated DNA damage response accompanied by increased sensitivity to DNA damage and replication stress as well as increased levels of persisting Rad52...... foci. Interestingly, the DNA damage sensitivity of an rfa1 mutant was suppressed by bur1 mutation, further underscoring a functional link between these two protein complexes. The transcription elongation factor Bur1-Bur2 interacts with RPA and maintains genome integrity during DNA replication stress....

Fractional Response Models - A Replication Exercise of Papke and Wooldridge (1996

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Harald Oberhofer

2012-09-01

Full Text Available This paper replicates the estimates of a fractional response model for share data reported in the seminal paper of Leslie E. Papke and Jeffrey M. Wooldridge published in the Journal of Applied Econometrics 11(6, 1996, pp.619-632. We have been able to replicate all of the reported estimation results concerning the determinants of employee participation rates in 401(k pension plans using the standard routines provided in Stata. As an alternative, we estimate a two-part model that is capable of coping with the excessive number of boundary values equalling one in the data. The estimated marginal effects are similar to those derived in the paper. A small-scale Monte Carlo simulation exercise suggests that the RESET tests proposed by Papke and Wooldridge in their robust form are useful for detecting neglected non-linearities in small samples.

Method of magnetic separation and apparatus therefore

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Oder, Robin R. (Inventor)

1991-01-01

An apparatus for magnetically separating and collecting particulate matter fractions of a raw sample according to relative magnetic susceptibilities of each fraction so collected is disclosed. The separation apparatus includes a splitter which is used in conjunction with a magnetic separator for achieving the desired fractionation.

Modeling DNA Replication.

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Bennett, Joan

1998-01-01

Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

Distinct functions of human RecQ helicases during DNA replication.

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Urban, Vaclav; Dobrovolna, Jana; Janscak, Pavel

2017-06-01

DNA replication is the most vulnerable process of DNA metabolism in proliferating cells and therefore it is tightly controlled and coordinated with processes that maintain genomic stability. Human RecQ helicases are among the most important factors involved in the maintenance of replication fork integrity, especially under conditions of replication stress. RecQ helicases promote recovery of replication forks being stalled due to different replication roadblocks of either exogenous or endogenous source. They prevent generation of aberrant replication fork structures and replication fork collapse, and are involved in proper checkpoint signaling. The essential role of human RecQ helicases in the genome maintenance during DNA replication is underlined by association of defects in their function with cancer predisposition. Copyright © 2016 Elsevier B.V. All rights reserved.

Evolution of complexity in RNA-like replicator systems

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Hogeweg Paulien

2008-03-01

Full Text Available Abstract Background The evolution of complexity is among the most important questions in biology. The evolution of complexity is often observed as the increase of genetic information or that of the organizational complexity of a system. It is well recognized that the formation of biological organization – be it of molecules or ecosystems – is ultimately instructed by the genetic information, whereas it is also true that the genetic information is functional only in the context of the organization. Therefore, to obtain a more complete picture of the evolution of complexity, we must study the evolution of both information and organization. Results Here we investigate the evolution of complexity in a simulated RNA-like replicator system. The simplicity of the system allows us to explicitly model the genotype-phenotype-interaction mapping of individual replicators, whereby we avoid preconceiving the functionality of genotypes (information or the ecological organization of replicators in the model. In particular, the model assumes that interactions among replicators – to replicate or to be replicated – depend on their secondary structures and base-pair matching. The results showed that a population of replicators, originally consisting of one genotype, evolves to form a complex ecosystem of up to four species. During this diversification, the species evolve through acquiring unique genotypes with distinct ecological functionality. The analysis of this diversification reveals that parasitic replicators, which have been thought to destabilize the replicator's diversity, actually promote the evolution of diversity through generating a novel "niche" for catalytic replicators. This also makes the current replicator system extremely stable upon the evolution of parasites. The results also show that the stability of the system crucially depends on the spatial pattern formation of replicators. Finally, the evolutionary dynamics is shown to

Molecular Mechanisms of DNA Replication Checkpoint Activation

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Bénédicte Recolin

2014-03-01

Full Text Available The major challenge of the cell cycle is to deliver an intact, and fully duplicated, genetic material to the daughter cells. To this end, progression of DNA synthesis is monitored by a feedback mechanism known as replication checkpoint that is untimely linked to DNA replication. This signaling pathway ensures coordination of DNA synthesis with cell cycle progression. Failure to activate this checkpoint in response to perturbation of DNA synthesis (replication stress results in forced cell division leading to chromosome fragmentation, aneuploidy, and genomic instability. In this review, we will describe current knowledge of the molecular determinants of the DNA replication checkpoint in eukaryotic cells and discuss a model of activation of this signaling pathway crucial for maintenance of genomic stability.

A new MCM modification cycle regulates DNA replication initiation.

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Wei, Lei; Zhao, Xiaolan

2016-03-01

The MCM DNA helicase is a central regulatory target during genome replication. MCM is kept inactive during G1, and it initiates replication after being activated in S phase. During this transition, the only known chemical change to MCM is the gain of multisite phosphorylation that promotes cofactor recruitment. Because replication initiation is intimately linked to multiple biological cues, additional changes to MCM can provide further regulatory points. Here, we describe a yeast MCM SUMOylation cycle that regulates replication. MCM subunits undergo SUMOylation upon loading at origins in G1 before MCM phosphorylation. MCM SUMOylation levels then decline as MCM phosphorylation levels rise, thus suggesting an inhibitory role of MCM SUMOylation during replication. Indeed, increasing MCM SUMOylation impairs replication initiation, partly through promoting the recruitment of a phosphatase that decreases MCM phosphorylation and activation. We propose that MCM SUMOylation counterbalances kinase-based regulation, thus ensuring accurate control of replication initiation.

A CI-Independent Form of Replicative Inhibition: Turn Off of Early Replication of Bacteriophage Lambda

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Hayes, Sidney; Horbay, Monique A.; Hayes, Connie

2012-01-01

Several earlier studies have described an unusual exclusion phenotype exhibited by cells with plasmids carrying a portion of the replication region of phage lambda. Cells exhibiting this inhibition phenotype (IP) prevent the plating of homo-immune and hybrid hetero-immune lambdoid phages. We have attempted to define aspects of IP, and show that it is directed to repλ phages. IP was observed in cells with plasmids containing a λ DNA fragment including oop, encoding a short OOP micro RNA, and part of the lambda origin of replication, oriλ, defined by iteron sequences ITN1-4 and an adjacent high AT-rich sequence. Transcription of the intact oop sequence from its promoter, pO is required for IP, as are iterons ITN3–4, but not the high AT-rich portion of oriλ. The results suggest that IP silencing is directed to theta mode replication initiation from an infecting repλ genome, or an induced repλ prophage. Phage mutations suppressing IP, i.e., Sip, map within, or adjacent to cro or in O, or both. Our results for plasmid based IP suggest the hypothesis that there is a natural mechanism for silencing early theta-mode replication initiation, i.e. the buildup of λ genomes with oop + oriλ+ sequence. PMID:22590552

Replication of cultured lung epithelial cells

International Nuclear Information System (INIS)

Guzowski, D.; Bienkowski, R.

1986-01-01

The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during the first week showed that the cells contained myofilaments, microvilli and lamellar bodies (markers for type 2 cells). The following changes were observed after the first week: increase in cell size; loss of lamellar bodies and appearance of multivesicular bodies; increase in rough endoplasmic reticulum and golgi; increase in tonafilaments and well-defined junctions. General cell morphology was good for up to 10 wk. Cells cultured on plastic surface degenerated after 1 wk. Cell replication was assayed by autoradiography of cultures exposed to ( 3 H)-thymidine and by direct cell counts. The cells did not replicate during the first week; however, between 2-10 wk the cells incorporated the label and went through approximately 6 population doublings. They have demonstrated that lung alveolar epithelial cells can replicate in culture if they are maintained on an appropriate substrate. The coincidence of ability to replicate and loss of markers for differentiation may reflect the dichotomy between growth and differentiation commonly observed in developing systems

Is dopamine D1 receptor availability related to social behavior? A positron emission tomography replication study.

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Pontus Plavén-Sigray

Full Text Available Associations between dopamine receptor levels and pro- and antisocial behavior have previously been demonstrated in human subjects using positron emission tomography (PET and self-rated measures of personality traits. So far, only one study has focused on the dopamine D1-receptor (D1-R, finding a positive correlation with the trait social desirability, which is characterized by low dominant and high affiliative behavior, while physical aggression showed a negative correlation. The aim of the present study was to replicate these previous findings using a new independent sample of subjects.Twenty-six healthy males were examined with the radioligand [11C]SCH-23390, and completed the Swedish universities Scales of Personality (SSP which includes measures of social desirability and physical trait aggression. The simplified reference tissue model with cerebellum as reference region was used to calculate BPND values in the whole striatum and limbic striatum. The two regions were selected since they showed strong association between D1-R availability and personality scores in the previous study. Pearson's correlation coefficients and replication Bayes factors were then employed to assess the replicability and robustness of previous results.There were no significant correlations (all p values > 0.3 between regional BPND values and personality scale scores. Replication Bayes factors showed strong to moderate evidence in favor no relationship between D1-receptor availability and social desirability (striatum BF01 = 12.4; limbic striatum BF01 = 7.2 or physical aggression scale scores (limbic striatum BF01 = 3.3, compared to the original correlations.We could not replicate the previous findings of associations between D1-R availability and either pro- or antisocial behavior as measured using the SSP. Rather, there was evidence in favor of failed replications of associations between BPND and scale scores. Potential reasons for these results are restrictive

Spacetime replication of continuous variable quantum information

International Nuclear Information System (INIS)

Hayden, Patrick; Nezami, Sepehr; Salton, Grant; Sanders, Barry C

2016-01-01

The theory of relativity requires that no information travel faster than light, whereas the unitarity of quantum mechanics ensures that quantum information cannot be cloned. These conditions provide the basic constraints that appear in information replication tasks, which formalize aspects of the behavior of information in relativistic quantum mechanics. In this article, we provide continuous variable (CV) strategies for spacetime quantum information replication that are directly amenable to optical or mechanical implementation. We use a new class of homologically constructed CV quantum error correcting codes to provide efficient solutions for the general case of information replication. As compared to schemes encoding qubits, our CV solution requires half as many shares per encoded system. We also provide an optimized five-mode strategy for replicating quantum information in a particular configuration of four spacetime regions designed not to be reducible to previously performed experiments. For this optimized strategy, we provide detailed encoding and decoding procedures using standard optical apparatus and calculate the recovery fidelity when finite squeezing is used. As such we provide a scheme for experimentally realizing quantum information replication using quantum optics. (paper)

COPI is required for enterovirus 71 replication.

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Jianmin Wang

Full Text Available Enterovirus 71 (EV71, a member of the Picornaviridae family, is found in Asian countries where it causes a wide range of human diseases. No effective therapy is available for the treatment of these infections. Picornaviruses undergo RNA replication in association with membranes of infected cells. COPI and COPII have been shown to be involved in the formation of picornavirus-induced vesicles. Replication of several picornaviruses, including poliovirus and Echovirus 11 (EV11, is dependent on COPI or COPII. Here, we report that COPI, but not COPII, is required for EV71 replication. Replication of EV71 was inhibited by brefeldin A and golgicide A, inhibitors of COPI activity. Furthermore, we found EV71 2C protein interacted with COPI subunits by co-immunoprecipitation and GST pull-down assay, indicating that COPI coatomer might be directed to the viral replication complex through viral 2C protein. Additionally, because the pathway is conserved among different species of enteroviruses, it may represent a novel target for antiviral therapies.

Identification and replication of loci involved in camptothecin-induced cytotoxicity using CEPH pedigrees.

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Venita Gresham Watson

2011-05-01

Full Text Available To date, the Centre d'Etude Polymorphism Humain (CEPH cell line model has only been used as a pharmacogenomic tool to evaluate which genes are responsible for the disparity in response to a single drug. The purpose of this study was demonstrate the model's ability to establish a specific pattern of quantitative trait loci (QTL related to a shared mechanism for multiple structurally related drugs, the camptothecins, which are Topoisomerase 1 inhibitors. A simultaneous screen of six camptothecin analogues for in vitro sensitivity in the CEPH cell lines resulted in cytotoxicity profiles and orders of potency which were in agreement with the literature. For all camptothecins studied, heritability estimates for cytotoxic response averaged 23.1 ± 2.6%. Nonparametric linkage analysis was used to identify a relationship between genetic markers and response to the camptothecins. Ten QTLs on chromosomes 1, 3, 5, 6, 11, 12, 16 and 20 were identified as shared by all six camptothecin analogues. In a separate validation experiment, nine of the ten QTLs were replicated at the significant and suggestive levels using three additional camptothecin analogues. To further refine this list of QTLs, another validation study was undertaken and seven of the nine QTLs were independently replicated for all nine camptothecin analogues. This is the first study using the CEPH cell lines that demonstrates that a specific pattern of QTLs could be established for a class of drugs which share a mechanism of action. Moreover, it is the first study to report replication of linkage results for drug-induced cytotoxicity using this model. The QTLs, which have been identified as shared by all camptothecins and replicated across multiple datasets, are of considerable interest; they harbor genes related to the shared mechanism of action for the camptothecins, which are responsible for variation in response.

DNA replication after mutagenic treatment in Hordeum vulgare.

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Kwasniewska, Jolanta; Kus, Arita; Swoboda, Monika; Braszewska-Zalewska, Agnieszka

2016-12-01

The temporal and spatial properties of DNA replication in plants related to DNA damage and mutagenesis is poorly understood. Experiments were carried out to explore the relationships between DNA replication, chromatin structure and DNA damage in nuclei from barley root tips. We quantitavely analysed the topological organisation of replication foci using pulse EdU labelling during the S phase and its relationship with the DNA damage induced by mutagenic treatment with maleic hydrazide (MH), nitroso-N-methyl-urea (MNU) and gamma ray. Treatment with mutagens did not change the characteristic S-phase patterns in the nuclei; however, the frequencies of the S-phase-labelled cells after treatment differed from those observed in the control cells. The analyses of DNA replication in barley nuclei were extended to the micronuclei induced by mutagens. Replication in the chromatin of the micronuclei was rare. The results of simultanous TUNEL reaction to identify cells with DNA strand breaks and the labelling of the S-phase cells with EdU revealed the possibility of DNA replication occurring in damaged nuclei. For the first time, the intensity of EdU fluorescence to study the rate of DNA replication was analysed. Copyright © 2016 Elsevier B.V. All rights reserved.

Replication labeling patterns and chromosome territories typical of mammalian nuclei are conserved in the early metazoan Hydra.

Science.gov (United States)

Alexandrova, Olga; Solovei, Irina; Cremer, Thomas; David, Charles N

2003-12-01

To investigate the evolutionary conservation of higher order nuclear architecture previously described for mammalian cells we have analyzed the nuclear architecture of the simple polyp Hydra. These diploblastic organisms have large nuclei (8-10 microm) containing about 3x10(9) bp of DNA organized in 15 chromosome pairs. They belong to the earliest metazoan phylum and are separated from mammals by at least 600 million years. Single and double pulse labeling with halogenated nucleotides (bromodeoxyuridine, iododeoxyuridine and chlorodeoxyuridine) revealed striking similarities to the known sequence of replication labeling patterns in mammalian nuclei. These patterns reflect a persistent nuclear arrangement of early, mid-, and late replicating chromatin foci that could be identified during all stages of interphase over at least 5-10 cell generations. Segregation of labeled chromatids led after several cell divisions to nuclei with single or a few labeled chromosome territories. In such nuclei distinct clusters of labeled chromatin foci were separated by extended nuclear areas with non-labeled chromatin, which is typical of a territorial arrangement of interphase chromosomes. Our results indicate the conservation of fundamental features of higher order chromatin arrangements throughout the evolution of metazoan animals and suggest the existence of conserved mechanism(s) controlling this architecture.

The Design of Finite State Machine for Asynchronous Replication Protocol

Science.gov (United States)

Wang, Yanlong; Li, Zhanhuai; Lin, Wei; Hei, Minglei; Hao, Jianhua

Data replication is a key way to design a disaster tolerance system and to achieve reliability and availability. It is difficult for a replication protocol to deal with the diverse and complex environment. This means that data is less well replicated than it ought to be. To reduce data loss and to optimize replication protocols, we (1) present a finite state machine, (2) run it to manage an asynchronous replication protocol and (3) report a simple evaluation of the asynchronous replication protocol based on our state machine. It's proved that our state machine is applicable to guarantee the asynchronous replication protocol running in the proper state to the largest extent in the event of various possible events. It also can helpful to build up replication-based disaster tolerance systems to ensure the business continuity.

An evaluation of a single-step extraction chromatography separation method for Sm-Nd isotope analysis of micro-samples of silicate rocks by high-sensitivity thermal ionization mass spectrometry

International Nuclear Information System (INIS)

Li Chaofeng; Li Xianhua; Li Qiuli; Guo Jinghui; Li Xianghui; Liu Tao

2011-01-01

Graphical abstract: Distribution curve of all eluting fractions for a BCR-2 (1-2-3.5-7 mg) on LN column using HCl and HF as eluting reagent. Highlights: → This analytical protocol affords a simple and rapid analysis for Sm and Nd isotope in minor rock samples. → The single-step separation method exhibits satisfactory separation effect for complex silicate samples. → Corrected 143 Nd/ 144 Nd data show excellent accuracy even if the 140 Ce 16 O + / 144 Nd 16 O + ratio reached to 0.03. - Abstract: A single-step separation scheme is presented for Sm-Nd radiogenic isotope system on very small samples (1-3 mg) of silicate rock. This method is based on Eichrom LN Spec chromatographic material and affords a straightforward separation of Sm-Nd from complex matrix with good purity and satisfactory blank levels, suitable for thermal ionization mass spectrometry (TIMS). This technique, characterized by high efficiency (single-step Sm-Nd separation) and high sensitivity (TIMS on NdO + ion beam), is able to process rapidly (3-4 h), with low procedure blanks ( 143 Nd/ 144 Nd ratios and Sm-Nd concentrations are presented for eleven international silicate rock reference materials, spanning a wide range of Sm-Nd contents and bulk compositions. The analytical results show a good agreement with recommended values within ±0.004% for the 143 Nd/ 144 Nd isotopic ratio and ±2% for Sm-Nd quantification at the 95% confidence level. It is noted that the uncertainty of this method is about 3 times larger than typical precision achievable with two-stage full separation followed by state-of-the-art conventional TIMS using Nd + ion beams which require much larger amounts of Nd. Hence, our single-step separation followed by NdO + ion beam technique is preferred to the analysis for microsamples.

Analytical strategies for discovery and replication of genetic effects in pharmacogenomic studies

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Kohler JR

2014-08-01

Full Text Available Jared R Kohler, Tobias Guennel, Scott L MarshallBioStat Solutions, Inc., Frederick, MD, USAAbstract: In the past decade, the pharmaceutical industry and biomedical research sector have devoted considerable resources to pharmacogenomics (PGx with the hope that understanding genetic variation in patients would deliver on the promise of personalized medicine. With the advent of new technologies and the improved collection of DNA samples, the roadblock to advancements in PGx discovery is no longer the lack of high-density genetic information captured on patient populations, but rather the development, adaptation, and tailoring of analytical strategies to effectively harness this wealth of information. The current analytical paradigm in PGx considers the single-nucleotide polymorphism (SNP as the genomic feature of interest and performs single SNP association tests to discover PGx effects – ie, genetic effects impacting drug response. While it can be straightforward to process single SNP results and to consider how this information may be extended for use in downstream patient stratification, the rate of replication for single SNP associations has been low and the desired success of producing clinically and commercially viable biomarkers has not been realized. This may be due to the fact that single SNP association testing is suboptimal given the complexities of PGx discovery in the clinical trial setting, including: 1 relatively small sample sizes; 2 diverse clinical cohorts within and across trials due to genetic ancestry (potentially impacting the ability to replicate findings; and 3 the potential polygenic nature of a drug response. Subsequently, a shift in the current paradigm is proposed: to consider the gene as the genomic feature of interest in PGx discovery. The proof-of-concept study presented in this manuscript demonstrates that genomic region-based association testing has the potential to improve the power of detecting single SNP or

The Alleged Crisis and the Illusion of Exact Replication.

Science.gov (United States)

Stroebe, Wolfgang; Strack, Fritz

2014-01-01

There has been increasing criticism of the way psychologists conduct and analyze studies. These critiques as well as failures to replicate several high-profile studies have been used as justification to proclaim a "replication crisis" in psychology. Psychologists are encouraged to conduct more "exact" replications of published studies to assess the reproducibility of psychological research. This article argues that the alleged "crisis of replicability" is primarily due to an epistemological misunderstanding that emphasizes the phenomenon instead of its underlying mechanisms. As a consequence, a replicated phenomenon may not serve as a rigorous test of a theoretical hypothesis because identical operationalizations of variables in studies conducted at different times and with different subject populations might test different theoretical constructs. Therefore, we propose that for meaningful replications, attempts at reinstating the original circumstances are not sufficient. Instead, replicators must ascertain that conditions are realized that reflect the theoretical variable(s) manipulated (and/or measured) in the original study. © The Author(s) 2013.

Comparison of Proteins in Whole Blood and Dried Blood Spot Samples by LC/MS/MS

Science.gov (United States)

Chambers, Andrew G.; Percy, Andrew J.; Hardie, Darryl B.; Borchers, Christoph H.

2013-09-01

Dried blood spot (DBS) sampling methods are desirable for population-wide biomarker screening programs because of their ease of collection, transportation, and storage. Immunoassays are traditionally used to quantify endogenous proteins in these samples but require a separate assay for each protein. Recently, targeted mass spectrometry (MS) has been proposed for generating highly-multiplexed assays for biomarker proteins in DBS samples. In this work, we report the first comparison of proteins in whole blood and DBS samples using an untargeted MS approach. The average number of proteins identified in undepleted whole blood and DBS samples by liquid chromatography (LC)/MS/MS was 223 and 253, respectively. Protein identification repeatability was between 77 %-92 % within replicates and the majority of these repeated proteins (70 %) were observed in both sample formats. Proteins exclusively identified in the liquid or dried fluid spot format were unbiased based on their molecular weight, isoelectric point, aliphatic index, and grand average hydrophobicity. In addition, we extended this comparison to include proteins in matching plasma and serum samples with their dried fluid spot equivalents, dried plasma spot (DPS), and dried serum spot (DSS). This work begins to define the accessibility of endogenous proteins in dried fluid spot samples for analysis by MS and is useful in evaluating the scope of this new approach.

Chaotic interactions of self-replicating RNA.

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Forst, C V

1996-03-01

A general system of high-order differential equations describing complex dynamics of replicating biomolecules is given. Symmetry relations and coordinate transformations of general replication systems leading to topologically equivalent systems are derived. Three chaotic attractors observed in Lotka-Volterra equations of dimension n = 3 are shown to represent three cross-sections of one and the same chaotic regime. Also a fractal torus in a generalized three-dimensional Lotka-Volterra Model has been linked to one of the chaotic attractors. The strange attractors are studied in the equivalent four-dimensional catalytic replicator network. The fractal torus has been examined in adapted Lotka-Volterra equations. Analytic expressions are derived for the Lyapunov exponents of the flow in the replicator system. Lyapunov spectra for different pathways into chaos has been calculated. In the generalized Lotka-Volterra system a second inner rest point--coexisting with (quasi)-periodic orbits--can be observed; with an abundance of different bifurcations. Pathways from chaotic tori, via quasi-periodic tori, via limit cycles, via multi-periodic orbits--emerging out of periodic doubling bifurcations--to "simple" chaotic attractors can be found.

Insights into the Initiation of Eukaryotic DNA Replication.

Science.gov (United States)

Bruck, Irina; Perez-Arnaiz, Patricia; Colbert, Max K; Kaplan, Daniel L

2015-01-01

The initiation of DNA replication is a highly regulated event in eukaryotic cells to ensure that the entire genome is copied once and only once during S phase. The primary target of cellular regulation of eukaryotic DNA replication initiation is the assembly and activation of the replication fork helicase, the 11-subunit assembly that unwinds DNA at a replication fork. The replication fork helicase, called CMG for Cdc45-Mcm2-7, and GINS, assembles in S phase from the constituent Cdc45, Mcm2-7, and GINS proteins. The assembly and activation of the CMG replication fork helicase during S phase is governed by 2 S-phase specific kinases, CDK and DDK. CDK stimulates the interaction between Sld2, Sld3, and Dpb11, 3 initiation factors that are each required for the initiation of DNA replication. DDK, on the other hand, phosphorylates the Mcm2, Mcm4, and Mcm6 subunits of the Mcm2-7 complex. Sld3 recruits Cdc45 to Mcm2-7 in a manner that depends on DDK, and recent work suggests that Sld3 binds directly to Mcm2-7 and also to single-stranded DNA. Furthermore, recent work demonstrates that Sld3 and its human homolog Treslin substantially stimulate DDK phosphorylation of Mcm2. These data suggest that the initiation factor Sld3/Treslin coordinates the assembly and activation of the eukaryotic replication fork helicase by recruiting Cdc45 to Mcm2-7, stimulating DDK phosphorylation of Mcm2, and binding directly to single-stranded DNA as the origin is melted.

Autonomous replication of plasmids bearing monkey DNA origin-enriched sequences

International Nuclear Information System (INIS)

Frappier, L.; Zannis-Hadjopoulos, M.

1987-01-01

Twelve clones of origin-enriched sequences (ORS) isolated from early replicating monkey (CV-1) DNA were examined for transient episomal replication in transfected CV-1, COS-7, and HeLa cells. Plasmid DNA was isolated at time intervals after transfection and screened by the Dpn I resistance assay or by the bromodeoxyuridine substitution assay to differentiate between input and replicated DNA. The authors have identified four monkey ORS (ORS3, -8, -9, and -12) that can support plasmid replication in mammalian cells. This replication is carried out in a controlled and semiconservative manner characteristic of mammalian replicons. ORS replication was most efficient in HeLa cells. Electron microscopy showed ORS8 and ORS12 plasmids of the correct size with replication bubbles. Using a unique restriction site in ORS12, we have mapped the replication bubble within the monkey DNA sequence

Materials Chemistry and Performance of Silicone-Based Replicating Compounds.

Energy Technology Data Exchange (ETDEWEB)

Brumbach, Michael T.; Mirabal, Alex James; Kalan, Michael; Trujillo, Ana B; Hale, Kevin

2014-11-01

Replicating compounds are used to cast reproductions of surface features on a variety of materials. Replicas allow for quantitative measurements and recordkeeping on parts that may otherwise be difficult to measure or maintain. In this study, the chemistry and replicating capability of several replicating compounds was investigated. Additionally, the residue remaining on material surfaces upon removal of replicas was quantified. Cleaning practices were tested for several different replicating compounds. For all replicating compounds investigated, a thin silicone residue was left by the replica. For some compounds, additional inorganic species could be identified in the residue. Simple solvent cleaning could remove some residue.

Does a single session of reading literary fiction prime enhanced mentalising performance? Four replication experiments of Kidd and Castano (2013).

Science.gov (United States)

Samur, Dalya; Tops, Mattie; Koole, Sander L

2018-02-01

Prior experiments indicated that reading literary fiction improves mentalising performance relative to reading popular fiction, non-fiction, or not reading. However, the experiments had relatively small sample sizes and hence low statistical power. To address this limitation, the present authors conducted four high-powered replication experiments (combined N = 1006) testing the causal impact of reading literary fiction on mentalising. Relative to the original research, the present experiments used the same literary texts in the reading manipulation; the same mentalising task; and the same kind of participant samples. Moreover, one experiment was pre-registered as a direct replication. In none of the experiments did reading literary fiction have any effect on mentalising relative to control conditions. The results replicate earlier findings that familiarity with fiction is positively correlated with mentalising. Taken together, the present findings call into question whether a single session of reading fiction leads to immediate improvements in mentalising.

Neurotoxic Antibodies against the Prion Protein Do Not Trigger Prion Replication.

Directory of Open Access Journals (Sweden)

Karl Frontzek

Full Text Available Prions are the infectious agents causing transmissible spongiform encephalopathies (TSE, progressive, inexorably lethal neurological diseases. Antibodies targeting the globular domain (GD of the cellular prion protein PrPC trigger a neurotoxic syndrome morphologically and molecularly similar to prion disease. This phenomenon raises the question whether such antibodies induce infectious prions de novo. Here we exposed cerebellar organotypic cultured slices (COCS to the neurotoxic antibody, POM1. We then inoculated COCS homogenates into tga20 mice, which overexpress PrPC and are commonly utilized as sensitive indicators of prion infectivity. None of the mice inoculated with COCS-derived lysates developed any signs of disease, and all mice survived for at least 200 days post-inoculation. In contrast, all mice inoculated with bona fide prions succumbed to TSE after 55-95 days. Post-mortem analyses did not reveal any signs of prion pathology in mice inoculated with POM1-COCS lysates. Also, lysates from POM1-exposed COCS were unable to convert PrP by quaking. Hence, anti-GD antibodies do not catalyze the generation of prion infectivity. These data indicate that prion replication can be separated from prion toxicity, and suggest that anti-GD antibodies exert toxicity by acting downstream of prion replication.

Large-scale replication study reveals a limit on probabilistic prediction in language comprehension.

Science.gov (United States)

Nieuwland, Mante S; Politzer-Ahles, Stephen; Heyselaar, Evelien; Segaert, Katrien; Darley, Emily; Kazanina, Nina; Von Grebmer Zu Wolfsthurn, Sarah; Bartolozzi, Federica; Kogan, Vita; Ito, Aine; Mézière, Diane; Barr, Dale J; Rousselet, Guillaume A; Ferguson, Heather J; Busch-Moreno, Simon; Fu, Xiao; Tuomainen, Jyrki; Kulakova, Eugenia; Husband, E Matthew; Donaldson, David I; Kohút, Zdenko; Rueschemeyer, Shirley-Ann; Huettig, Falk

2018-04-03

Do people routinely pre-activate the meaning and even the phonological form of upcoming words? The most acclaimed evidence for phonological prediction comes from a 2005 Nature Neuroscience publication by DeLong, Urbach and Kutas, who observed a graded modulation of electrical brain potentials (N400) to nouns and preceding articles by the probability that people use a word to continue the sentence fragment ('cloze'). In our direct replication study spanning 9 laboratories ( N =334), pre-registered replication-analyses and exploratory Bayes factor analyses successfully replicated the noun-results but, crucially, not the article-results. Pre-registered single-trial analyses also yielded a statistically significant effect for the nouns but not the articles. Exploratory Bayesian single-trial analyses showed that the article-effect may be non-zero but is likely far smaller than originally reported and too small to observe without very large sample sizes. Our results do not support the view that readers routinely pre-activate the phonological form of predictable words. © 2018, Nieuwland et al.

Bayesian tests to quantify the result of a replication attempt

NARCIS (Netherlands)

Verhagen, J.; Wagenmakers, E.-J.

2014-01-01

Replication attempts are essential to the empirical sciences. Successful replication attempts increase researchers’ confidence in the presence of an effect, whereas failed replication attempts induce skepticism and doubt. However, it is often unclear to what extent a replication attempt results in

A nanosilver-based spectrophotometric method for determination of malachite green in surface water samples.

Science.gov (United States)

Sahraei, R; Farmany, A; Mortazavi, S S; Noorizadeh, H

2013-07-01

A new spectrophotometric method is reported for the determination of nanomolar level of malachite green in surface water samples. The method is based on the catalytic effect of silver nanoparticles on the oxidation of malachite green by hexacyanoferrate (III) in acetate-acetic acid medium. The absorbance is measured at 610 nm with the fixed-time method. Under the optimum conditions, the linear range was 8.0 × 10(-9)-2.0 × 10(-7) mol L(-1) malachite green with a correlation coefficient of 0.996. The limit of detection (S/N = 3) was 2.0 × 10(-9) mol L(-1). Relative standard deviation for ten replicate determinations of 1.0 × 10(-8) mol L(-1) malachite green was 1.86%. The method is featured with good accuracy and reproducibility for malachite green determination in surface water samples without any pre-concentration and separation step.

Spatio-temporal re-organization of replication foci accompanies replication domain consolidation during human pluripotent stem cell lineage specification

Science.gov (United States)

Wilson, Korey A.; Elefanty, Andrew G.; Stanley, Edouard G.; Gilbert, David M.

2016-01-01

ABSTRACT Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial consolidation of chromosome domains and temporal consolidation of their replication timing. Replication timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear chromatin compartments throughout all cell cycle transitions. We find that, as human PSCs differentiate, the length of S phase devoted to replication of spatially clustered replication foci increases, coincident with global compartmentalization of domains into temporally clustered blocks of chromatin. Importantly, re-localization and anchorage of domains was completed prior to the onset of S phase, even in the context of an abbreviated PSC G1 phase. This approach can also be employed to investigate cell fate transitions in single PSCs, which could be seen to differentiate preferentially from G1 phase. Together, our results establish real-time, live-cell imaging methods for tracking cell cycle transitions during human PSC differentiation that can be applied to study chromosome domain consolidation and other aspects of lineage specification. PMID:27433885

Unreliable Yet Still Replicable: A Comment on LeBel and Paunonen (2011)

Science.gov (United States)

De Schryver, Maarten; Hughes, Sean; Rosseel, Yves; De Houwer, Jan

2016-01-01

Lebel and Paunonen (2011) highlight that despite their importance and popularity in both theoretical and applied research, many implicit measures continue to be plagued by a persistent and troublesome issue—low reliability. In their paper, they offer a conceptual analysis of the relationship between reliability, power and replicability, and then provide a series of recommendations for researchers interested in using implicit measures in an experimental setting. At the core of their account is the idea that reliability can be equated with statistical power, such that “lower levels of reliability are associated with decreasing probabilities of detecting a statistically significant effect, given one exists in the population” (p. 573). They also take the additional step of equating reliability and replicability. In our commentary, we draw attention to the fact that there is no direct, fixed or one-to-one relation between reliability and power or replicability. More specifically, we argue that when adopting an experimental (rather than a correlational) approach, researchers strive to minimize inter-individual variation, which has a direct impact on sample based reliability estimates. We evaluate the strengths and weaknesses of the LeBel and Paunonen's recommendations and refine them where appropriate. PMID:26793150

Sampling efficiency for species composition assessments using the ...

African Journals Online (AJOL)

A pilot survey to determine the sampling efficiency of the wheel-point method, using the nearest plant method, to assess species composition (using replicate similarity related to sampling intensity, and total sampling time) was conducted on three plot sizes (20 x 20m, 30 x 30m, 40 x 40m) at two sites in a semi-arid savanna.

Magnetic separations in biotechnology.

Science.gov (United States)

Borlido, L; Azevedo, A M; Roque, A C A; Aires-Barros, M R

2013-12-01

Magnetic separations are probably one of the most versatile separation processes in biotechnology as they are able to purify cells, viruses, proteins and nucleic acids directly from crude samples. The fast and gentle process in combination with its easy scale-up and automation provide unique advantages over other separation techniques. In the midst of this process are the magnetic adsorbents tailored for the envisioned target and whose complex synthesis spans over multiple fields of science. In this context, this article reviews both the synthesis and tailoring of magnetic adsorbents for bioseparations as well as their ultimate application. Copyright © 2013 Elsevier Inc. All rights reserved.

Synchronous separation, seaming, sealing and sterilization (S4) using brazing for sample containerization and planetary protection

Science.gov (United States)

Bar-Cohen, Yoseph; Badescu, Mircea; Sherrit, Stewart; Bao, Xiaoqi; Lindsey, Cameron; Kutzer, Thomas; Salazar, Eduardo

2018-03-01

The return of samples back to Earth in future missions would require protection of our planet from the risk of bringing uncontrolled biological materials back with the samples. This protection would require "breaking the chain of contact (BTC)", where any returned material reaching Earth for further analysis would have to be sealed inside a container with extremely high confidence. Therefore, the acquired samples would need to be contained while destroying any potential biological materials that may contaminate the external surface of the container. A novel process that could be used to contain returning samples has been developed and demonstrated in a quarter scale size. The process consists of brazing using non-contact induction heating that synchronously separates, seams, seals and sterilizes (S4) the container. The use of brazing involves melting at temperatures higher than 500°C and this level of heating assures sterilization of the exposed areas since all carbon bonds (namely, organic materials) are broken at this temperature. The mechanism consists of a double wall container with inner and outer shells having Earth-clean interior surfaces. The process consists of two-steps, Step-1: the double wall container halves are fabricated and brazed (equivalent to production on Earth); and Step-2 is the S4 process and it is the equivalent to the execution on-orbit around Mars. In a potential future mission, the double wall container would be split into two halves and prepared on Earth. The potential on-orbit execution would consist of inserting the orbiting sample (OS) container into one of the halves and then mated to the other half and brazed. The latest results of this effort will be described and discussed in this manuscript.

A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation.

Science.gov (United States)

Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

2018-01-01

Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an "alien" impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types.

A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation

DEFF Research Database (Denmark)

Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

2018-01-01

" impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication......Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an "alien......-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types....

Structural properties of replication origins in yeast DNA sequences

International Nuclear Information System (INIS)

Cao Xiaoqin; Zeng Jia; Yan Hong

2008-01-01

Sequence-dependent DNA flexibility is an important structural property originating from the DNA 3D structure. In this paper, we investigate the DNA flexibility of the budding yeast (S. Cerevisiae) replication origins on a genome-wide scale using flexibility parameters from two different models, the trinucleotide and the tetranucleotide models. Based on analyzing average flexibility profiles of 270 replication origins, we find that yeast replication origins are significantly rigid compared with their surrounding genomic regions. To further understand the highly distinctive property of replication origins, we compare the flexibility patterns between yeast replication origins and promoters, and find that they both contain significantly rigid DNAs. Our results suggest that DNA flexibility is an important factor that helps proteins recognize and bind the target sites in order to initiate DNA replication. Inspired by the role of the rigid region in promoters, we speculate that the rigid replication origins may facilitate binding of proteins, including the origin recognition complex (ORC), Cdc6, Cdt1 and the MCM2-7 complex

Establishing a coherent and replicable measurement model of the Edinburgh Postnatal Depression Scale.

Science.gov (United States)

Martin, Colin R; Redshaw, Maggie

2018-06-01

The 10-item Edinburgh Postnatal Depression Scale (EPDS) is an established screening tool for postnatal depression. Inconsistent findings in factor structure and replication difficulties have limited the scope of development of the measure as a multi-dimensional tool. The current investigation sought to robustly determine the underlying factor structure of the EPDS and the replicability and stability of the most plausible model identified. A between-subjects design was used. EPDS data were collected postpartum from two independent cohorts using identical data capture methods. Datasets were examined with confirmatory factor analysis, model invariance testing and systematic evaluation of relational and internal aspects of the measure. Participants were two samples of postpartum women in England assessed at three months (n = 245) and six months (n = 217). The findings showed a three-factor seven-item model of the EPDS offered an excellent fit to the data, and was observed to be replicable in both datasets and invariant as a function of time point of assessment. Some EPDS sub-scale scores were significantly higher at six months. The EPDS is multi-dimensional and a robust measurement model comprises three factors that are replicable. The potential utility of the sub-scale components identified requires further research to identify a role in contemporary screening practice. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

From structure to mechanism—understanding initiation of DNA replication

Science.gov (United States)

Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L. Maximilian; Schneider, Sarah; Speck, Christian

2017-01-01

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2–7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. PMID:28717046

MYC and the Control of DNA Replication

Science.gov (United States)

Dominguez-Sola, David; Gautier, Jean

2014-01-01

The MYC oncogene is a multifunctional protein that is aberrantly expressed in a significant fraction of tumors from diverse tissue origins. Because of its multifunctional nature, it has been difficult to delineate the exact contributions of MYC’s diverse roles to tumorigenesis. Here, we review the normal role of MYC in regulating DNA replication as well as its ability to generate DNA replication stress when overexpressed. Finally, we discuss the possible mechanisms by which replication stress induced by aberrant MYC expression could contribute to genomic instability and cancer. PMID:24890833

Organization of Replication of Ribosomal DNA in Saccharomyces cerevisiae

NARCIS (Netherlands)

Linskens, Maarten H.K.; Huberman, Joel A.

1988-01-01

Using recently developed replicon mapping techniques, we have analyzed the replication of the ribosomal DNA in Saccharomyces cerevisiae. The results show that (i) the functional origin of replication colocalizes with an autonomously replicating sequence element previously mapped to the

The Reliability and Validity of the Outcome Rating Scale: A Replication Study of a Brief Clinical Measure

National Research Council Canada - National Science Library

Bringhurst, David L; Watson, Curtis W; Miller, Scott D; Duncan, Barry L

2004-01-01

... (Miller, Duncan, Brown, Sparks, AND Claud, 2003). This article reports the findings of an independent replication study evaluating the reliability and concurrent validity of the ORS as studied in a non-clinical sample...

An Investigation of Abstract Construal on Impression Formation: A Multi-Lab Replication of McCarthy and Skowronski (2011

Directory of Open Access Journals (Sweden)

Randy J. McCarthy

2018-05-01

Full Text Available Perceivers often view individuals described as “warm” to be generally positive and individuals described as “cold” to be generally negative. Consistent with the tenets of Construal Level Theory, McCarthy and Skowronski (2011 demonstrated this difference was larger among perceivers who were instructed the information was psychologically distant rather than psychologically near; however, those results have never been subjected to replication attempts. To test the replicability of those results, we closely replicated the methods of McCarthy and Skowronski (2011 Study 1b at eight separate data collection sites and pooled the results into a random-effects meta-analysis. Within the replication attempts, the overall effect was not significantly different from zero (d = 0.10, 95% CI [–0.01, 0.22] and an equivalence test confirmed this effect was smaller than our smallest effect size of interest. However, when the original study was incorporated into the meta-analysis, the overall effect was significantly different from zero in the theoretically-consistent direction (d = 0.13, 95% CI [0.02, 0.24]. The weight of the overall evidence suggests the traits “warm” and “cold” are more influential among participants who were presented with information that was psychologically distant; however, this effect is small. Future research should try to identify more potent moderators, which would make the effect more affordable to detect.

Cyclophilin B facilitates the replication of Orf virus.

Science.gov (United States)

Zhao, Kui; Li, Jida; He, Wenqi; Song, Deguang; Zhang, Ximu; Zhang, Di; Zhou, Yanlong; Gao, Feng

2017-06-15

Viruses interact with host cellular factors to construct a more favourable environment for their efficient replication. Expression of cyclophilin B (CypB), a cellular peptidyl-prolyl cis-trans isomerase (PPIase), was found to be significantly up-regulated. Recently, a number of studies have shown that CypB is important in the replication of several viruses, including Japanese encephalitis virus (JEV), hepatitis C virus (HCV) and human papillomavirus type 16 (HPV 16). However, the function of cellular CypB in ORFV replication has not yet been explored. Suppression subtractive hybridization (SSH) technique was applied to identify genes differentially expressed in the ORFV-infected MDBK cells at an early phase of infection. Cellular CypB was confirmed to be significantly up-regulated by quantitative reverse transcription-PCR (qRT-PCR) analysis and Western blotting. The role of CypB in ORFV infection was further determined using Cyclosporin A (CsA) and RNA interference (RNAi). Effect of CypB gene silencing on ORFV replication by 50% tissue culture infectious dose (TCID 50 ) assay and qRT-PCR detection. In the present study, CypB was found to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection. Cyclosporin A (CsA) exhibited suppressive effects on ORFV replication through the inhibition of CypB. Silencing of CypB gene inhibited the replication of ORFV in MDBK cells. In conclusion, these data suggest that CypB is critical for the efficient replication of the ORFV genome. Cellular CypB was confirmed to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection, which could effectively facilitate the replication of ORFV.

Autophagy Facilitates Salmonella Replication in HeLa Cells

Science.gov (United States)

Yu, Hong B.; Croxen, Matthew A.; Marchiando, Amanda M.; Ferreira, Rosana B. R.; Cadwell, Ken; Foster, Leonard J.; Finlay, B. Brett

2014-01-01

ABSTRACT Autophagy is a process whereby a double-membrane structure (autophagosome) engulfs unnecessary cytosolic proteins, organelles, and invading pathogens and delivers them to the lysosome for degradation. We examined the fate of cytosolic Salmonella targeted by autophagy and found that autophagy-targeted Salmonella present in the cytosol of HeLa cells correlates with intracellular bacterial replication. Real-time analyses revealed that a subset of cytosolic Salmonella extensively associates with autophagy components p62 and/or LC3 and replicates quickly, whereas intravacuolar Salmonella shows no or very limited association with p62 or LC3 and replicates much more slowly. Replication of cytosolic Salmonella in HeLa cells is significantly decreased when autophagy components are depleted. Eventually, hyperreplication of cytosolic Salmonella potentiates cell detachment, facilitating the dissemination of Salmonella to neighboring cells. We propose that Salmonella benefits from autophagy for its cytosolic replication in HeLa cells. PMID:24618251

Separation of atropisomers by chiral liquid chromatography and thermodynamic analysis of separation mechanism

Directory of Open Access Journals (Sweden)

Ling Zhang

2017-06-01

Full Text Available In the pharmaceutical industry, the analysis of atropisomers is of considerable interest from a scientific and regulatory perspective. The compound of interest contains two stereogenic axes due to the hindered rotation around the single bonds connecting the aryl groups, which results in four potential configurational isomers (atropisomers. The separation of the four atropisomers is achieved on a derivatized β-cyclodextrin bonded stationary phase. Further investigation shows that low temperature conditions, including sample preparation (−70 °C, sample storage (−70 °C, and chromatographic separation (6 °C, were critical to preventing interconversion. LC-UV-Laser Polarimetric analysis identified peak 1/2 as a pair of enantiomers and peak 3/4 as another. Thermodynamic analysis of the retention data indicated that the separation of the pairs of enantiomers is primarily enthalpy controlled as indicated by the positive slope of the van’t Huff plot. The difference in absolute Δ (Δ H, ranged from 2.20 kJ/mol to 2.42 kJ/mol.

Use of an isotope separator at the INEL

International Nuclear Information System (INIS)

Anderl, R.A.

1977-01-01

An electromagnetic isotope separator with a retardation lens as a collector was used to prepare highly enriched samples of Nd-143, -144, -145, -146, -148, -150, Sm-147, -149; Eu-151, -152, -153, -154. The 50 μg to 75 μg samples, deposited on 1 mil nickel foil or 0.5 mil vanadium foil, are part of a sample set to be irradiated in EBR-II as part of an integral-capture cross-section measurement program at the INEL. The isotope separator and the apparatus used for the sample preparation are described

Stabilization of Reversed Replication Forks by Telomerase Drives Telomere Catastrophe.

Science.gov (United States)

Margalef, Pol; Kotsantis, Panagiotis; Borel, Valerie; Bellelli, Roberto; Panier, Stephanie; Boulton, Simon J

2018-01-25

Telomere maintenance critically depends on the distinct activities of telomerase, which adds telomeric repeats to solve the end replication problem, and RTEL1, which dismantles DNA secondary structures at telomeres to facilitate replisome progression. Here, we establish that reversed replication forks are a pathological substrate for telomerase and the source of telomere catastrophe in Rtel1 -/- cells. Inhibiting telomerase recruitment to telomeres, but not its activity, or blocking replication fork reversal through PARP1 inhibition or depleting UBC13 or ZRANB3 prevents the rapid accumulation of dysfunctional telomeres in RTEL1-deficient cells. In this context, we establish that telomerase binding to reversed replication forks inhibits telomere replication, which can be mimicked by preventing replication fork restart through depletion of RECQ1 or PARG. Our results lead us to propose that telomerase inappropriately binds to and inhibits restart of reversed replication forks within telomeres, which compromises replication and leads to critically short telomeres. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Chronic DNA Replication Stress Reduces Replicative Lifespan of Cells by TRP53-Dependent, microRNA-Assisted MCM2-7 Downregulation.

Directory of Open Access Journals (Sweden)

Gongshi Bai

2016-01-01

Full Text Available Circumstances that compromise efficient DNA replication, such as disruptions to replication fork progression, cause a state known as DNA replication stress (RS. Whereas normally proliferating cells experience low levels of RS, excessive RS from intrinsic or extrinsic sources can trigger cell cycle arrest and senescence. Here, we report that a key driver of RS-induced senescence is active downregulation of the Minichromosome Maintenance 2-7 (MCM2-7 factors that are essential for replication origin licensing and which constitute the replicative helicase core. Proliferating cells produce high levels of MCM2-7 that enable formation of dormant origins that can be activated in response to acute, experimentally-induced RS. However, little is known about how physiological RS levels impact MCM2-7 regulation. We found that chronic exposure of primary mouse embryonic fibroblasts (MEFs to either genetically-encoded or environmentally-induced RS triggered gradual MCM2-7 repression, followed by inhibition of replication and senescence that could be accelerated by MCM hemizygosity. The MCM2-7 reduction in response to RS is TRP53-dependent, and involves a group of Trp53-dependent miRNAs, including the miR-34 family, that repress MCM expression in replication-stressed cells before they undergo terminal cell cycle arrest. miR-34 ablation partially rescued MCM2-7 downregulation and genomic instability in mice with endogenous RS. Together, these data demonstrate that active MCM2-7 repression is a physiologically important mechanism for RS-induced cell cycle arrest and genome maintenance on an organismal level.

Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

Science.gov (United States)

Stoeck, Ina Karen; Lee, Ji-Young; Tabata, Keisuke; Romero-Brey, Inés; Paul, David; Schult, Philipp; Lohmann, Volker; Kaderali, Lars; Bartenschlager, Ralf

2018-01-01

Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell

Separation and preconcentration of copper in environmental samples on Amberlite XAD-8 resin after complexation with a carbothioamide derivative

Directory of Open Access Journals (Sweden)

Hamide Elvan

2013-01-01

Full Text Available A new solid phase extraction (SPE method has been developed for the selective separation and preconcentration of Cu (II ions in food and water samples prior to its flame atomic absorption spectrometry determination. The method is based on the adsorption of the Cu(II - 2-{[4-Amino-3-(4-methylphenyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]acetyl}-N-phenyl hydrazinecarbothioamide complex on Amberlite XAD-8 resin. The metal complex retained on the resin was eluted with 7.5 mL of 2.0 mol L-1 HCl in acetone. The optimum conditions for the SPE of Cu(II ions were investigated, and the method was subsequently applied to sea water, stream water, rice, tea, and tobacco samples for the determination of Cu(II levels.

Geminin: a major DNA replication safeguard in higher eukaryotes

DEFF Research Database (Denmark)

Melixetian, Marina; Helin, Kristian

2004-01-01

Eukaryotes have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. These mechanisms prevent relicensing of origins of replication after initiation of DNA replication in S phase until the end of mitosis. Most of our knowledge of mechanisms controlling prereplication...

Sample preparation for phosphoproteomic analysis of circadian time series in Arabidopsis thaliana.

Science.gov (United States)

Krahmer, Johanna; Hindle, Matthew M; Martin, Sarah F; Le Bihan, Thierry; Millar, Andrew J

2015-01-01

Systems biological approaches to study the Arabidopsis thaliana circadian clock have mainly focused on transcriptomics while little is known about the proteome, and even less about posttranslational modifications. Evidence has emerged that posttranslational protein modifications, in particular phosphorylation, play an important role for the clock and its output. Phosphoproteomics is the method of choice for a large-scale approach to gain more knowledge about rhythmic protein phosphorylation. Recent plant phosphoproteomics publications have identified several thousand phosphopeptides. However, the methods used in these studies are very labor-intensive and therefore not suitable to apply to a well-replicated circadian time series. To address this issue, we present and compare different strategies for sample preparation for phosphoproteomics that are compatible with large numbers of samples. Methods are compared regarding number of identifications, variability of quantitation, and functional categorization. We focus on the type of detergent used for protein extraction as well as methods for its removal. We also test a simple two-fraction separation of the protein extract. © 2015 Elsevier Inc. All rights reserved.

Optical tweezers reveal how proteins alter replication

Science.gov (United States)

Chaurasiya, Kathy

Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic

Genome-wide association analysis and replication of coronary artery disease in South Korea suggests a causal variant common to diverse populations

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Cho, Eun Young; Jang, Yangsoo; Shin, Eun Soon; Jang, Hye Yoon; Yoo, Yeon-Kyeong; Kim, Sook; Jang, Ji Hyun; Lee, Ji Yeon; Yun, Min Hye; Park, Min Young; Chae, Jey Sook; Lim, Jin Woo; Shin, Dong Jik; Park, Sungha; Lee, Jong Ho; Han, Bok Ghee; Rae, Kim Hyung; Cardon, Lon R; Morris, Andrew P; Lee, Jong Eun; Clarke, Geraldine M

2010-01-01

Background Recent genome-wide association (GWA) studies have identified and replicated several genetic loci associated with the risk of development of coronary artery disease (CAD) in samples from populations of Caucasian and Asian descent. However, only chromosome 9p21 has been confirmed as a major susceptibility locus conferring risk for development of CAD across multiple ethnic groups. The authors aimed to find evidence of further similarities and differences in genetic risk of CAD between Korean and other populations. Methods The authors performed a GWA study comprising 230 cases and 290 controls from a Korean population typed on 490 032 single nucleotide polymorphisms (SNPs). A total of 3148 SNPs were taken forward for genotyping in a subsequent replication study using an independent sample of 1172 cases and 1087 controls from the same population. Results The association previously observed on chromosome 9p21 was independently replicated (p=3.08e–07). Within this region, the same risk haplotype was observed in samples from both Korea and of Western European descent, suggesting that the causal mutation carried on this background occurred on a single ancestral allele. Other than 9p21, the authors were unable to replicate any of the previously reported signals for association with CAD. Furthermore, no evidence of association was found at chromosome 1q41 for risk of myocardial infarction, previously identified as conferring risk in a Japanese population. Conclusion A common causal variant is likely to be responsible for risk of CAD in Korean and Western European populations at chromosome 9p21.3. Further investigations are required to confirm non-replication of any other cross-race genetic risk factors. PMID:27325954

From structure to mechanism-understanding initiation of DNA replication.

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Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L Maximilian; Schneider, Sarah; Speck, Christian

2017-06-01

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. © 2017 Riera et al.; Published by Cold Spring Harbor Laboratory Press.

Cytoplasmic ATR Activation Promotes Vaccinia Virus Genome Replication

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Antonio Postigo

2017-05-01

Full Text Available In contrast to most DNA viruses, poxviruses replicate their genomes in the cytoplasm without host involvement. We find that vaccinia virus induces cytoplasmic activation of ATR early during infection, before genome uncoating, which is unexpected because ATR plays a fundamental nuclear role in maintaining host genome integrity. ATR, RPA, INTS7, and Chk1 are recruited to cytoplasmic DNA viral factories, suggesting canonical ATR pathway activation. Consistent with this, pharmacological and RNAi-mediated inhibition of canonical ATR signaling suppresses genome replication. RPA and the sliding clamp PCNA interact with the viral polymerase E9 and are required for DNA replication. Moreover, the ATR activator TOPBP1 promotes genome replication and associates with the viral replisome component H5. Our study suggests that, in contrast to long-held beliefs, vaccinia recruits conserved components of the eukaryote DNA replication and repair machinery to amplify its genome in the host cytoplasm.

The DNA Replication Stress Hypothesis of Alzheimer’s Disease

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Yuri B. Yurov

2011-01-01

Full Text Available A well-recognized theory of Alzheimer’s disease (AD pathogenesis suggests ectopic cell cycle events to mediate neurodegeneration. Vulnerable neurons of the AD brain exhibit biomarkers of cell cycle progression and DNA replication suggesting a reentry into the cell cycle. Chromosome reduplication without proper cell cycle completion and mitotic division probably causes neuronal cell dysfunction and death. However, this theory seems to require some inputs in accordance with the generally recognized amyloid cascade theory as well as to explain causes and consequences of genomic instability (aneuploidy in the AD brain. We propose that unscheduled and incomplete DNA replication (replication stress destabilizes (epigenomic landscape in the brain and leads to DNA replication “catastrophe” causing cell death during the S phase (replicative cell death. DNA replication stress can be a key element of the pathogenetic cascade explaining the interplay between ectopic cell cycle events and genetic instabilities in the AD brain. Abnormal cell cycle reentry and somatic genome variations can be used for updating the cell cycle theory introducing replication stress as a missing link between cell genetics and neurobiology of AD.

Preparation of 1-(2-pyridylazo)-2-naphthol functionalized benzophenone/naphthalene and their uses in solid phase extractive preconcentration/separation of uranium(VI)

International Nuclear Information System (INIS)

Preetha, C.R.; Prasada Rao, T.

2003-01-01

The preparation of solid reagent 1-(2-pyridylazo)-2-naphthol functionalized benzophenone/naphthalene for preconcentration/separation of uranium(VI) is described. These reagents enriches uranium(VI) quantitatively from dilute aqueous solutions in the pH range 10.5-11.0. The solid mixture consisting of the enriched metal ion along with solid phase extractant were dissolved in 2 ml of acetone and uranium(VI) content was established spectrophotometrically by using Arsenazo III procedure. Calibration graphs were rectilinear over the uranium(VI) concentration in the range 0.002-0.1 μg cm -3 . Five replicate determinations of 40 μg of uranium present in 1 dm 3 of sample solution gave a mean absorbance of 0.185 with a relative standard deviation of 1.4%. The detection limit (corresponding to 3 times the standard deviation of the blank) and the enrichment factor were found to be 2 μg dm -3 and 500 respectively. Further the possible separation of uranium(VI) from several bivalent, trivalent and tetravalent elements was also established. In addition to validating the developed method by successfully analysing marine sediment reference material (MESS-3), uranium content was established in soil, river and marine sediment samples by the developed method and compared with standard inductively coupled plasma mass spectrometry (ICP-MS) values. (orig.)

CHROMATOGRAPHIC SEPARATION AND SPECTRO ...

African Journals Online (AJOL)

Four different fractions having colours yellow, grey, orange and ... Two different dyes vis a vis: methylene blue and malachite green dyes have been separated .... hydrogen carbonate of alkaline and alkaline earth metals in the samples.

Regulated eukaryotic DNA replication origin firing with purified proteins.

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Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

2015-03-26

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

DNA replication stress and cancer chemotherapy.

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Kitao, Hiroyuki; Iimori, Makoto; Kataoka, Yuki; Wakasa, Takeshi; Tokunaga, Eriko; Saeki, Hiroshi; Oki, Eiji; Maehara, Yoshihiko

2018-02-01

DNA replication is one of the fundamental biological processes in which dysregulation can cause genome instability. This instability is one of the hallmarks of cancer and confers genetic diversity during tumorigenesis. Numerous experimental and clinical studies have indicated that most tumors have experienced and overcome the stresses caused by the perturbation of DNA replication, which is also referred to as DNA replication stress (DRS). When we consider therapeutic approaches for tumors, it is important to exploit the differences in DRS between tumor and normal cells. In this review, we introduce the current understanding of DRS in tumors and discuss the underlying mechanism of cancer therapy from the aspect of DRS. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Quantification of fatty acids as methyl esters and phospholipids in cheese samples after separation of triacylglycerides and phospholipids

Energy Technology Data Exchange (ETDEWEB)

Hauff, Simone [University of Hohenheim, Institute of Food Chemistry, Garbenstrasse 28, D-70599 Stuttgart (Germany); Vetter, Walter [University of Hohenheim, Institute of Food Chemistry, Garbenstrasse 28, D-70599 Stuttgart (Germany)], E-mail: w-vetter@uni-hohenheim.de

2009-03-23

Determination of the individual fatty acid composition of neutral- and phospholipids as well as the phospholipid content of dairy food and other foodstuffs are important tasks in life sciences. For these purposes, a method was developed for the separation of lipids (standards of triolein and diacylphosphatidylcholines as well as three cheese samples) by solid-phase extraction using a self-packed column filled with partly deactivated silica. Non-halogenated solvents were used for the elution of the lipid classes. Cyclohexane/ethyl acetate (1:1, v/v) served for the elution of neutral lipids, while polar lipids were eluted with three solvents (ethyl acetate/methanol, methanol, and methanol/water) into one fraction. The separated lipid fractions were transesterified and the individual fatty acids were quantified by using gas chromatography coupled to electron ionization mass spectrometry (GC/EI-MS) in the selected ion monitoring (SIM) mode. The recovery rate for standard phosphatidylcholines was {approx}90% and cross-contamination from neutral lipids was negligible. The method was applied to cheese samples. Quantitative amounts of individual fatty acids in the phospholipid fraction were <0.002-0.29% of total lipids from camembert, <0.002-0.12% of total lipids from mozzarella, and <0.002-0.18% of total lipids in a goat cream cheese. Differences in the fatty acid pattern of neutral and polar lipids were detected. The quantity of the fatty acids determined in the phospholipid fraction was divided by the factor 0.7 in order to convert the fatty acid content into the phospholipid content of the cheese samples. This factor is based on the contribution of 16:0 to dipalmitoylphosphatidylcholine (DPPC). The resulting DPPC equivalents (DPPC{sub eq}) were found to be representative for the average contribution of fatty acids to all classes of phospholipids in dairy products. Using this approach, the phospholipid content of lipids from mozzarella, camembert, and goat cream cheese

Termination of DNA replication forks: "Breaking up is hard to do".

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Bailey, Rachael; Priego Moreno, Sara; Gambus, Agnieszka

2015-01-01

To ensure duplication of the entire genome, eukaryotic DNA replication initiates from thousands of replication origins. The replication forks move through the chromatin until they encounter forks from neighboring origins. During replication fork termination forks converge, the replisomes disassemble and topoisomerase II resolves the daughter DNA molecules. If not resolved efficiently, terminating forks result in genomic instability through the formation of pathogenic structures. Our recent findings shed light onto the mechanism of replisome disassembly upon replication fork termination. We have shown that termination-specific polyubiquitylation of the replicative helicase component - Mcm7, leads to dissolution of the active helicase in a process dependent on the p97/VCP/Cdc48 segregase. The inhibition of terminating helicase disassembly resulted in a replication termination defect. In this extended view we present hypothetical models of replication fork termination and discuss remaining and emerging questions in the DNA replication termination field.

Changes in nucleosome repeat lengths precede replication in the early replicating metallothionein II gene region of cells synchronized in early S phase

International Nuclear Information System (INIS)

D'Anna, J.A.; Tobey, R.A.

1989-01-01

Previous investigations showed that inhibition of DNA synthesis by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine produced large changes in the composition and nucleosome repeat lengths of bulk chromatin. There the authors report results of investigations to determine whether the changes in nucleosome repeat lengths might be localized in the initiated replicons, as postulated. In most experiments, Chinese hamster (line CHO) cells were synchronized in G1, or they were synchronized in early S phase by allowing G1 cells to enter S phase in medium containing 1 mM hydroxyurea or 5 μg mL -1 aphidicolin, a procedure believed to produce an accumulation of initiated replicons that arise from normally early replicating DNA. Measurements of nucleosome repeat lengths of bulk chromatin, the early replicating unexpressed metallothionein II (MTII) gene region, and a later replicating repeated sequence indicate that the changes in repeat lengths occur preferentially in the early replicating MTII gene region as G1 cells enter and become synchronized in early S phase. During that time, the MTII gene region is not replicated nor is there any evidence for induction of MTII messenger RNA. Thus, the results are consistent with the hypothesis that changes in chromatin structure occur preferentially in the early replicating (presumably initiated) replicons at initiation or that changes in chromatin structure can precede replication during inhibition of DNA synthesis. The shortened repeat lengths that precede MTII replication are, potentially, reversible, because they become elongated when the synchronized early S-phase cells are released to resume cell cycle progression

Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices: Sample preparation methods and liquid chromatography tandem mass spectrometric separations.

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Tölgyesi, Ádám; Barta, Enikő; Simon, Andrea; McDonald, Thomas J; Sharma, Virender K

2017-10-25

Veterinary drugs containing synthetic anabolic steroid and nitroimidazole active agents are not allowed for their applications in livestock of the European Union (EU). This paper presents analyses of twelve selected steroids and six nitroimidazole antibiotics at low levels (1.56μg/L-4.95μg/L and 0.17μg/kg-2.14μg/kg, respectively) in body fluids and egg incurred samples. Analyses involved clean-up procedures, high performance liquid chromatography (HPLC) separation, and tandem mass spectrometric screening and confirmatory methods. Target steroids and nitroimidazoles in samples were cleaned by two independent supported liquid extraction and solid phase extraction procedures. Separation of the selected compounds was conducted on Kinetex XB C-18 HPLC column using gradient elution. The screening methods utilised supported liquid extraction that enabled fast and cost effective clean-up. The confirmatory methods were improved by extending the number of matrices and compounds, and by introducing an isotope dilution mass spectrometry for nitroimidazoles. The new methods were validated according to the recommendation of the European Union Reference Laboratories and the performance characteristics evaluated met fully the criteria. The methods were applied to incurred samples in the proficiency tests. The obtained results of Z-scores demonstrated the applicability of developed protocols of the methods to real samples. The confirmatory methods were applied to the national monitoring program and natural contamination of prednisolone could be detected in urine at low concentration in few samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Nano clay Ni/NiO nanocomposite new sorbent for separation and preconcentration dibenzothiophene from crude prior to UV–vis spectrophotometery determination

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Amineh Mashkuri

2017-06-01

Full Text Available In this work, Ni/Ni nanoparticles were immobilized on modified nanoclay then, the resultant nanocomposite was used as a sorbent for separation of dibenzothiophene. The concentration of dibenzothiophene was determined with UV–vis spectrophotometry at 287 nm. Different parameters affecting the separation of dibenzothiophene such as: the ratio of Ni/NiO nanoparticles to nanoclay, amounts of Ni/NiO nanoparticles and nanoclay, stirring time of dibenzothiophene solution on the sorbent, type of eluent, eluent volume, elution time, stirring time for preparing the sorbent were optimized. The obtained results were shown quantitative desorption of dibenzothiophene performed using ethanol as eluent. A linear concentration range of 0.5–15 mg/L was obtained with a detection limit of 0.047 mg/mL. The sorbent capacity was found to be 32 mg/g. Relative standard deviation was ±1.4 for six replicated determination of 5 mg/g of dibenzothiophene. The optimized procedure was successfully used for a crude oil sample.

Chromatin Constrains the Initiation and Elongation of DNA Replication.

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Devbhandari, Sujan; Jiang, Jieqing; Kumar, Charanya; Whitehouse, Iestyn; Remus, Dirk

2017-01-05