Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

Science.gov (United States)

Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-ichiro M.

2013-01-01

Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds. PMID:23737447

[Late-replicating regions in salivary gland polytene chromosomes of Drosophila melanogaster].

Science.gov (United States)

Kolesnikov, T D; Andreenkova, N G; Beliaeva, E S; Goncharov, F P; Zykova, T Iu; Boldyreva, L V; Pokholkova, g V; Zhimulev, I F

2013-01-01

About 240 specific regions that are replicated at the very end of the S-phase have been identified in D. melanogaster polytene chromosomes. These regions have a repressive chromatine state, low gene density, long intergenic distances and are enriched in tissue specific genes. In polytene chromosomes, about a quarter of these regions have no enough time to complete replication. As a result, underreplication zones represented by fewer DNA copy number, appear. We studied 60 chromosome regions that demonstrated the most pronounced under-replication. By comparing the location of these regions on a molecular map with syntenic blocks found earlier for Drosophila species by von Grotthuss et al., 2010, we have shown that across the genus Drosophila, these regions tend to have conserved gene order. This forces us to assume the existence of evolutionary mechanisms aimed at maintaining the integrity of these regions.

Going ill to work--what personal circumstances, attitudes and work-related factors are associated with sickness presenteeism?

DEFF Research Database (Denmark)

Hansen, C. D.; Andersen, JH

2008-01-01

The aim of this study is to assess the impact of a broad range of possible factors relating to work, personal circumstances and attitudes towards sickness absence on a person's decision to go to work despite feeling ill, a phenomenon that has been termed sickness presence (SP), or 'presenteeism...... period. This means that SP is just as prevalent a phenomenon as sickness absence. Many of the results from earlier studies of SP were replicated and new factors were discovered: for example time pressure (having a supervisory role and/or working more than 45 h per week) and relationship with colleagues...... (measured by working in a small company, having non-standard hours and degree of cooperation) both increase the likelihood of SP. However, personal circumstances and attitudes, e.g. treating work as home (cf. Hochschild's thesis) and being over-committed to work, were also found to lead to higher levels...

Replicative age induces mitotic recombination in the ribosomal RNA gene cluster of Saccharomyces cerevisiae.

Directory of Open Access Journals (Sweden)

Derek L Lindstrom

2011-03-01

Full Text Available Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array. As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells.

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.

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…

Chromatin Heterogeneity and Distribution of Regulatory Elements in the Late-Replicating Intercalary Heterochromatin Domains of Drosophila melanogaster Chromosomes.

Directory of Open Access Journals (Sweden)

Varvara A Khoroshko

Full Text Available Late-replicating domains (intercalary heterochromatin in the Drosophila genome display a number of features suggesting their organization is quite unique. Typically, they are quite large and encompass clusters of functionally unrelated tissue-specific genes. They correspond to the topologically associating domains and conserved microsynteny blocks. Our study aims at exploring further details of molecular organization of intercalary heterochromatin and has uncovered surprising heterogeneity of chromatin composition in these regions. Using the 4HMM model developed in our group earlier, intercalary heterochromatin regions were found to host chromatin fragments with a particular epigenetic profile. Aquamarine chromatin fragments (spanning 0.67% of late-replicating regions are characterized as a class of sequences that appear heterogeneous in terms of their decompactization. These fragments are enriched with enhancer sequences and binding sites for insulator proteins. They likely mark the chromatin state that is related to the binding of cis-regulatory proteins. Malachite chromatin fragments (11% of late-replicating regions appear to function as universal transitional regions between two contrasting chromatin states. Namely, they invariably delimit intercalary heterochromatin regions from the adjacent active chromatin of interbands. Malachite fragments also flank aquamarine fragments embedded in the repressed chromatin of late-replicating regions. Significant enrichment of insulator proteins CP190, SU(HW, and MOD2.2 was observed in malachite chromatin. Neither aquamarine nor malachite chromatin types appear to correlate with the positions of highly conserved non-coding elements (HCNE that are typically replete in intercalary heterochromatin. Malachite chromatin found on the flanks of intercalary heterochromatin regions tends to replicate earlier than the malachite chromatin embedded in intercalary heterochromatin. In other words, there exists a

Optimal HIV testing and earlier care: the way forward in Europe

DEFF Research Database (Denmark)

Coenen, T; Lundgren, J; Lazarus, Jeff

2008-01-01

The articles in this supplement were developed from a recent pan-European conference entitled 'HIV in Europe 2007: Working together for optimal testing and earlier care', which took place on 26-27 November in Brussels, Belgium. The conference, organized by a multidisciplinary group of experts rep...

A Systems Approach to Effectiveness in Catholic Elementary Schools: A Replication and Extension

Science.gov (United States)

Mitchell, Roxanne M.; Tarter, C. John

2011-01-01

This study replicated an earlier study conducted by Tarter and Hoy (2004) in which an open systems model was used to test a series of hypotheses that explained elements of school performance. Four internal system elements (structure, individual, culture, and politics) of the school were used to explain two sets of school outcomes (student…

Checkpoint independence of most DNA replication origins in fission yeast.

Science.gov (United States)

Mickle, Katie L; Ramanathan, Sunita; Rosebrock, Adam; Oliva, Anna; Chaudari, Amna; Yompakdee, Chulee; Scott, Donna; Leatherwood, Janet; Huberman, Joel A

2007-12-19

In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2). Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (approximately 3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint

Checkpoint independence of most DNA replication origins in fission yeast

Science.gov (United States)

Mickle, Katie L; Ramanathan, Sunita; Rosebrock, Adam; Oliva, Anna; Chaudari, Amna; Yompakdee, Chulee; Scott, Donna; Leatherwood, Janet; Huberman, Joel A

2007-01-01

Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2). Results Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in

Checkpoint independence of most DNA replication origins in fission yeast

Directory of Open Access Journals (Sweden)

Scott Donna

2007-12-01

Full Text Available Abstract Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU, which limits the pool of deoxyribonucleoside triphosphates (dNTPs and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR or cds1 (which encodes the fission yeast homologue of Chk2. Results Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3% behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild

USP7/HAUSP: A SUMO deubiquitinase at the heart of DNA replication.

Science.gov (United States)

Smits, Veronique A J; Freire, Raimundo

2016-09-01

DNA replication is both highly conserved and controlled. Problematic DNA replication can lead to genomic instability and therefore carcinogenesis. Numerous mechanisms work together to achieve this tight control and increasing evidence suggests that post-translational modifications (phosphorylation, ubiquitination, SUMOylation) of DNA replication proteins play a pivotal role in this process. Here we discuss such modifications in the light of a recent article that describes a novel role for the deubiquitinase (DUB) USP7/HAUSP in the control of DNA replication. USP7 achieves this function by an unusual and novel mechanism, namely deubiquitination of SUMOylated proteins at the replication fork, making USP7 also a SUMO DUB (SDUB). This work extends previous observations of increased levels of SUMO and low levels of ubiquitin at the on-going replication fork. Here, we discuss this novel study, its contribution to the DNA replication and genomic stability field and what questions arise from this work. © 2016 WILEY Periodicals, Inc.

Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing.

Science.gov (United States)

Senkevich, Tatiana G; Bruno, Daniel; Martens, Craig; Porcella, Stephen F; Wolf, Yuri I; Moss, Bernard

2015-09-01

Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNA-primed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.

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

Three attempts to replicate the moral licensing effect

NARCIS (Netherlands)

Blanken, I.; van de Ven, N.; Zeelenberg, M.; Meijers, Marijn H. C.

2014-01-01

The present work includes three attempts to replicate the moral licensing effect by Sachdeva, Iliev, and Medin (2009). The original authors found that writing about positive traits led to lower donations to charity and decreased cooperative behavior. The first two replication attempts (student

The Reputational Consequences of Failed Replications and Wrongness Admission among Scientists.

Directory of Open Access Journals (Sweden)

Adam K Fetterman

Full Text Available Scientists are dedicating more attention to replication efforts. While the scientific utility of replications is unquestionable, the impact of failed replication efforts and the discussions surrounding them deserve more attention. Specifically, the debates about failed replications on social media have led to worry, in some scientists, regarding reputation. In order to gain data-informed insights into these issues, we collected data from 281 published scientists. We assessed whether scientists overestimate the negative reputational effects of a failed replication in a scenario-based study. Second, we assessed the reputational consequences of admitting wrongness (versus not as an original scientist of an effect that has failed to replicate. Our data suggests that scientists overestimate the negative reputational impact of a hypothetical failed replication effort. We also show that admitting wrongness about a non-replicated finding is less harmful to one's reputation than not admitting. Finally, we discovered a hint of evidence that feelings about the replication movement can be affected by whether replication efforts are aimed one's own work versus the work of another. Given these findings, we then present potential ways forward in these discussions.

The Reputational Consequences of Failed Replications and Wrongness Admission among Scientists.

Science.gov (United States)

Fetterman, Adam K; Sassenberg, Kai

2015-01-01

Scientists are dedicating more attention to replication efforts. While the scientific utility of replications is unquestionable, the impact of failed replication efforts and the discussions surrounding them deserve more attention. Specifically, the debates about failed replications on social media have led to worry, in some scientists, regarding reputation. In order to gain data-informed insights into these issues, we collected data from 281 published scientists. We assessed whether scientists overestimate the negative reputational effects of a failed replication in a scenario-based study. Second, we assessed the reputational consequences of admitting wrongness (versus not) as an original scientist of an effect that has failed to replicate. Our data suggests that scientists overestimate the negative reputational impact of a hypothetical failed replication effort. We also show that admitting wrongness about a non-replicated finding is less harmful to one's reputation than not admitting. Finally, we discovered a hint of evidence that feelings about the replication movement can be affected by whether replication efforts are aimed one's own work versus the work of another. Given these findings, we then present potential ways forward in these discussions.

Replication of Chilo iridescent virus in the cotton boll weevil, Anthonomus grandis, and development of an infectivity assay.

Science.gov (United States)

Henderson, C W; Johnson, C L; Lodhi, S A; Bilimoria, S L

2001-01-01

The boll weevil, Anthonomus grandis Boheman, is a devastating pest of cotton. Chemical pesticides are problematic due to relative lack of target specificity and resistance. Microbial pesticides may provide viable alternatives because of their narrow host range. Chilo iridescent virus (CIV) is the type species for genus Iridovirus, family Iridoviridae: large, icosahedral cytoplasmic viruses containing a double-stranded DNA genome. Earlier work suggested that CIV replicated in the boll weevil; however, efficiency or production of infectious virus was not established. We showed that CIV undergoes a productive cycle in A. grandis. CIV DNA levels in boll weevil pupae increased significantly from 0 to 3 days post infection. Moreover, virogenic stromata and complete virus particles were observed in the cytoplasm by 7 days. An endpoint dilution assay using viral DNA replication as indicator suggested a 10(5)-fold increase in infectious virus titer over 7 days. This is the first such demonstration in larval infections with genus Iridovirus. Our study establishes that CIV undergoes a productive cycle in the boll weevil and provides an important and useful model system for replication at the organismal level. These results have important implications for the potential of CIV and its components in boll weevil control.

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.

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.

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.

Genetic moderation of effects of maternal sensitivity on girl's age of menarche: Replication of the Manuck et al. study.

Science.gov (United States)

Hartman, Sarah; Widaman, Keith F; Belsky, Jay

2015-08-01

Manuck, Craig, Flory, Halder, and Ferrell (2011) reported that a theoretically anticipated effect of family rearing on girls' menarcheal age was genetically moderated by two single nucleotide polymorphisms (SNPs) of the estrogen receptor-α gene. We sought to replicate and extend these findings, studying 210 White females followed from birth. The replication was general because a different measure of the rearing environment was used in this inquiry (i.e., maternal sensitivity) than in the prior one (i.e., family cohesion). Extensions of the work included prospective rather than retrospective measurements of the rearing environment, reports of first menstruation within a year of its occurrence rather than decades later, accounting for some heritability of menarcheal age by controlling for maternal age of menarche, and using a new model-fitting approach to competitively compare diathesis-stress versus differential-susceptibility models of Gene × Environment interaction. The replication/extension effort proved successful in the case of both estrogen receptor-α SNPs, with the Gene × Environment interactions principally reflecting diathesis-stress: lower levels of maternal sensitivity predicted earlier age of menarche for girls homozygous for the minor alleles of either SNP but not for girls carrying other genotypes. Results are discussed in light of the new analytic methods adopted.

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 ...

Earlier time to aerobic exercise is associated with faster recovery following acute sport concussion.

Science.gov (United States)

Lawrence, David Wyndham; Richards, Doug; Comper, Paul; Hutchison, Michael G

2018-01-01

To determine whether earlier time to initiation of aerobic exercise following acute concussion is associated with time to full return to (1) sport and (2) school or work. A retrospective stratified propensity score survival analysis of acute (≤14 days) concussion was used to determine whether time (days) to initiation of aerobic exercise post-concussion was associated with, both, time (days) to full return to (1) sport and (2) school or work. A total of 253 acute concussions [median (IQR) age, 17.0 (15.0-20.0) years; 148 (58.5%) males] were included in this study. Multivariate Cox regression models identified that earlier time to aerobic exercise was associated with faster return to sport and school/work adjusting for other covariates, including quintile propensity strata. For each successive day in delay to initiation of aerobic exercise, individuals had a less favourable recovery trajectory. Initiating aerobic exercise at 3 and 7 days following injury was associated with a respective 36.5% (HR, 0.63; 95% CI, 0.53-0.76) and 73.2% (HR, 0.27; 95% CI, 0.16-0.45) reduced probability of faster full return to sport compared to within 1 day; and a respective 45.9% (HR, 0.54; 95% CI, 0.44-0.66) and 83.1% (HR, 0.17; 95% CI, 0.10-0.30) reduced probability of faster full return to school/work. Additionally, concussion history, symptom severity, LOC deleteriously influenced concussion recovery. Earlier initiation of aerobic exercise was associated with faster full return to sport and school or work. This study provides greater insight into the benefits and safety of aerobic exercise within the first week of the injury.

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...

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....

Is Implicit Theory of Mind a Real and Robust Phenomenon? Results From a Systematic Replication Study.

Science.gov (United States)

Kulke, Louisa; von Duhn, Britta; Schneider, Dana; Rakoczy, Hannes

2018-06-01

Recently, theory-of-mind research has been revolutionized by findings from novel implicit tasks suggesting that at least some aspects of false-belief reasoning develop earlier in ontogeny than previously assumed and operate automatically throughout adulthood. Although these findings are the empirical basis for far-reaching theories, systematic replications are still missing. This article reports a preregistered large-scale attempt to replicate four influential anticipatory-looking implicit theory-of-mind tasks using original stimuli and procedures. Results showed that only one of the four paradigms was reliably replicated. A second set of studies revealed, further, that this one paradigm was no longer replicated once confounds were removed, which calls its validity into question. There were also no correlations between paradigms, and thus, no evidence for their convergent validity. In conclusion, findings from anticipatory-looking false-belief paradigms seem less reliable and valid than previously assumed, thus limiting the conclusions that can be drawn from them.

[The effects of TorR protein on initiation of DNA replication in Escherichia coli].

Science.gov (United States)

Yuan, Yao; Jiaxin, Qiao; Jing, Li; Hui, Li; Morigen, Morigen

2015-03-01

The two-component systems, which could sense and respond to environmental changes, widely exist in bacteria as a signal transduction pathway. The bacterial CckA/CtrA, ArcA/ArcB and PhoP/PhoQ two-component systems are associated with initiation of DNA replication and cell division, however, the effects of the TorS/TorR system on cell cycle and DNA replication remains unknown. The TorS/TorR system in Escherichia coli can sense changes in trimethylamine oxide (TMAO) concentration around the cells. However, it is unknown if it also affects initiation of DNA replication. We detected DNA replication patterns in ΔtorS and ΔtorR mutant strains by flow cytometry. We found that the average number of replication origins (oriCs) per cell and doubling time in ΔtorS mutants were the same while the average number of oriCs in ΔtorR mutants was increased compared with that in wild-type cells. These results indicated that absence of TorR led to an earlier initiation of DNA replication than that in wild-type cells. Strangely, neither overexpression of TorR nor co-expression of TorR and TorS could restore ΔtorR mutant phenotype to the wild type. However, overexpression of SufD in both wild type and ΔtorR mutants promoted initiation of DNA replication, while mutation of SufD delayed it in ΔtorR mutants. Thus, TorR may affect initiation of DNA replication indirectly through regulating gene expression of sufD.

Muslim woman seeking work: An English case study with a dutch comparison, of discrimination and achievement

OpenAIRE

Bagley, C; Abubaker, M

2017-01-01

© 2016 by the author.The measurement of discrimination in employment is a key variable in understanding dynamics in the nature of, and change in "race relations". Measuring such discrimination using 'situation' and 'correspondence' tests was influenced by John Rex's sociological analyses, and earlier work, begun in America, was continued in England in the 1960s, and further replicated in Europe and America in later decades. This literature is reviewed, and the methodologies of testing for emp...

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

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

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.

Fragile genomic sites are associated with origins of replication.

Science.gov (United States)

Di Rienzi, Sara C; Collingwood, David; Raghuraman, M K; Brewer, Bonita J

2009-09-09

Genome rearrangements are mediators of evolution and disease. Such rearrangements are frequently bounded by transfer RNAs (tRNAs), transposable elements, and other repeated elements, suggesting a functional role for these elements in creating or repairing breakpoints. Though not well explored, there is evidence that origins of replication also colocalize with breakpoints. To investigate a potential correlation between breakpoints and origins, we analyzed evolutionary breakpoints defined between Saccharomyces cerevisiae and Kluyveromyces waltii and S. cerevisiae and a hypothetical ancestor of both yeasts, as well as breakpoints reported in the experimental literature. We find that origins correlate strongly with both evolutionary breakpoints and those described in the literature. Specifically, we find that origins firing earlier in S phase are more strongly correlated with breakpoints than are later-firing origins. Despite origins being located in genomic regions also bearing tRNAs and Ty elements, the correlation we observe between origins and breakpoints appears to be independent of these genomic features. This study lays the groundwork for understanding the mechanisms by which origins of replication may impact genome architecture and disease.

THE PERSONAS BEING (BAKHTIN’S CONCEPT OF THE HUMAN PERSONAS PRESENTED IN HIS EARLIER PHILOSOPHICAL WORKS OF THE 1920S

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J. KOLESNICHENKO

2011-08-01

Full Text Available The author attempts a detailed analysis of the earlier philosophical works of Bakhtin which in themselves are somewhat open to question and often full of contradictions. Her aim is to try to determine an all-comprehensive key term which formed the core of the philosopher’s early thought. By means of various methods of analysis, the author concludes that this key term is «personality» («lichnost». For Bakhtin, Personalityhas not only a social dimension but contains a broader scope: it refl ects Being (the substantiality per se. It is the contention of the author that Bakhtin’s early studies should be read against the background of this central key-term of «lichnost». As a result, a new non-contradictory theory of personality emerges. The author emphasizes that Bakhtin’s theory of personality is one example of a new, avant-garde type of philosophical paradigm which arose during the 1920’s.

Synchronization of DNA array replication kinetics

Science.gov (United States)

Manturov, Alexey O.; Grigoryev, Anton V.

2016-04-01

In the present work we discuss the features of the DNA replication kinetics at the case of multiplicity of simultaneously elongated DNA fragments. The interaction between replicated DNA fragments is carried out by free protons that appears at the every nucleotide attachment at the free end of elongated DNA fragment. So there is feedback between free protons concentration and DNA-polymerase activity that appears as elongation rate dependence. We develop the numerical model based on a cellular automaton, which can simulate the elongation stage (growth of DNA strands) for DNA elongation process with conditions pointed above and we study the possibility of the DNA polymerases movement synchronization. The results obtained numerically can be useful for DNA polymerase movement detection and visualization of the elongation process in the case of massive DNA replication, eg, under PCR condition or for DNA "sequencing by synthesis" sequencing devices evaluation.

De novo identification of replication-timing domains in the human genome by deep learning.

Science.gov (United States)

Liu, Feng; Ren, Chao; Li, Hao; Zhou, Pingkun; Bo, Xiaochen; Shu, Wenjie

2016-03-01

The de novo identification of the initiation and termination zones-regions that replicate earlier or later than their upstream and downstream neighbours, respectively-remains a key challenge in DNA replication. Building on advances in deep learning, we developed a novel hybrid architecture combining a pre-trained, deep neural network and a hidden Markov model (DNN-HMM) for the de novo identification of replication domains using replication timing profiles. Our results demonstrate that DNN-HMM can significantly outperform strong, discriminatively trained Gaussian mixture model-HMM (GMM-HMM) systems and other six reported methods that can be applied to this challenge. We applied our trained DNN-HMM to identify distinct replication domain types, namely the early replication domain (ERD), the down transition zone (DTZ), the late replication domain (LRD) and the up transition zone (UTZ), using newly replicated DNA sequencing (Repli-Seq) data across 15 human cells. A subsequent integrative analysis revealed that these replication domains harbour unique genomic and epigenetic patterns, transcriptional activity and higher-order chromosomal structure. Our findings support the 'replication-domain' model, which states (1) that ERDs and LRDs, connected by UTZs and DTZs, are spatially compartmentalized structural and functional units of higher-order chromosomal structure, (2) that the adjacent DTZ-UTZ pairs form chromatin loops and (3) that intra-interactions within ERDs and LRDs tend to be short-range and long-range, respectively. Our model reveals an important chromatin organizational principle of the human genome and represents a critical step towards understanding the mechanisms regulating replication timing. Our DNN-HMM method and three additional algorithms can be freely accessed at https://github.com/wenjiegroup/DNN-HMM The replication domain regions identified in this study are available in GEO under the accession ID GSE53984. shuwj@bmi.ac.cn or boxc

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.

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.

Essential 'ways-of-working' features of 'configuration management for new-build 2011' and 'issue-resolved replication'

International Nuclear Information System (INIS)

Schneider, B.

2011-01-01

The 'Ways-of-Working Foundation' of the Competencies most vital to 'New-Build' are provided. Examples are provided of local CM disasters and I-R.R success stories from else-where. [Configuration Management for New-Build 2011 - State-of-Art Specifications at Every Level - driven by Best-Available OpEx - based on SofA Expertise - subjected to Transparent, Independent Review]. It is the position of CMC2011 that weak CM is at the root of much 'less-than-adequate performance' (especially refurb programs - since ∼1986). For New-Build, the 'Foundation' of Configuration-Management is 'State-of-Art Specifications for All Systems, Equipment, Materials, and Processes' - supported by 'State-of-Art Service-Provider Expertise' - and Audit-able via 'Transparent, Third-Party, Issue-Review-and-Resolution Programs'. CM must be executed by the Architecture-Service-Providers - but it must be demanded by the Utility-Owners. [Issue-Resolved, Configuration-Managed, Replication - Rigorous, Configuration-Managed Replication for the Capture of the 85% that is Proven - plus - Parallel, Transparent, Separately-Managed, Contractually-Facilitated, Side-Stream Programs for the Identification and Resolution of the highly-troublesome 15% that remains Un-Resolved]. For New-Build in 2011, as well as for Refurb and Ops/Outage Support at any time, the last thing the Owner-Utilities want is innovation - or invention - or anything else for which substantial operating-experience and reliability-verification cannot be provided. I-R.R is discussed as - i) 'Rigorous Replication' and ii) 'Issue-Identification and Resolution'. What we refer to as 'Side-Stream, Independently-Driven, Issue-Identification and Resolution' is already practiced by utility 'SIR Teams' - i.e. - 'go-anywhere, do-anything, flying-squads' - for whom the watch-word is 'expect the un-anticipated'. Issue-Resolution must be provided as part of every project. It must be demanded (and contractually-facilitated) up-front by the Utility

Early manifestations of replicative aging in the yeast Saccharomyces cerevisiae

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Maksim I. Sorokin

2014-01-01

Full Text Available The yeast Saccharomyces cerevisiae is successfully used as a model organism to find genes responsible for lifespan control of higher organisms. As functional decline of higher eukaryotes can start as early as one quarter of the average lifespan, we asked whether S. cerevisiae can be used to model this manifestation of aging. While the average replicative lifespan of S. cerevisiae mother cells ranges between 15 and 30 division cycles, we found that resistances to certain stresses start to decrease much earlier. Looking into the mechanism, we found that knockouts of genes responsible for mitochondriato-nucleus (retrograde signaling, RTG1 or RTG3, significantly decrease the resistance of cells that generated more than four daughters, but not of the younger ones. We also found that even young mother cells frequently contain mitochondria with heterogeneous transmembrane potential and that the percentage of such cells correlates with replicative age. Together, these facts suggest that retrograde signaling starts to malfunction in relatively young cells, leading to accumulation of heterogeneous mitochondria within one cell. The latter may further contribute to a decline in stress resistances.

Multiprocessor Real-Time Locking Protocols for Replicated Resources

Science.gov (United States)

2016-07-01

assignment problem, the ac- tual identities of the allocated replicas must be known. When locking protocols are used, tasks may experience delays due to both...Multiprocessor Real-Time Locking Protocols for Replicated Resources ∗ Catherine E. Jarrett1, Kecheng Yang1, Ming Yang1, Pontus Ekberg2, and James H...replicas to execute. In prior work on replicated resources, k-exclusion locks have been used, but this restricts tasks to lock only one replica at a time. To

Guided Self-Help Treatment for Recurrent Binge Eating: Replication and Extension

Science.gov (United States)

DeBar, Lynn L.; Striegel-Moore, Ruth H.; Wilson, G. Terence; Perrin, Nancy; Yarborough, Bobbi Jo; Dickerson, John; Lynch, Frances; Rosselli, Francine; Kraemer, Helena C.

2014-01-01

Objective The aim of this study was to replicate and extend results of a previous blended efficacy and effectiveness trial of a low-intensity, manual-based guided self-help form of cognitive-behavioral therapy (CBT-GSH) for the treatment of binge eating disorders in a large health maintenance organization (HMO) and to compare them with usual care. Methods To extend earlier findings, the investigators modified earlier recruitment and assessment approaches and conducted a randomized clinical trial to better reflect procedures that may be reasonably carried out in real-world practices. The intervention was delivered by master’s-level interventionists to 160 female members of a health maintenance organization who met diagnostic criteria for recurrent binge eating. Data collected at baseline, immediately posttreatment, and at six- and 12-month follow-ups were used in intent-to-treat analyses. Results At the 12-month follow-up, CBT-GSH resulted in greater remission from binge eating (35%, N=26) than usual care (14%, N=10) (number needed to treat=5). The CBT-GSH group also demonstrated greater improvements in dietary restraint (d=.71) and eating, shape, and weight concerns (d=1.10, 1.24, and .98, respectively) but not weight change. Conclusions Replication of the pattern of previous findings suggests that CBT-GSH is a robust treatment for patients with recurrent binge eating. The magnitude of changes was significantly smaller than in the original study, however, suggesting that patients recruited and assessed with less intensive procedures may respond differently from their counterparts enrolled in trials requiring more comprehensive procedures. PMID:21459987

Do positive psychology exercises work? A replication of Seligman et al. (2005).

Science.gov (United States)

Mongrain, Myriam; Anselmo-Matthews, Tracy

2012-04-01

The current work replicated a landmark study conducted by Seligman and colleagues (2005) that demonstrated the long-term benefits of positive psychology exercises (PPEs). In the original study, two exercises administered over 1 week ("Three Good Things" and "Using your Signature Strengths in a New Way") were found to have long-lasting effects on depression and happiness (Seligman, Steen, Park, & Peterson, 2005). These exercises were tested here using the same methodology except for improvements to the control condition, and the addition of a second "positive placebo" to isolate the common factor of accessing positive, self-relevant constructs. This component control design was meant to assess the effect of expectancies for success (expectancy control), as well the cognitive access of positive information about the self (positive placebo). Repeated measures analyses showed that the PPEs led to lasting increases in happiness, as did the positive placebo. The PPEs did not exceed the control condition in producing changes in depression over time. Brief, positive psychology interventions may boost happiness through a common factor involving the activation of positive, self-relevant information rather than through other specific mechanisms. Finally, the effects of PPEs on depression may be more modest than previously assumed. © 2012 Wiley Periodicals, Inc.

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.

Inhibition of rotavirus replication by downregulation of fatty acid synthesis.

Science.gov (United States)

Gaunt, Eleanor R; Cheung, Winsome; Richards, James E; Lever, Andrew; Desselberger, Ulrich

2013-06-01

Recently the recruitment of lipid droplets (LDs) to sites of rotavirus (RV) replication was reported. LDs are polymorphic organelles that store triacylglycerols, cholesterol and cholesterol esters. The neutral fats are derived from palmitoyl-CoA, synthesized via the fatty acid biosynthetic pathway. RV-infected cells were treated with chemical inhibitors of the fatty acid biosynthetic pathway, and the effects on viral replication kinetics were assessed. Treatment with compound C75, an inhibitor of the fatty acid synthase enzyme complex (FASN), reduced RV infectivity 3.2-fold (P = 0.07) and modestly reduced viral RNA synthesis (1.2-fold). Acting earlier in the fatty acid synthesis pathway, TOFA [5-(Tetradecyloxy)-2-furoic acid] inhibits the enzyme acetyl-CoA carboxylase 1 (ACC1). TOFA reduced the infectivity of progeny RV 31-fold and viral RNA production 6-fold. The effect of TOFA on RV infectivity and RNA replication was dose-dependent, and infectivity was reduced by administering TOFA up to 4 h post-infection. Co-treatment of RV-infected cells with C75 and TOFA synergistically reduced viral infectivity. Knockdown by siRNA of FASN and ACC1 produced findings similar to those observed by inhibiting these proteins with the chemical compounds. Inhibition of fatty acid synthesis using a range of approaches uniformly had a more marked impact on viral infectivity than on viral RNA yield, inferring a role for LDs in virus assembly and/or egress. Specific inhibitors of fatty acid metabolism may help pinpoint the critical structural and biochemical features of LDs that are essential for RV replication, and facilitate the development of antiviral therapies.

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.

Science.gov (United States)

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.

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

Science.gov (United States)

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).

Polyomavirus BK replication in renal transplant recipients: combined monitoring of viremia and VP1 mRNA in urine

Directory of Open Access Journals (Sweden)

Sara Astegiano

2010-06-01

Full Text Available Introduction. Human polyomavirus BK (BKV is worldwide distributed, with a seroprevalence rate of 70–90% in the adults. Following primary infection, BK remains latent in the renourinary tract as the epidemiologically most relevant latency site, and in B cell, brain, spleen and probably other tissues. Reactivation may occur in both immunocompetent subjects and immunocompromised patients. In renal transplantation, in the context of intense immunosuppression, viral replication may determine BKV-associated nephropathy (BKVAN with interstitial nephritis and/or ureteral stenosis in 1–10% of the patients and leading to graft failure and return to haemodialysis in 30 to 80% of the cases (5. Screening of BKV replication represents the basic strategy to predict early the onset of BKVAN and may allow for earlier intervention with reduced allograft loss (3, 4. Nowadays, replication of BKV is monitored by quantification of BKV-DNA in serum and urine (2. The aim of this study was to evaluated the role of BKV VP1 mRNA in urine as a marker of viral replication in renal transplant recipients.

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.)

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...

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

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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.

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

Science.gov (United States)

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.

Making Connections: Replicating and Extending the Utility Value Intervention in the Classroom

Science.gov (United States)

Hulleman, Chris S.; Kosovich, Jeff J.; Barron, Kenneth E.; Daniel, David B.

2017-01-01

We replicated and extended prior research investigating a theoretically guided intervention based on expectancy-value theory designed to enhance student learning outcomes (e.g., Hulleman & Harackiewicz, 2009). First, we replicated prior work by demonstrating that the utility value intervention, which manipulated whether students made…

Live Replication of Paravirtual Machines

OpenAIRE

Stodden, Daniel

2009-01-01

Virtual machines offer a fair degree of system state encapsulation, which promotes practical advances in fault tolerance, system debugging, profiling and security applications. This work investigates deterministic replay and semi-active replication for system paravirtualization, a software discipline trading guest kernel binar compatibility for reduced dependency on costly trap-and-emulate techniques. A primary contribution is evidence that trace capturing under a piecewise deterministic exec...

The scenario on the origin of translation in the RNA world: in principle of replication parsimony

Directory of Open Access Journals (Sweden)

Ma Wentao

2010-11-01

to aid the binding of proto-tRNAs and proto-mRNAs, allowing the reduction of base pairs between them (ultimately resulting in the triplet anticodon/codon pair, thus further saving the replication cost. In this context, the replication cost saved would allow the appearance of more and longer functional peptides and, finally, proteins. The hypothesis could be called "DRT-RP" ("RP" for "replication parsimony". Testing the hypothesis The scenario described here is open for experimental work at some key scenes, including the compact DRT mechanism, the development of adaptors from aa-aptamers, the synthesis of peptides by proto-tRNAs and proto-mRNAs without the participation of proto-rRNAs, etc. Interestingly, a recent computer simulation study has demonstrated the plausibility of one of the evolving processes driven by replication parsimony in the scenario. Implication of the hypothesis An RNA-based proto-translation system could arise gradually from the DRT mechanism according to the principle of "replication parsimony" --- to save the replication cost of RNA templates for functional peptides. A surprising side deduction along the logic of the hypothesis is that complex, biosynthetic amino acids might have entered the genetic code earlier than simple, prebiotic amino acids, which is opposite to the common sense. Overall, the present discussion clarifies the blurry scenario concerning the origin of translation with a major clue, which shows vividly how life could "manage" to exploit potential chemical resources in nature, eventually in an efficient way over evolution. Reviewers This article was reviewed by Eugene V. Koonin, Juergen Brosius, and Arcady Mushegian.

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

Dynamics of DNA replication during premeiosis and early meiosis in wheat.

Science.gov (United States)

Rey, María-Dolores; Prieto, Pilar

2014-01-01

Meiosis is a specialised cell division that involves chromosome replication, two rounds of chromosome segregation and results in the formation of the gametes. Meiotic DNA replication generally precedes chromosome pairing, recombination and synapsis in sexually developing eukaryotes. In this work, replication has been studied during premeiosis and early meiosis in wheat using flow cytometry, which has allowed the quantification of the amount of DNA in wheat anther in each phase of the cell cycle during premeiosis and each stage of early meiosis. Flow cytometry has been revealed as a suitable and user-friendly tool to detect and quantify DNA replication during early meiosis in wheat. Chromosome replication was detected in wheat during premeiosis and early meiosis until the stage of pachytene, when chromosomes are associated in pairs to further recombine and correctly segregate in the gametes. In addition, the effect of the Ph1 locus, which controls chromosome pairing and affects replication in wheat, was also studied by flow cytometry. Here we showed that the Ph1 locus plays an important role on the length of meiotic DNA replication in wheat, particularly affecting the rate of replication during early meiosis in wheat.

Synchronous contextual irregularities affect early scene processing: replication and extension.

Science.gov (United States)

Mudrik, Liad; Shalgi, Shani; Lamy, Dominique; Deouell, Leon Y

2014-04-01

Whether contextual regularities facilitate perceptual stages of scene processing is widely debated, and empirical evidence is still inconclusive. Specifically, it was recently suggested that contextual violations affect early processing of a scene only when the incongruent object and the scene are presented a-synchronously, creating expectations. We compared event-related potentials (ERPs) evoked by scenes that depicted a person performing an action using either a congruent or an incongruent object (e.g., a man shaving with a razor or with a fork) when scene and object were presented simultaneously. We also explored the role of attention in contextual processing by using a pre-cue to direct subjects׳ attention towards or away from the congruent/incongruent object. Subjects׳ task was to determine how many hands the person in the picture used in order to perform the action. We replicated our previous findings of frontocentral negativity for incongruent scenes that started ~ 210 ms post stimulus presentation, even earlier than previously found. Surprisingly, this incongruency ERP effect was negatively correlated with the reaction times cost on incongruent scenes. The results did not allow us to draw conclusions about the role of attention in detecting the regularity, due to a weak attention manipulation. By replicating the 200-300 ms incongruity effect with a new group of subjects at even earlier latencies than previously reported, the results strengthen the evidence for contextual processing during this time window even when simultaneous presentation of the scene and object prevent the formation of prior expectations. We discuss possible methodological limitations that may account for previous failures to find this an effect, and conclude that contextual information affects object model selection processes prior to full object identification, with semantic knowledge activation stages unfolding only later on. Copyright © 2014 Elsevier Ltd. All rights reserved.

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…

Amplified Self-replication of DNA Origami Nanostructures through Multi-cycle Fast-annealing Process

Science.gov (United States)

Zhou, Feng; Zhuo, Rebecca; He, Xiaojin; Sha, Ruojie; Seeman, Nadrian; Chaikin, Paul

We have developed a non-biological self-replication process using templated reversible association of components and irreversible linking with annealing and UV cycles. The current method requires a long annealing time, up to several days, to achieve the specific self-assembly of DNA nanostructures. In this work, we accomplished the self-replication with a shorter time and smaller replication rate per cycle. By decreasing the ramping time, we obtained the comparable replication yield within 90 min. Systematic studies show that the temperature and annealing time play essential roles in the self-replication process. In this manner, we can amplify the self-replication process to a factor of 20 by increasing the number of cycles within the same amount of time.

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.

Hydroxyurea-Induced Replication Stress

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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.

Replicating animal mitochondrial DNA

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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.

Alzheimer's and Dementia Testing for Earlier Diagnosis

Science.gov (United States)

... for Earlier Diagnosis What if we could diagnose Alzheimer's before symptoms started? The hope is, future treatments ... diagnosis is among the most active areas in Alzheimer's science, and funding from the Alzheimer's Association has ...

Replication of an Experimental Study Investigating the Efficacy of a Multisyllabic Word Reading Intervention With and Without Motivational Beliefs Training for Struggling Readers.

Science.gov (United States)

Toste, Jessica R; Capin, Philip; Williams, Kelly J; Cho, Eunsoo; Vaughn, Sharon

2018-05-01

This randomized control trial examined the efficacy of an intervention aimed at improving multisyllabic word reading (MWR) skills among fourth- and fifth-grade struggling readers ( n = 109, 48.6% male), as well as the relative effects of an embedded motivational beliefs training component. This study was a closely aligned replication of our earlier work. The intervention was replicated with a three-condition design: MWR only, MWR with a motivational beliefs component, and business-as-usual control. Students were tutored in small groups for 40 lessons (four 40-min lessons each week). When we combined performance of students in both MWR conditions, intervention students significantly outperformed controls on proximal measures of affix reading and MWR, as well as standardized measures of decoding, spelling, and text comprehension. Furthermore, there was a noted interaction between English learner status and treatment on spelling performance. There were no statistically significant main effects between the MWR groups on proximal or standardized measures of interest. Findings are discussed in terms of their relevance to MWR instruction for students with persistent reading difficulties and considerations for future research related to the malleability of motivation.

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.

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

Science.gov (United States)

Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

2016-05-17

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Quality of topographical micro replication in injection moulding

DEFF Research Database (Denmark)

Arlø, Uffe Rolf; Hansen, Hans Nørgaard; Bariani, Paolo

2003-01-01

The quality of tool-to-part rough surface topography replication in injection moulding has been investigated. Quantitative descriptors suitable for detecting process conditions induced topography changes have been identified using a statistical criterion. The experimental work is based on a tool ...

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.

Promotion of Hendra Virus Replication by MicroRNA 146a

Science.gov (United States)

Marsh, Glenn A.; Jenkins, Kristie A.; Gantier, Michael P.; Tizard, Mark L.; Middleton, Deborah; Lowenthal, John W.; Haining, Jessica; Izzard, Leonard; Gough, Tamara J.; Deffrasnes, Celine; Stambas, John; Robinson, Rachel; Heine, Hans G.; Pallister, Jackie A.; Foord, Adam J.; Bean, Andrew G.; Wang, Lin-Fa

2013-01-01

Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease. PMID:23345523

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....

How and why multiple MCMs are loaded at origins of DNA replication.

Science.gov (United States)

Das, Shankar P; Rhind, Nicholas

2016-07-01

Recent work suggests that DNA replication origins are regulated by the number of multiple mini-chromosome maintenance (MCM) complexes loaded. Origins are defined by the loading of MCM - the replicative helicase which initiates DNA replication and replication kinetics determined by origin's location and firing times. However, activation of MCM is heterogeneous; different origins firing at different times in different cells. Also, more MCMs are loaded in G1 than are used in S phase. These aspects of MCM biology are explained by the observation that multiple MCMs are loaded at origins. Having more MCMs at early origins makes them more likely to fire, effecting differences in origin efficiency that define replication timing. Nonetheless, multiple MCM loading raises new questions, such as how they are loaded, where these MCMs reside at origins, and how their presence affects replication timing. In this review, we address these questions and discuss future avenues of research. © 2016 WILEY Periodicals, Inc.

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.

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...

Gefitinib and pyrrolidine dithiocarbamate decrease viral replication and cytokine production in dengue virus infected human monocyte cultures.

Science.gov (United States)

Duran, Anyelo; Valero, Nereida; Mosquera, Jesús; Fuenmayor, Edgard; Alvarez-Mon, Melchor

2017-12-15

The epidermal growth factor receptor (EGFR) and nucleotide-binding and oligomerization-domain containing 2 (NOD2) are important in cancer and in microbial recognition, respectively. These molecules trigger intracellular signaling pathways inducing the expression of inflammatory genes by NF-kB translocation. Gefitinib (GBTC) and pyrrolidine dithiocarbamate (PDTC) are capable of inhibiting EGFR/NOD2 and NF-kB, respectively. In earlier stages of dengue virus (DENV) infection, monocytes are capable of sustaining viral replication and increasing cytokine production, suggesting that monocyte/macrophages play an important role in early DENV replication. GBTC and PDTC have not been used to modify the pathogenesis of DENV in infected cells. This study was aimed to determine the effect of GBTC and PDTC on viral replication and cytokine production in DENV serotype 2 (DENV2)-infected human monocyte cultures. GBTC and PDTC were used to inhibit EGFR/NOD2 and NF-kB, respectively. Cytokine production was measured by ELISA and viral replication by plaque forming unit assay. Increased DENV2 replication and anti-viral cytokine production (IFN-α/β, TNF-α, IL-12 and IL-18) in infected cultures were found. These parameters were decreased after EGFR/NOD2 or NF-kB inhibitions. The inhibitory effects of GBTC and PDTC on viral replication and cytokine production can be beneficial in the treatment of patients infected by dengue and suggest a possible role of EGFR/NOD2 receptors and NF-kB in dengue pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

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…

Registered Replication Report: Testing Disruptive Effects of Irrelevant Speech on Visual-Spatial Working Memory

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Tatiana Kvetnaya

2018-04-01

Full Text Available A Partial Replication of “Functional Equivalence of Verbal and Spatial Information in Serial Short-Term Memory (Jones, Farrand, Stuart, & Morris, 1995; Experiment 4” The irrelevant speech effect (ISE—the phenomenon that background speech impairs serial recall of visually presented material—has been widely used for examining the structure of short-term memory. In Experiment 4, Jones, Farrand, Stuart, and Morris (1995 employed the ISE to demonstrate that impairment of performance is determined by the changing-state characteristics of the material, rather than its modality of origin. The present study directly replicated the spatial condition of Experiment 4 with 'N' = 40 German participants. In contrast to the original findings, no main effect of sound type was observed, 'F'(2, 78 = 0.81, 'p' = .450, η2'p' = .02. The absence of an ISE in the spatial domain does not support the changing state hypothesis.

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.

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…

Registered Replication Report

DEFF Research Database (Denmark)

Bouwmeester, S.; Verkoeijen, P. P.J.L.; Aczel, B.

2017-01-01

and colleagues. The results of studies using time pressure have been mixed, with some replication attempts observing similar patterns (e.g., Rand et al., 2014) and others observing null effects (e.g., Tinghög et al., 2013; Verkoeijen & Bouwmeester, 2014). This Registered Replication Report (RRR) assessed...... the size and variability of the effect of time pressure on cooperative decisions by combining 21 separate, preregistered replications of the critical conditions from Study 7 of the original article (Rand et al., 2012). The primary planned analysis used data from all participants who were randomly assigned...

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...

Surface topography analysis for dimensional quality control of replication at the micrometre scale

DEFF Research Database (Denmark)

Balcon, M.; Marinello, F.; Tosello, Guido

2011-01-01

Replication of geometrical features and surfaces are present at different production levels, from realization of moulds to final product. Geometrical features must be reproduced within specification limits, to ensure product functionality . In order to control the replication quality, mould...... and replica surfaces must be quantitatively analysed and compared. In the present work, reference simulated surfaces were considered and studied in order to evaluate the effectiveness and traceability of different analysis tools for replication quality control. Topographies were analysed simulating different...

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.

Insight Into Illness and Cognition in Schizophrenia in Earlier and Later Life.

Science.gov (United States)

Gerretsen, Philip; Voineskos, Aristotle N; Graff-Guerrero, Ariel; Menon, Mahesh; Pollock, Bruce G; Mamo, David C; Mulsant, Benoit H; Rajji, Tarek K

2017-04-01

Impaired insight into illness in schizophrenia is associated with illness severity and deficits in premorbid intellectual function, executive function, and memory. A previous study of patients aged 60 years and older found that illness severity and premorbid intellectual function accounted for variance in insight impairment. As such, we aimed to test whether similar relationships would be observed in earlier life. A retrospective analysis was performed on 1 large sample of participants (n = 171) with a DSM-IV-TR diagnosis of schizophrenia aged 19 to 79 years acquired from 2 studies: (1) a psychosocial intervention trial for older persons with schizophrenia (June 2008 to May 2014) and (2) a diffusion tensor imaging and genetics study of psychosis across the life span (February 2007 to December 2013). We assessed insight into illness using the Positive and Negative Syndrome Scale (PANSS) item G12 and explored its relationship to illness severity (PANSS total modified), premorbid intellectual function (Wechsler Test of Adult Reading [WTAR]), and cognition. Insight impairment was more severe in later life (≥ 60 years) than in earlier years (t = -3.75, P insight was explained by PANSS total modified (Exp[B] = 1.070, P insight, they did not independently contribute to its variance. However, the relationships between impaired insight and illness severity and between impaired insight and cognition, particularly working memory, were stronger in later life than in earlier life. These results suggest an opportunity for intervention may exist with cognitive-enhancing neurostimulation or medications to improve insight into illness in schizophrenia across the life span. Original study registered on ClinicalTrials.gov (identifier: NCT00832845). © Copyright 2017 Physicians Postgraduate Press, Inc.

An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies.

Science.gov (United States)

Komatsu, Tetsuro; Nagata, Kyosuke; Wodrich, Harald

2016-02-01

/or subsequent viral DNA replication. Here we performed a detailed analyses of the spatiotemporal distribution of incoming adenoviral genome complexes and found, in contrast to the expectation, that an adenoviral DNA replication factor, but not incoming genomes, targets PML-NBs. Thus, our findings may explain why adenoviral genomes could be observed at PML-NBs in earlier reports but argue against a generalized role for PML-NBs in targeting invading viral genomes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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.

Assumption and program of the earlier stage construction of L/ILW disposal site

International Nuclear Information System (INIS)

Li Xuequn; Chen Shi; Li Xinbang

1993-01-01

The authors analysed the production and treatment of low- and intermediate-level radwastes (L/ILW) in China. Some problems and situation in this field are introduced. Over the past ten years, preliminary efforts have been made by CNNC (China National Nuclear Corporation) in policy, law and rules, developing program, management system, siting, engineering techniques, and safety assessment for radwaste disposal. The investment of the earlier stage work of L/ILW disposal site construction is estimated, the program and assumption to disposal site construction of the L/ILW are reviewed

Diversity climate enhances work outcomes through trust and openness in workgroup communication.

Science.gov (United States)

Hofhuis, Joep; van der Rijt, Pernill G A; Vlug, Martijn

2016-01-01

Diversity climate, defined as an organizational climate characterized by openness towards and appreciation of individual differences, has been shown to enhance outcomes in culturally diverse teams. To date, it remains unclear which processes are responsible for these findings. This paper presents two quantitative studies (n = 91; 246) that identify trust and openness in workgroup communication as possible mediators. We replicate earlier findings that perceived diversity climate positively relates to job satisfaction, sense of inclusion, work group identification and knowledge sharing in teams. In study 1, trust is shown to mediate the effects of perceived diversity climate on team members' sense of inclusion. In study 2, trust mediates the relationship between perceived diversity climate and workgroup identification and openness mediates its relationship with knowledge sharing.

DATABASE OF MIGRATION AND REPLICATION WITH ORACLE GOLDEN GATE

Directory of Open Access Journals (Sweden)

Suharjito Suharjito

2014-10-01

Full Text Available The main goal of this research is to analyze and design a database configuration of migration and replication in PT Metro Batavia. Research methodologies used in this research are data collecting, analysis and design model. Data collecting method is conducted with library research and direct survey in the company. Analysis method is conducted by analyzing hangar system, migration and reflection process and the available problems. Design method is conducted by designing a prototype for migration process with the implementation of Oracle SQL Developer and replication process with implementation of Oracle Golden Gate. The result of this research is a prototype for configuration of migration and replication process by using Oracle Golden Gate, which can produce two sets of identical data for the purpose of backup and recovery, and also design a simple tool that is expected to help active-active or active-passive replication process. The conclusion of this research is migration process of MySQL database to Oracle database by using Oracle Golden Gate hasn’t been conducted, because Oracle Golden Gate still has bug related to binary log, so database of migration is conducted by using Oracle Golden Gate. However, replication of bi-directional in between database of Oracle by using Oracle SQL Developer can guarantee data availability and reduce work burden from primary database.

Tracking replicability as a method of post-publication open evaluation

Directory of Open Access Journals (Sweden)

Joshua eHartshorne

2012-03-01

Full Text Available Recent reports have suggested that many published results are unreliable. To increase the reliability and accuracy of published papers, multiple changes have been proposed, such as changes in statistical methods. We support such reforms. However, we believe that the incentive structure of scientific publishing must change for such reforms to be successful. Under the current system, the quality of individual scientists is judged on the basis of their number of publications and citations, with journals similarly judged via numbers of citations. Neither of these measures takes into account the replicability of the published findings, as false or controversial results are often particularly widely cited. We propose tracking replications as a means of post-publication evaluation, both to help researchers identify reliable findings and to incentivize the publication of reliable results.Tracking replications requires a database linking published studies that replicate one another. As any such database is limited by the number of replication attempts published, we propose establishing an open-access journal dedicated to publishing replication attempts. Data quality of both the database and the affiliated journal would be ensured through a combination of crowd-sourcing and peer review. As reports in the database are aggregated, ultimately it will be possible to calculate replicability scores, which may be used alongside citation counts to evaluate the quality of work published in individual journals. In this paper, we lay out a detailed description of how this system could be implemented, including mechanisms for compiling the information, ensuring data quality, and incentivizing the research community to participate.

In-utero cigarette smoke exposure and the risk of earlier menopause

NARCIS (Netherlands)

Honorato, Talita C; Haadsma, Maaike L; Land, Jolande A; Boezen, Marike H; Hoek, Annemieke; Groen, Henk

2018-01-01

OBJECTIVE: Cigarette smoking is a risk factor for earlier menopause. Animal studies show that in-utero smoke exposure is toxic to developing ovaries. Our aim was to evaluate whether in-utero smoke exposed women reach menopause earlier compared with nonexposed women. METHODS: This is a cohort study

DNA Replication Profiling Using Deep Sequencing.

Science.gov (United States)

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.

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.)

Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

International Nuclear Information System (INIS)

Diaz, Jason; Wang, Xin; Tsang, Sabrina H.; Jiao, Jing; You, Jianxin

2014-01-01

Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells

Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

Energy Technology Data Exchange (ETDEWEB)

Diaz, Jason; Wang, Xin; Tsang, Sabrina H. [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States); Jiao, Jing [Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104 (United States); You, Jianxin, E-mail: jianyou@mail.med.upenn.edu [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States)

2014-07-08

Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.

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...

Derivatives. Replication and (auto)plagiarism in the social sciences

NARCIS (Netherlands)

R. Tweehuysen (Rolandt ); J. den Haan (Joost); K. Berkhout (Karel ); P.A.G. van Bergeijk (Peter)

2012-01-01

textabstractThis working paper reports on the travelling exhibition “Derivatives”. This exhibition investigates the issue of originality in the context of (self) plagiarism and replication. The different views in the Arts and the scientific discourse form the point of departure for discovering how

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.

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…

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.

A study of an adaptive replication framework for orchestrated composite web services.

Science.gov (United States)

Mohamed, Marwa F; Elyamany, Hany F; Nassar, Hamed M

2013-01-01

Replication is considered one of the most important techniques to improve the Quality of Services (QoS) of published Web Services. It has achieved impressive success in managing resource sharing and usage in order to moderate the energy consumed in IT environments. For a robust and successful replication process, attention should be paid to suitable time as well as the constraints and capabilities in which the process runs. The replication process is time-consuming since outsourcing some new replicas into other hosts is lengthy. Furthermore, nowadays, most of the business processes that might be implemented over the Web are composed of multiple Web services working together in two main styles: Orchestration and Choreography. Accomplishing a replication over such business processes is another challenge due to the complexity and flexibility involved. In this paper, we present an adaptive replication framework for regular and orchestrated composite Web services. The suggested framework includes a number of components for detecting unexpected and unhappy events that might occur when consuming the original published web services including failure or overloading. It also includes a specific replication controller to manage the replication process and select the best host that would encapsulate a new replica. In addition, it includes a component for predicting the incoming load in order to decrease the time needed for outsourcing new replicas, enhancing the performance greatly. A simulation environment has been created to measure the performance of the suggested framework. The results indicate that adaptive replication with prediction scenario is the best option for enhancing the performance of the replication process in an online business environment.

Replication of Holograms with Corn Syrup by Rubbing

Directory of Open Access Journals (Sweden)

Arturo Olivares-Pérez

2012-08-01

Full Text Available Corn syrup films are used to replicate holograms in order to fabricate micro-structural patterns without the toxins commonly found in photosensitive salts and dyes. We use amplitude and relief masks with lithographic techniques and rubbing techniques in order to transfer holographic information to corn syrup material. Holographic diffraction patterns from holographic gratings and computer Fourier holograms fabricated with corn syrup are shown. We measured the diffraction efficiency parameter in order to characterize the film. The versatility of this material for storage information is promising. Holographic gratings achieved a diffraction efficiency of around 8.4% with an amplitude mask and 36% for a relief mask technique. Preliminary results using corn syrup as an emulsion for replicating holograms are also shown in this work.

Replication of Holograms with Corn Syrup by Rubbing

Science.gov (United States)

Mejias-Brizuela, Nildia Y.; Olivares-Pérez, Arturo; Ortiz-Gutiérrez, Mauricio

2012-01-01

Corn syrup films are used to replicate holograms in order to fabricate micro-structural patterns without the toxins commonly found in photosensitive salts and dyes. We use amplitude and relief masks with lithographic techniques and rubbing techniques in order to transfer holographic information to corn syrup material. Holographic diffraction patterns from holographic gratings and computer Fourier holograms fabricated with corn syrup are shown. We measured the diffraction efficiency parameter in order to characterize the film. The versatility of this material for storage information is promising. Holographic gratings achieved a diffraction efficiency of around 8.4% with an amplitude mask and 36% for a relief mask technique. Preliminary results using corn syrup as an emulsion for replicating holograms are also shown in this work.

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

Science.gov (United States)

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.

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

Sensitivity to Audiovisual Temporal Asynchrony in Children with a History of Specific Language Impairment and Their Peers with Typical Development: A Replication and Follow-Up Study

Science.gov (United States)

Kaganovich, Natalya

2017-01-01

Purpose: Earlier, my colleagues and I showed that children with a history of specific language impairment (H-SLI) are significantly less able to detect audiovisual asynchrony compared with children with typical development (TD; Kaganovich & Schumaker, 2014). Here, I first replicate this finding in a new group of children with H-SLI and TD and…

Spacetime Replication of Quantum Information Using (2 , 3) Quantum Secret Sharing and Teleportation

Science.gov (United States)

Wu, Yadong; Khalid, Abdullah; Davijani, Masoud; Sanders, Barry

The aim of this work is to construct a protocol to replicate quantum information in any valid configuration of causal diamonds and assess resources required to physically realize spacetime replication. We present a set of codes to replicate quantum information along with a scheme to realize these codes using continuous-variable quantum optics. We use our proposed experimental realizations to determine upper bounds on the quantum and classical resources required to simulate spacetime replication. For four causal diamonds, our implementation scheme is more efficient than the one proposed previously. Our codes are designed using a decomposition algorithm for complete directed graphs, (2 , 3) quantum secret sharing, quantum teleportation and entanglement swapping. These results show the simulation of spacetime replication of quantum information is feasible with existing experimental methods. Alberta Innovates, NSERC, China's 1000 Talent Plan and the Institute for Quantum Information and Matter, which is an NSF Physics Frontiers Center (NSF Grant PHY-1125565) with support of the Gordon and Betty Moore Foundation (GBMF-2644).

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

Effect of deoxyribonucleic acid replication inhibitors on bacterial recombination

International Nuclear Information System (INIS)

Canosi, U.; Siccardi, A.G.; Falaschi, A.; Mazza, G.

1976-01-01

Two inhibitors of replicative deoxyribonucleic acid (DNA) synthesis, nalidixic acid (NAL) and 6-(p-hydroxyphenylazo)-uracil (HPUra), showed different effects on genetic recombination and DNA repair in Bacillus subtilis. Previous work (Pedrini et al., 1972) showed that NAL does not interfere with the transformation process of B. subtilis. The results reported in this work demonstrated that the drug was also without effect on the transfection SPP1 or SPO-1 phage DNA (a process that requires a recombination event). The drug was also ineffective on the host cell reactivation of ultraviolet-irradiated SPP1 phage, as well as on transfection with ultraviolet-irradiated DNA of the same phage. HPUra instead markedly reduced the transformation process, as well as transfection, by SPO-1 DNA, but it did not affect the host cell reactivation of SPO-1 phage. In conclusion, whereas the NAL target seems to be specific for replicative DNA synthesis, the HPUra target (i.e., the DNA polymerase III of B. subtilis) seems to be involved also in recombination, but not in the excision repair process. The mutations conferring NAL and HPUra resistance used in this work were mapped by PBS-1 transduction

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.

Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

Science.gov (United States)

van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

2015-01-01

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

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....

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

OpenAIRE

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

2015-01-01

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 cellula...

Initiation of chromosomal replication in predatory bacterium Bdellovibrio bacteriovorus

Directory of Open Access Journals (Sweden)

Lukasz Makowski

2016-11-01

Full Text Available Bdellovibrio bacteriovorus is a small Gram-negative predatory bacterium that attacks other Gram-negative bacteria, including many animal, human, and plant pathogens. This bacterium exhibits a peculiar biphasic life cycle during which two different types of cells are produced: non-replicating highly motile cells (the free-living phase and replicating cells (the intracellular-growth phase. The process of chromosomal replication in B. bacteriovorus must therefore be temporally and spatially regulated to ensure that it is coordinated with cell differentiation and cell cycle progression. Recently, B. bacteriovorus has received considerable research interest due to its intriguing life cycle and great potential as a prospective antimicrobial agent. Although we know that chromosomal replication in bacteria is mainly regulated at the initiation step, no data exists about this process in B. bacteriovorus. We report the first characterization of key elements of initiation of chromosomal replication – DnaA protein and oriC region from the predatory bacterium, B. bacteriovorus. In vitro studies using different approaches demonstrate that the B. bacteriovorus oriC (BdoriC is specifically bound and unwound by the DnaA protein. Sequence comparison of the DnaA-binding sites enabled us to propose a consensus sequence for the B. bacteriovorus DnaA box (5’-NN(A/TTCCACA-3’. Surprisingly, in vitro analysis revealed that BdoriC is also bound and unwound by the host DnaA proteins (relatively distantly related from B. bacteriovorus. We compared the architecture of the DnaA–oriC complexes (orisomes in homologous (oriC and DnaA from B. bacteriovorus and heterologous (BdoriC and DnaA from prey, E. coli or P. aeruginosa systems. This work provides important new entry points toward improving our understanding of the initiation of chromosomal replication in this predatory bacterium.

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.

Prescription stimulant use is associated with earlier onset of psychosis.

Science.gov (United States)

Moran, Lauren V; Masters, Grace A; Pingali, Samira; Cohen, Bruce M; Liebson, Elizabeth; Rajarethinam, R P; Ongur, Dost

2015-12-01

A childhood history of attention deficit hyperactivity disorder (ADHD) is common in psychotic disorders, yet prescription stimulants may interact adversely with the physiology of these disorders. Specifically, exposure to stimulants leads to long-term increases in dopamine release. We therefore hypothesized that individuals with psychotic disorders previously exposed to prescription stimulants will have an earlier onset of psychosis. Age of onset of psychosis (AOP) was compared in individuals with and without prior exposure to prescription stimulants while controlling for potential confounding factors. In a sample of 205 patients recruited from an inpatient psychiatric unit, 40% (n = 82) reported use of stimulants prior to the onset of psychosis. Most participants were prescribed stimulants during childhood or adolescence for a diagnosis of ADHD. AOP was significantly earlier in those exposed to stimulants (20.5 vs. 24.6 years stimulants vs. no stimulants, p drugs of abuse, and family history of a first-degree relative with psychosis, the association between stimulant exposure and earlier AOP remained significant. There was a significant gender × stimulant interaction with a greater reduction in AOP for females, whereas the smaller effect of stimulant use on AOP in males did not reach statistical significance. In conclusion, individuals with psychotic disorders exposed to prescription stimulants had an earlier onset of psychosis, and this relationship did not appear to be mediated by IQ or cannabis. Copyright © 2015 Elsevier Ltd. All rights reserved.

DNA replication: stalling a fork for imprinting and switching

DEFF Research Database (Denmark)

Egel, Richard

2004-01-01

Mating-type switching in fission yeast has long been known to be directed by a DNA 'imprint'. This imprint has now been firmly characterized as a protected site-specific and strand-specific nick. New work also links the widely conserved Swi1-Swi3 complex to the protection of stalled replication...

Cardiac Complications, Earlier Treatment, and Initial Disease Severity in Kawasaki Disease.

Science.gov (United States)

Abrams, Joseph Y; Belay, Ermias D; Uehara, Ritei; Maddox, Ryan A; Schonberger, Lawrence B; Nakamura, Yosikazu

2017-09-01

To assess if observed higher observed risks of cardiac complications for patients with Kawasaki disease (KD) treated earlier may reflect bias due to confounding from initial disease severity, as opposed to any negative effect of earlier treatment. We used data from Japanese nationwide KD surveys from 1997 to 2004. Receipt of additional intravenous immunoglobulin (IVIG) (data available all years) or any additional treatment (available for 2003-2004) were assessed as proxies for initial disease severity. We determined associations between earlier or later IVIG treatment (defined as receipt of IVIG on days 1-4 vs days 5-10 of illness) and cardiac complications by stratifying by receipt of additional treatment or by using logistic modeling to control for the effect of receiving additional treatment. A total of 48 310 patients with KD were included in the analysis. In unadjusted analysis, earlier IVIG treatment was associated with a higher risk for 4 categories of cardiac complications, including all major cardiac complications (risk ratio, 1.10; 95% CI, 1.06-1.15). Stratifying by receipt of additional treatment removed this association, and earlier IVIG treatment became protective against all major cardiac complications when controlling for any additional treatment in logistic regressions (OR, 0.90; 95% CI, 0.80-1.00). Observed higher risks of cardiac complications among patients with KD receiving IVIG treatment on days 1-4 of the illness are most likely due to underlying higher initial disease severity, and patients with KD should continue to be treated with IVIG as early as possible. Published by Elsevier Inc.

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

Science.gov (United States)

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.

Sample preparation composite and replicate strategy case studies for assay of solid oral drug products.

Science.gov (United States)

Nickerson, Beverly; Harrington, Brent; Li, Fasheng; Guo, Michele Xuemei

2017-11-30

Drug product assay is one of several tests required for new drug products to ensure the quality of the product at release and throughout the life cycle of the product. Drug product assay testing is typically performed by preparing a composite sample of multiple dosage units to obtain an assay value representative of the batch. In some cases replicate composite samples may be prepared and the reportable assay value is the average value of all the replicates. In previously published work by Harrington et al. (2014) [5], a sample preparation composite and replicate strategy for assay was developed to provide a systematic approach which accounts for variability due to the analytical method and dosage form with a standard error of the potency assay criteria based on compendia and regulatory requirements. In this work, this sample preparation composite and replicate strategy for assay is applied to several case studies to demonstrate the utility of this approach and its application at various stages of pharmaceutical drug product development. Copyright © 2017 Elsevier B.V. All rights reserved.

Nordihydroguaiaretic acid (NDGA) inhibits replication and viral morphogenesis of dengue virus.

Science.gov (United States)

Soto-Acosta, Rubén; Bautista-Carbajal, Patricia; Syed, Gulam H; Siddiqui, Aleem; Del Angel, Rosa M

2014-09-01

Dengue is the most common mosquito borne viral disease in humans. The infection with any of the 4 dengue virus serotypes (DENV) can either be asymptomatic or manifest in two clinical forms, the mild dengue fever or the more severe dengue hemorrhagic fever that may progress into dengue shock syndrome. A DENV replicative cycle relies on host lipid metabolism; specifically, DENV infection modulates cholesterol and fatty acid synthesis, generating a lipid-enriched cellular environment necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with antioxidant and anti-inflammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for 24 and 48 h with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated with different concentrations of NDGA. NDGA treatment significantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment also led to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number during DENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection by targeting genome replication and viral assembly. Copyright © 2014 Elsevier B.V. All rights reserved.

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

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.

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)

Multiscale analysis of replication technique efficiency for 3D roughness characterization of manufactured surfaces

Science.gov (United States)

Jolivet, S.; Mezghani, S.; El Mansori, M.

2016-09-01

The replication of topography has been generally restricted to optimizing material processing technologies in terms of statistical and single-scale features such as roughness. By contrast, manufactured surface topography is highly complex, irregular, and multiscale. In this work, we have demonstrated the use of multiscale analysis on replicates of surface finish to assess the precise control of the finished replica. Five commercial resins used for surface replication were compared. The topography of five standard surfaces representative of common finishing processes were acquired both directly and by a replication technique. Then, they were characterized using the ISO 25178 standard and multiscale decomposition based on a continuous wavelet transform, to compare the roughness transfer quality at different scales. Additionally, atomic force microscope force modulation mode was used in order to compare the resins’ stiffness properties. The results showed that less stiff resins are able to replicate the surface finish along a larger wavelength band. The method was then tested for non-destructive quality control of automotive gear tooth surfaces.

Jnk2 effects on tumor development, genetic instability and replicative stress in an oncogene-driven mouse mammary tumor model.

Directory of Open Access Journals (Sweden)

Peila Chen

2010-05-01

Full Text Available Oncogenes induce cell proliferation leading to replicative stress, DNA damage and genomic instability. A wide variety of cellular stresses activate c-Jun N-terminal kinase (JNK proteins, but few studies have directly addressed the roles of JNK isoforms in tumor development. Herein, we show that jnk2 knockout mice expressing the Polyoma Middle T Antigen transgene developed mammary tumors earlier and experienced higher tumor multiplicity compared to jnk2 wildtype mice. Lack of jnk2 expression was associated with higher tumor aneuploidy and reduced DNA damage response, as marked by fewer pH2AX and 53BP1 nuclear foci. Comparative genomic hybridization further confirmed increased genomic instability in PyV MT/jnk2-/- tumors. In vitro, PyV MT/jnk2-/- cells underwent replicative stress and cell death as evidenced by lower BrdU incorporation, and sustained chromatin licensing and DNA replication factor 1 (CDT1 and p21(Waf1 protein expression, and phosphorylation of Chk1 after serum stimulation, but this response was not associated with phosphorylation of p53 Ser15. Adenoviral overexpression of CDT1 led to similar differences between jnk2 wildtype and knockout cells. In normal mammary cells undergoing UV induced single stranded DNA breaks, JNK2 localized to RPA (Replication Protein A coated strands indicating that JNK2 responds early to single stranded DNA damage and is critical for subsequent recruitment of DNA repair proteins. Together, these data support that JNK2 prevents replicative stress by coordinating cell cycle progression and DNA damage repair mechanisms.

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...

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.)

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....

Lattice gas simulations of replicating domains

International Nuclear Information System (INIS)

Dawson, S.P.; Hasslacher, B.; Pearson, J.E.

1993-01-01

We use the lattice gas cellular automation (LGCA) developed to simulate a process of pattern-formation recently observed in reaction-diffusion systems. We study the reaction mechanism, which is an extension of the Selkov model for glycolytic oscillations. We are able to reproduce the self-replicating domains observed in this work. We use the LGCA simulation to estimate the smallest length-scale on which this process can occur under conditions encountered in the cell. These estimates are similar to those obtained for Turing patterns in the same setting

Lattice gas simulations of replicating domains

Energy Technology Data Exchange (ETDEWEB)

Dawson, S.P.; Hasslacher, B.; Pearson, J.E.

1993-12-31

We use the lattice gas cellular automation (LGCA) developed to simulate a process of pattern-formation recently observed in reaction-diffusion systems. We study the reaction mechanism, which is an extension of the Selkov model for glycolytic oscillations. We are able to reproduce the self-replicating domains observed in this work. We use the LGCA simulation to estimate the smallest length-scale on which this process can occur under conditions encountered in the cell. These estimates are similar to those obtained for Turing patterns in the same setting.

Reasoning=working Memoryattention

Science.gov (United States)

Buehner, M.; Krumm, S.; Pick, M.

2005-01-01

The purpose of this study was to clarify the relationship between attention, components of working memory, and reasoning. Therefore, twenty working memory tests, two attention tests, and nine intelligence subtests were administered to 135 students. Using structural equation modeling, we were able to replicate a functional model of working memory…

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...

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...

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.

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

Science.gov (United States)

Kunkel, Thomas A; Burgers, Peter M J

2017-08-01

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

Biological consequences of earlier snowmelt from desert dust deposition in alpine landscapes.

Science.gov (United States)

Steltzer, Heidi; Landry, Chris; Painter, Thomas H; Anderson, Justin; Ayres, Edward

2009-07-14

Dust deposition to mountain snow cover, which has increased since the late 19(th) century, accelerates the rate of snowmelt by increasing the solar radiation absorbed by the snowpack. Snowmelt occurs earlier, but is decoupled from seasonal warming. Climate warming advances the timing of snowmelt and early season phenological events (e.g., the onset of greening and flowering); however, earlier snowmelt without warmer temperatures may have a different effect on phenology. Here, we report the results of a set of snowmelt manipulations in which radiation-absorbing fabric and the addition and removal of dust from the surface of the snowpack advanced or delayed snowmelt in the alpine tundra. These changes in the timing of snowmelt were superimposed on a system where the timing of snowmelt varies with topography and has been affected by increased dust loading. At the community level, phenology exhibited a threshold response to the timing of snowmelt. Greening and flowering were delayed before seasonal warming, after which there was a linear relationship between the date of snowmelt and the timing of phenological events. Consequently, the effects of earlier snowmelt on phenology differed in relation to topography, which resulted in increasing synchronicity in phenology across the alpine landscape with increasingly earlier snowmelt. The consequences of earlier snowmelt from increased dust deposition differ from climate warming and include delayed phenology, leading to synchronized growth and flowering across the landscape and the opportunity for altered species interactions, landscape-scale gene flow via pollination, and nutrient cycling.

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...

Genome-wide Association for Major Depression Through Age at Onset Stratification: Major Depressive Disorder Working Group of the Psychiatric Genomics Consortium.

Science.gov (United States)

Power, Robert A; Tansey, Katherine E; Buttenschøn, Henriette Nørmølle; Cohen-Woods, Sarah; Bigdeli, Tim; Hall, Lynsey S; Kutalik, Zoltán; Lee, S Hong; Ripke, Stephan; Steinberg, Stacy; Teumer, Alexander; Viktorin, Alexander; Wray, Naomi R; Arolt, Volker; Baune, Bernard T; Boomsma, Dorret I; Børglum, Anders D; Byrne, Enda M; Castelao, Enrique; Craddock, Nick; Craig, Ian W; Dannlowski, Udo; Deary, Ian J; Degenhardt, Franziska; Forstner, Andreas J; Gordon, Scott D; Grabe, Hans J; Grove, Jakob; Hamilton, Steven P; Hayward, Caroline; Heath, Andrew C; Hocking, Lynne J; Homuth, Georg; Hottenga, Jouke J; Kloiber, Stefan; Krogh, Jesper; Landén, Mikael; Lang, Maren; Levinson, Douglas F; Lichtenstein, Paul; Lucae, Susanne; MacIntyre, Donald J; Madden, Pamela; Magnusson, Patrik K E; Martin, Nicholas G; McIntosh, Andrew M; Middeldorp, Christel M; Milaneschi, Yuri; Montgomery, Grant W; Mors, Ole; Müller-Myhsok, Bertram; Nyholt, Dale R; Oskarsson, Hogni; Owen, Michael J; Padmanabhan, Sandosh; Penninx, Brenda W J H; Pergadia, Michele L; Porteous, David J; Potash, James B; Preisig, Martin; Rivera, Margarita; Shi, Jianxin; Shyn, Stanley I; Sigurdsson, Engilbert; Smit, Johannes H; Smith, Blair H; Stefansson, Hreinn; Stefansson, Kari; Strohmaier, Jana; Sullivan, Patrick F; Thomson, Pippa; Thorgeirsson, Thorgeir E; Van der Auwera, Sandra; Weissman, Myrna M; Breen, Gerome; Lewis, Cathryn M

2017-02-15

Major depressive disorder (MDD) is a disabling mood disorder, and despite a known heritable component, a large meta-analysis of genome-wide association studies revealed no replicable genetic risk variants. Given prior evidence of heterogeneity by age at onset in MDD, we tested whether genome-wide significant risk variants for MDD could be identified in cases subdivided by age at onset. Discovery case-control genome-wide association studies were performed where cases were stratified using increasing/decreasing age-at-onset cutoffs; significant single nucleotide polymorphisms were tested in nine independent replication samples, giving a total sample of 22,158 cases and 133,749 control subjects for subsetting. Polygenic score analysis was used to examine whether differences in shared genetic risk exists between earlier and adult-onset MDD with commonly comorbid disorders of schizophrenia, bipolar disorder, Alzheimer's disease, and coronary artery disease. We identified one replicated genome-wide significant locus associated with adult-onset (>27 years) MDD (rs7647854, odds ratio: 1.16, 95% confidence interval: 1.11-1.21, p = 5.2 × 10 -11 ). Using polygenic score analyses, we show that earlier-onset MDD is genetically more similar to schizophrenia and bipolar disorder than adult-onset MDD. We demonstrate that using additional phenotype data previously collected by genetic studies to tackle phenotypic heterogeneity in MDD can successfully lead to the discovery of genetic risk factor despite reduced sample size. Furthermore, our results suggest that the genetic susceptibility to MDD differs between adult- and earlier-onset MDD, with earlier-onset cases having a greater genetic overlap with schizophrenia and bipolar disorder. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Multiple determinants controlling activation of yeast replication origins late in S phase.

Science.gov (United States)

Friedman, K L; Diller, J D; Ferguson, B M; Nyland, S V; Brewer, B J; Fangman, W L

1996-07-01

Analysis of a 131-kb segment of the left arm of yeast chromosome XIV beginning 157 kb from the telomere reveals four highly active origins of replication that initiate replication late in S phase. Previous work has shown that telomeres act as determinants for late origin activation. However, at least two of the chromosome XIV origins maintain their late activation time when located on large circular plasmids, indicating that late replication is independent of telomeres. Analysis of the replication time of plasmid derivatives containing varying amounts of chromosome XIV DNA show that a minimum of three chromosomal elements, distinct from each tested origin, contribute to late activation time. These late determinants are functionally equivalent, because duplication of one set of contributing sequences can compensate for the removal of another set. Furthermore, insertion of an origin that is normally early activated into this domain results in a shift to late activation, suggesting that the chromosome XIV origins are not unique in their ability to respond to the late determinants.

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.

DBR1 siRNA inhibition of HIV-1 replication

Directory of Open Access Journals (Sweden)

Naidu Yathi

2005-10-01

Full Text Available Abstract Background HIV-1 and all retroviruses are related to retroelements of simpler organisms such as the yeast Ty elements. Recent work has suggested that the yeast retroelement Ty1 replicates via an unexpected RNA lariat intermediate in cDNA synthesis. The putative genomic RNA lariat intermediate is formed by a 2'-5' phosphodiester bond, like that found in pre-mRNA intron lariats and it facilitates the minus-strand template switch during cDNA synthesis. We hypothesized that HIV-1 might also form a genomic RNA lariat and therefore that siRNA-mediated inhibition of expression of the human RNA lariat de-branching enzyme (DBR1 expression would specifically inhibit HIV-1 replication. Results We designed three short interfering RNA (siRNA molecules targeting DBR1, which were capable of reducing DBR1 mRNA expression by 80% and did not significantly affect cell viability. We assessed HIV-1 replication in the presence of DBR1 siRNA and found that DBR1 knockdown led to decreases in viral cDNA and protein production. These effects could be reversed by cotransfection of a DBR1 cDNA indicating that the inhibition of HIV-1 replication was a specific effect of DBR1 underexpression. Conclusion These data suggest that DBR1 function may be needed to debranch a putative HIV-1 genomic RNA lariat prior to completion of reverse transcription.

DNA moves sequentially towards the nuclear matrix during DNA replication in vivo

Directory of Open Access Journals (Sweden)

Aranda-Anzaldo Armando

2011-01-01

Full Text Available Abstract Background In the interphase nucleus of metazoan cells DNA is organized in supercoiled loops anchored to a nuclear matrix (NM. There is varied evidence indicating that DNA replication occurs in replication factories organized upon the NM and that DNA loops may correspond to the actual replicons in vivo. In normal rat liver the hepatocytes are arrested in G0 but they synchronously re-enter the cell cycle after partial-hepatectomy leading to liver regeneration in vivo. We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops. Results In the present work we tracked down the movement relative to the NM of DNA sequences located at different points within such five structural DNA loops during the S phase and after the return to cellular quiescence during liver regeneration. Our results indicate that looped DNA moves sequentially towards the NM during replication and then returns to its original position in newly quiescent cells, once the liver regeneration has been achieved. Conclusions Looped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated. These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.

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

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...

The role of cooperation and parasites in non-linear replicator delayed extinctions

International Nuclear Information System (INIS)

Sardanyes, Josep; Sole, Ricard V.

2007-01-01

In the present work we study the role of cooperation and parasites on extinction delayed transitions for self-replicating species with catalytic activity. We first use a one-dimensional continuous equation to study the dynamics of both single autocatalytic replicator and symmetric two-member hypercycles, where two well-defined phases involving survival and extinction of replicators are shown to exist. Extinction dynamics is analyzed numerically and analytically and under both deterministic and stochastic scenarios. A ghost is also found for the single autocatalytic replicator and for the asymmetric hypercycle, with an extinction time delay following the square-root scaling law near bifurcation threshold. We find that the extinction delay is longer for the two-member hypercycle than for the single autocatalytic species, indicating that cooperation among replicators might involve to spend a longer time in the bottle-neck region of the ghost. The asymmetry of the network is shown to prolong the extinction time. We also show that an attached parasite decreases the time spent in the bottle-neck region of the ghost, thus accelerating extinction in these systems of replicators. Nevertheless the effect of the parasite is not so important when replicators catalytically cooperate, being the two-member hypercycle less sensitive to the parasite than the autocatalytic species. Here the hypercycle asymmetry can also significantly increase the delaying capacity. These features make the hypercycle to undergo a longer extinction delay, thus increasing the memory effect of the ghost. We finally explore the role of the ghost in fluctuating media, where the extinction delayed transition is shown to increase the survival probability of cooperating catalytic species

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)

Replication of clinical innovations in multiple medical practices.

Science.gov (United States)

Henley, N S; Pearce, J; Phillips, L A; Weir, S

1998-11-01

Many clinical innovations had been successfully developed and piloted in individual medical practice units of Kaiser Permanente in North Carolina during 1995 and 1996. Difficulty in replicating these clinical innovations consistently throughout all 21 medical practice units led to development of the interdisciplinary Clinical Innovation Implementation Team, which was formed by using existing resources from various departments across the region. REPLICATION MODEL: Based on a model of transfer of best practices, the implementation team developed a process and tools (master schedule and activity matrix) to quickly replicate successful pilot projects throughout all medical practice units. The process involved the following steps: identifying a practice and delineating its characteristics and measures (source identification); identifying a team to receive the (new) practice; piloting the practice; and standardizing, including the incorporation of learnings. The model includes the following components for each innovation: sending and receiving teams, an innovation coordinator role, an innovation expert role, a location expert role, a master schedule, and a project activity matrix. Communication depended on a partnership among the location experts (local knowledge and credibility), the innovation coordinator (process expertise), and the innovation experts (content expertise). Results after 12 months of working with the 21 medical practice units include integration of diabetes care team services into the practices, training of more than 120 providers in the use of personal computers and an icon-based clinical information system, and integration of a planwide self-care program into the medical practices--all with measurable improved outcomes. The model for sequential replication and the implementation team structure and function should be successful in other organizational settings.

Dynamics of Escherichia coli Chromosome Segregation during Multifork Replication

DEFF Research Database (Denmark)

Nielsen, Henrik Jørck; Youngren, Brenda; Hansen, Flemming G.

2007-01-01

Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division, the chro......Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division......, the chromosomes contain multiple replication forks and must be segregated while this complex pattern of replication is still ongoing. Here, we show that replication and segregation continue in step, starting at the origin and progressing to the replication terminus. Thus, early-replicated markers on the multiple......-branched chromosomes continue to separate soon after replication to form separate protonucleoids, even though they are not segregated into different daughter cells until later generations. The segregation pattern follows the pattern of chromosome replication and does not follow the cell division cycle. No extensive...

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.

Field testing existence values for riparian ecosystems

Science.gov (United States)

John W. Duffield; Chris J. Neher; David A. Patterson; Patricia A. Champ

2007-01-01

This paper presents preliminary findings on a cash and contingent valuation (cv) experiment. The study replicates major elements of an earlier (1990) experiment, which solicited hypothetical and actual donations to benefit instream flows for Montana fisheries. Extensions of the earlier work include: repeat contacts to increase response rate, follow-up of the contingent...

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

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...

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.

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

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.

Blurring of emotional and non-emotional memories by taxing working memory during recall.

Science.gov (United States)

van den Hout, Marcel A; Eidhof, Marloes B; Verboom, Jesse; Littel, Marianne; Engelhard, Iris M

2014-01-01

Memories that are recalled while working memory (WM) is taxed, e.g., by making eye movements (EM), become blurred during the recall + EM and later recall, without EM. This may help to explain the effects of Eye Movement and Desensitisation and Reprocessing (EMDR) in the treatment of post-traumatic stress disorder (PTSD) in which patients make EM during trauma recall. Earlier experimental studies on recall + EM have focused on emotional memories. WM theory suggests that recall + EM is superior to recall only but is silent about effects of memory emotionality. Based on the emotion and memory literature, we examined whether recall + EM has superior effects in blurring emotional memories relative to neutral memories. Healthy volunteers recalled negative or neutral memories, matched for vividness, while visually tracking a dot that moved horizontally ("recall + EM") or remained stationary ("recall only"). Compared to a pre-test, a post-test (without concentrating on the dot) replicated earlier findings: negative memories are rated as less vivid after "recall + EM" but not after "recall only". This was not found for neutral memories. Emotional memories are more taxing than neutral memories, which may explain the findings. Alternatively, transient arousal induced by recall of aversive memories may promote reconsolidation of the blurred memory image that is provoked by EM.

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).

Effects of ultraviolet irradiation on the rate and sequence of DNA replication in synchronized Chinese hamster cells

International Nuclear Information System (INIS)

Meyn, R.E.; Hewitt, R.R.; Thomson, L.F.; Humphrey, R.M.

1976-01-01

The effects of ultraviolet light (uv) irradiation on the rate of DNA replication in synchronized Chinese hamster ovary (CHO) cells were investigated. A technique for measuring semiconservative DNA replication was employed that involved growing the cells in medium containing 5-bromodeoxyuridine and subsequently determining the amount of DNA that acquired hybrid buoyant density in CsCl density gradients. One of the advantages of this technique was that it allowed a characterization of the extent of DNA replication as well as rate after irradiation. It was found that while there was a dose-dependent reduction in the rate of DNA replication following uv-irradiation, doses of up to 10 J/m 2 (which produce many dimers per replicon) did not prevent the ultimate replication of the entire genome. Hence, we conclude that dimers cannot be absolute blocks to DNA replication. In order to account for the total genome replication observed, a mechanism must exist that allows genome replication between dimers. The degree of reduction in the rate of replication by uv was the same whether the cells were irradiated at the Gl-S boundary or 1 h into S-phase. Previous work had shown that cells in early S-phase are considerably more sensitive to uv than cells at the G1-S boundary. Experiments specifically designed to test for reiterative replication showed that uv does not induce a second round of DNA replication within the same S-phase

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

Non‐Canonical Replication Initiation: You’re Fired!

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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

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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.

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.

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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

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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.

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

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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.

Does working memory capacity predict cross-modally induced failures of awareness?

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Kreitz, Carina; Furley, Philip; Simons, Daniel J; Memmert, Daniel

2016-01-01

People often fail to notice unexpected stimuli when they are focusing attention on another task. Most studies of this phenomenon address visual failures induced by visual attention tasks (inattentional blindness). Yet, such failures also occur within audition (inattentional deafness), and people can even miss unexpected events in one sensory modality when focusing attention on tasks in another modality. Such cross-modal failures are revealing because they suggest the existence of a common, central resource limitation. And, such central limits might be predicted from individual differences in cognitive capacity. We replicated earlier evidence, establishing substantial rates of inattentional deafness during a visual task and inattentional blindness during an auditory task. However, neither individual working memory capacity nor the ability to perform the primary task predicted noticing in either modality. Thus, individual differences in cognitive capacity did not predict failures of awareness even though the failures presumably resulted from central resource limitations. Copyright © 2015 Elsevier Inc. All rights reserved.

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

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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.

The Acid Test for Biological Science: STAP Cells, Trust, and Replication.

Science.gov (United States)

Lancaster, Cheryl

2016-02-01

In January 2014, a letter and original research article were published in Nature describing a process whereby somatic mouse cells could be converted into stem cells by subjecting them to stress. These "stimulus-triggered acquisition of pluripotency" (STAP) cells were shown to be capable of contributing to all cell types of a developing embryo, and extra-embryonic tissues. The lead author of the publications, Haruko Obokata, became an overnight celebrity in Japan, where she was dubbed the new face of Japanese science. However, in the weeks that followed publication of the research, issues arose. Other laboratories and researchers (including authors on the original papers) found that they were unable to replicate Obokata et al.'s work. Closer scrutiny of the papers by the scientific community also suggested that there was manipulation of images that had been published, and Obokata was accused of misconduct. Those who should have been supervising her work (also her co-authors on the publications) were also heavily criticised. The STAP cell saga of 2014 is used as an example to highlight the importance of trust and replication in twenty-first century biological science. The role of trust in the scientific community is highlighted, and the effects on interactions between science and the public examined. Similarly, this essay aims to highlight the importance of replication, and how this is understood by researchers, the media, and the public. The expected behaviour of scientists in the twenty-first century is now more closely scrutinised.

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.

Later endogenous circadian temperature nadir relative to an earlier wake time in older people

Science.gov (United States)

Duffy, J. F.; Dijk, D. J.; Klerman, E. B.; Czeisler, C. A.

1998-01-01

The contribution of the circadian timing system to the age-related advance of sleep-wake timing was investigated in two experiments. In a constant routine protocol, we found that the average wake time and endogenous circadian phase of 44 older subjects were earlier than that of 101 young men. However, the earlier circadian phase of the older subjects actually occurred later relative to their habitual wake time than it did in young men. These results indicate that an age-related advance of circadian phase cannot fully account for the high prevalence of early morning awakening in healthy older people. In a second study, 13 older subjects and 10 young men were scheduled to a 28-h day, such that they were scheduled to sleep at many circadian phases. Self-reported awakening from scheduled sleep episodes and cognitive throughput during the second half of the wake episode varied markedly as a function of circadian phase in both groups. The rising phase of both rhythms was advanced in the older subjects, suggesting an age-related change in the circadian regulation of sleep-wake propensity. We hypothesize that under entrained conditions, these age-related changes in the relationship between circadian phase and wake time are likely associated with self-selected light exposure at an earlier circadian phase. This earlier exposure to light could account for the earlier clock hour to which the endogenous circadian pacemaker is entrained in older people and thereby further increase their propensity to awaken at an even earlier time.

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

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.

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...

Personal and Interpersonal Motivation for Group Projects: Replications of an Attributional Analysis

Science.gov (United States)

Peterson, Sarah E.; Schreiber, James B.

2012-01-01

We report the results of two replication studies using attribution theory to analyze personal and interpersonal motivation for collaborative projects. Undergraduate students responded to questionnaires containing hypothetical vignettes depicting success or failure outcomes due to ability or effort for dyads working on a group project. Dependent…

Perceptual sensitivity to spectral properties of earlier sounds during speech categorization.

Science.gov (United States)

Stilp, Christian E; Assgari, Ashley A

2018-02-28

Speech perception is heavily influenced by surrounding sounds. When spectral properties differ between earlier (context) and later (target) sounds, this can produce spectral contrast effects (SCEs) that bias perception of later sounds. For example, when context sounds have more energy in low-F 1 frequency regions, listeners report more high-F 1 responses to a target vowel, and vice versa. SCEs have been reported using various approaches for a wide range of stimuli, but most often, large spectral peaks were added to the context to bias speech categorization. This obscures the lower limit of perceptual sensitivity to spectral properties of earlier sounds, i.e., when SCEs begin to bias speech categorization. Listeners categorized vowels (/ɪ/-/ɛ/, Experiment 1) or consonants (/d/-/g/, Experiment 2) following a context sentence with little spectral amplification (+1 to +4 dB) in frequency regions known to produce SCEs. In both experiments, +3 and +4 dB amplification in key frequency regions of the context produced SCEs, but lesser amplification was insufficient to bias performance. This establishes a lower limit of perceptual sensitivity where spectral differences across sounds can bias subsequent speech categorization. These results are consistent with proposed adaptation-based mechanisms that potentially underlie SCEs in auditory perception. Recent sounds can change what speech sounds we hear later. This can occur when the average frequency composition of earlier sounds differs from that of later sounds, biasing how they are perceived. These "spectral contrast effects" are widely observed when sounds' frequency compositions differ substantially. We reveal the lower limit of these effects, as +3 dB amplification of key frequency regions in earlier sounds was enough to bias categorization of the following vowel or consonant sound. Speech categorization being biased by very small spectral differences across sounds suggests that spectral contrast effects occur

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.

Smoking is associated with earlier time to revision of total knee arthroplasty.

Science.gov (United States)

Lim, Chin Tat; Goodman, Stuart B; Huddleston, James I; Harris, Alex H S; Bhowmick, Subhrojyoti; Maloney, William J; Amanatullah, Derek F

2017-10-01

Smoking is associated with early postoperative complications, increased length of hospital stay, and an increased risk of revision after total knee arthroplasty (TKA). However, the effect of smoking on time to revision TKA is unknown. A total of 619 primary TKAs referred to an academic tertiary center for revision TKA were retrospectively stratified according to the patient smoking status. Smoking status was then analyzed for associations with time to revision TKA using a Chi square test. The association was also analyzed according to the indication for revision TKA. Smokers (37/41, 90%) have an increased risk of earlier revision for any reason compared to non-smokers (274/357, 77%, p=0.031). Smokers (37/41, 90%) have an increased risk of earlier revision for any reason compared to ex-smokers (168/221, 76%, p=0.028). Subgroup analysis did not reveal a difference in indication for revision TKA (p>0.05). Smokers are at increased risk of earlier revision TKA when compared to non-smokers and ex-smokers. The risk for ex-smokers was similar to that of non-smokers. Smoking appears to have an all-or-none effect on earlier revision TKA as patients who smoked more did not have higher risk of early revision TKA. These results highlight the need for clinicians to urge patients not to begin smoking and encourage smokers to quit smoking prior to primary TKA. Copyright © 2017 Elsevier B.V. All rights reserved.

Escherichia coli DNA polymerase I can disrupt G-quadruplex structures during DNA replication.

Science.gov (United States)

Teng, Fang-Yuan; Hou, Xi-Miao; Fan, San-Hong; Rety, Stephane; Dou, Shuo-Xing; Xi, Xu-Guang

2017-12-01

Non-canonical four-stranded G-quadruplex (G4) DNA structures can form in G-rich sequences that are widely distributed throughout the genome. The presence of G4 structures can impair DNA replication by hindering the progress of replicative polymerases (Pols), and failure to resolve these structures can lead to genetic instability. In the present study, we combined different approaches to address the question of whether and how Escherichia coli Pol I resolves G4 obstacles during DNA replication and/or repair. We found that E. coli Pol I-catalyzed DNA synthesis could be arrested by G4 structures at low protein concentrations and the degree of inhibition was strongly dependent on the stability of the G4 structures. Interestingly, at high protein concentrations, E. coli Pol I was able to overcome some kinds of G4 obstacles without the involvement of other molecules and could achieve complete replication of G4 DNA. Mechanistic studies suggested that multiple Pol I proteins might be implicated in G4 unfolding, and the disruption of G4 structures requires energy derived from dNTP hydrolysis. The present work not only reveals an unrealized function of E. coli Pol I, but also presents a possible mechanism by which G4 structures can be resolved during DNA replication and/or repair in E. coli. © 2017 Federation of European Biochemical Societies.

Evidence of Ebola Virus Replication and High Concentration in Semen of a Patient During Recovery.

Science.gov (United States)

Barnes, Kayla G; Kindrachuk, Jason; Lin, Aaron E; Wohl, Shirlee; Qu, James; Tostenson, Samantha D; Dorman, William R; Busby, Michele; Siddle, Katherine J; Luo, Cynthia Y; Matranga, Christian B; Davey, Richard T; Sabeti, Pardis C; Chertow, Daniel S

2017-10-15

In one patient over time, we found that concentration of Ebola virus RNA in semen during recovery is remarkably higher than blood at peak illness. Virus in semen is replication-competent with no change in viral genome over time. Presence of sense RNA suggests replication in cells present in semen. Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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...

Semiotic systems with duality of patterning and the issue of cultural replicators.

Science.gov (United States)

Schaden, Gerhard; Patin, Cédric

2017-11-14

Two major works in recent evolutionary biology have in different ways touched upon the issue of cultural replicators in language, namely Dawkins' Selfish Gene and Maynard Smith and Szathmáry's Major Transitions in Evolution. In the latter, the emergence of language is referred to as the last major transition in evolution (for the time being), a claim we argue to be derived from a crucial property of language, called Duality of Patterning. Prima facie, this property makes natural language look like a structural equivalent to DNA, and its peer in terms of expressive power. We will argue that, if one takes seriously Maynard Smith and Szathmáry's outlook and examines what has been proposed as linguistic replicators, amongst others phonemes and words, the analogy meme-gene becomes problematic. A key issue is the fact that genes and memes are assumed to carry and transmit information, while what has been described as the best candidate for replicatorhood in language, i.e. the phoneme, does by definition not carry meaning. We will argue that semiotic systems with Duality of Pattering (like natural languages) force us to reconsider either the analogy between replicators in the biological and the cultural domain, or what it is to be a replicator in linguistics.

High-fidelity DNA replication in Mycobacterium tuberculosis relies on a trinuclear zinc center.

Science.gov (United States)

Baños-Mateos, Soledad; van Roon, Anne-Marie M; Lang, Ulla F; Maslen, Sarah L; Skehel, J Mark; Lamers, Meindert H

2017-10-11

High-fidelity DNA replication depends on a proofreading 3'-5' exonuclease that is associated with the replicative DNA polymerase. The replicative DNA polymerase DnaE1 from the major pathogen Mycobacterium tuberculosis (Mtb) uses its intrinsic PHP-exonuclease that is distinct from the canonical DEDD exonucleases found in the Escherichia coli and eukaryotic replisomes. The mechanism of the PHP-exonuclease is not known. Here, we present the crystal structure of the Mtb DnaE1 polymerase. The PHP-exonuclease has a trinuclear zinc center, coordinated by nine conserved residues. Cryo-EM analysis reveals the entry path of the primer strand in the PHP-exonuclease active site. Furthermore, the PHP-exonuclease shows a striking similarity to E. coli endonuclease IV, which provides clues regarding the mechanism of action. Altogether, this work provides important insights into the PHP-exonuclease and reveals unique properties that make it an attractive target for novel anti-mycobacterial drugs.The polymerase and histidinol phosphatase (PHP) domain in the DNA polymerase DnaE1 is essential for mycobacterial high-fidelity DNA replication. Here, the authors determine the DnaE1 crystal structure, which reveals the PHP-exonuclease mechanism that can be exploited for antibiotic development.

A comparison of methods for teaching receptive labeling to children with autism spectrum disorders: a systematic replication.

Science.gov (United States)

Grow, Laura L; Kodak, Tiffany; Carr, James E

2014-01-01

Previous research has demonstrated that the conditional-only method (starting with a multiple-stimulus array) is more efficient than the simple-conditional method (progressive incorporation of more stimuli into the array) for teaching receptive labeling to children with autism spectrum disorders (Grow, Carr, Kodak, Jostad, & Kisamore,). The current study systematically replicated the earlier study by comparing the 2 approaches using progressive prompting with 2 boys with autism. The results showed that the conditional-only method was a more efficient and reliable teaching procedure than the simple-conditional method. The results further call into question the practice of teaching simple discriminations to facilitate acquisition of conditional discriminations. © Society for the Experimental Analysis of Behavior.

Replication in hydroxyurea: it's a matter of time.

Science.gov (United States)

Alvino, Gina M; Collingwood, David; Murphy, John M; Delrow, Jeffrey; Brewer, Bonita J; Raghuraman, M K

2007-09-01

Hydroxyurea (HU) is a DNA replication inhibitor that negatively affects both the elongation and initiation phases of replication and triggers the "intra-S phase checkpoint." Previous work with budding yeast has shown that, during a short exposure to HU, MEC1/RAD53 prevent initiation at some late S phase origins. In this study, we have performed microarray experiments to follow the fate of all origins over an extended exposure to HU. We show that the genome-wide progression of DNA synthesis, including origin activation, follows the same pattern in the presence of HU as in its absence, although the time frames are very different. We find no evidence for a specific effect that excludes initiation from late origins. Rather, HU causes S phase to proceed in slow motion; all temporal classes of origins are affected, but the order in which they become active is maintained. We propose a revised model for the checkpoint response to HU that accounts for the continued but slowed pace of the temporal program of origin activation.

Wine glass size and wine sales: a replication study in two bars.

Science.gov (United States)

Pechey, Rachel; Couturier, Dominique-Laurent; Hollands, Gareth J; Mantzari, Eleni; Zupan, Zorana; Marteau, Theresa M

2017-08-01

Wine glass size may influence perceived volume and subsequently purchasing and consumption. Using a larger glass to serve the same portions of wine was found to increase wine sales by 9.4% (95% CI 1.9, 17.5) in a recent study conducted in one bar. The current study aimed to replicate this previous work in two other bars using a wider range of glass sizes. To match the previous study, a repeated multiple treatment reversal design, during which wine was served in glasses of the same design but different sizes, was used. The study was conducted in two bars in Cambridge, England, using glass sizes of 300, 370, 510 ml (Bar 1) and 300 and 510 ml (Bar 2). Customers purchased their choice of a 750 ml bottle, or standard UK measures of 125, 175 or 250 ml of wine, each of which was served with the same glass. Bar 1 Daily wine volume (ml) purchased was 10.5% (95% CI 1.0, 20.9) higher when sold in 510 ml compared to 370 ml glasses; but sales were not significantly higher with 370 ml versus 300 ml glasses (6.5%, 95% CI -5.2, 19.6). Bar 2 Findings were inconclusive as to whether daily wine purchased differed when using 510 ml versus 300 ml glasses (-1.1%, 95% CI -12.6, 11.9). These results provide a partial replication of previous work showing that introducing larger glasses (without manipulating portion size) increases purchasing. Understanding the mechanisms by which wine glass size influences consumption may elucidate when the effect can be expected and when not. Trial registration This study is a replication study, based on the procedure set out in the trial registration for the study that it attempts to replicate (ISRCTN registry: ISRCTN12018175).

DNA Copy-Number Control through Inhibition of Replication Fork Progression

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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.

When Is a Failure to Replicate Not a Type II Error?

Science.gov (United States)

Vasconcelos, Marco; Urcuioli, Peter J.; Lionello-DeNolf, Karen M.

2007-01-01

Zentall and Singer (2007) challenge our conclusion that the work-ethic effect reported by Clement, Feltus, Kaiser, and Zentall (2000) may have been a Type I error by arguing that (a) the effect has been extensively replicated and (b) the amount of overtraining our pigeons received may not have been sufficient to produce it. We believe that our…

Lean approach in knowledge work

Directory of Open Access Journals (Sweden)

Hanna Kropsu-Vehkapera

2018-05-01

Full Text Available Purpose: Knowledge work productivity is a key area of improvement for many organisations. Lean approach is a sustainable way to achieve operational excellence and can be applied in many areas. The purpose of this novel study is to examine the potential of using lean approach for improving knowledge work practices. Design/methodology/approach: A systematic literature review has been carried out to study how lean approach is realised in knowledge work. The research is conceptual in nature and draws upon earlier research findings. Findings: This study shows that lean studies’ in knowledge work is an emerging research area. This study documents the methods and practices implemented in knowledge work to date, and presents a knowledge work continuum, which is an essential framework for effective lean approach deployment and to frame future research focus in knowledge work productivity. Research limitations/implications: This study structures the concept of knowledge work and outlines a concrete concept derived from earlier literature. The study summarises the literature on lean in knowledge work and highlights, which methods are used. More research is needed to understand how lean can be implemented in complex knowledge work environment and not only on the repetitive knowledge work. The limitations of this research are due to the limited availability of previous research. Practical implications: To analyse the nature of knowledge work, we implicate the areas where lean methods especially apply to improving knowledge work productivity. When applying lean in knowledge work context the focus should be using the people better and improving information flow. Originality/value: This study focuses on adapting lean methods into a knowledge work context and summarises earlier research done in this field. The study discusses the potential to improve knowledge work productivity by implementing lean methods and presents a unique knowledge work continuum to

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.

Biological evolution of replicator systems: towards a quantitative approach.

Science.gov (United States)

Martin, Osmel; Horvath, J E

2013-04-01

The aim of this work is to study the features of a simple replicator chemical model of the relation between kinetic stability and entropy production under the action of external perturbations. We quantitatively explore the different paths leading to evolution in a toy model where two independent replicators compete for the same substrate. To do that, the same scenario described originally by Pross (J Phys Org Chem 17:312-316, 2004) is revised and new criteria to define the kinetic stability are proposed. Our results suggest that fast replicator populations are continually favored by the effects of strong stochastic environmental fluctuations capable to determine the global population, the former assumed to be the only acting evolution force. We demonstrate that the process is continually driven by strong perturbations only, and that population crashes may be useful proxies for these catastrophic environmental fluctuations. As expected, such behavior is particularly enhanced under very large scale perturbations, suggesting a likely dynamical footprint in the recovery patterns of new species after mass extinction events in the Earth's geological past. Furthermore, the hypothesis that natural selection always favors the faster processes may give theoretical support to different studies that claim the applicability of maximum principles like the Maximum Metabolic Flux (MMF) or Maximum Entropy Productions Principle (MEPP), seen as the main goal of biological evolution.

Ultrastructural Characterization of Zika Virus Replication Factories

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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

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.

LightLeaves: computer controlled kinetic reflection hologram installation and a brief discussion of earlier work

International Nuclear Information System (INIS)

Connors Chen, Betsy

2013-01-01

LightLeaves is an installation combining leaf shaped, white light reflection holograms of landscape images with a special kinetic lighting device that houses a lamp and moving leaf shaped masks. The masks are controlled by an Arduino microcontroller and servomotors that position the masks in front of the illumination source of the holograms. The work is the most recent in a long series of landscapes that combine multi-hologram installations with computer controlled devices that play with the motion of the holograms, the light, sound or other elements in the work. LightLeaves was first exhibited at the Peabody Essex Museum in Salem, Massachusetts in a show titled E ye Spy: Playing with Perception .

LightLeaves: computer controlled kinetic reflection hologram installation and a brief discussion of earlier work

Energy Technology Data Exchange (ETDEWEB)

Connors Chen, Betsy, E-mail: acmeholo@gmail.com [ACME Holography, Somerville, Massachusetts USA 02144 (United States)

2013-02-22

LightLeaves is an installation combining leaf shaped, white light reflection holograms of landscape images with a special kinetic lighting device that houses a lamp and moving leaf shaped masks. The masks are controlled by an Arduino microcontroller and servomotors that position the masks in front of the illumination source of the holograms. The work is the most recent in a long series of landscapes that combine multi-hologram installations with computer controlled devices that play with the motion of the holograms, the light, sound or other elements in the work. LightLeaves was first exhibited at the Peabody Essex Museum in Salem, Massachusetts in a show titled {sup E}ye Spy: Playing with Perception{sup .}.

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

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.

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.

Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum

International Nuclear Information System (INIS)

Seetharam, S.; Protic-Sabljic, M.; Seidman, M.M.; Kraemer, K.H.

1987-01-01

A shuttle vector plasmid, pZ189, was utilized to assess the types of mutations that cells from a patient with xeroderma pigmentosum, complementation group D, introduce into ultraviolet (UV) damaged, replicating DNA. Patients with xeroderma pigmentosum have clinical and cellular UV hypersensitivity, increased frequency of sun-induced skin cancer, and deficient DNA repair. In comparison to UV-treated pZ189 replicated in DNA repair-proficient cells, there were fewer surviving plasmids, a higher frequency of plasmids with mutations, fewer plasmids with two or more mutations in the marker gene, and a new mutagenic hotspot. The major type of base substitution mutation was the G:C to A:T transition with both cell lines. These results, together with similar findings published earlier with cells from a xeroderma pigmentosum patient in complementation group A, suggest that isolated G:C to A:T somatic mutations may be particularly important in generation of human skin cancer by UV radiation

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

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.

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.

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.

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.

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

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.

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…

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...

Enzymes involved in organellar DNA replication in photosynthetic eukaryotes.

Science.gov (United States)

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.

The earlier identification of the seedless characteristic of the wampee

African Journals Online (AJOL)

Jane

2010-12-13

Dec 13, 2010 ... the production of new seedless wampee cultivars in recent years were operated. ... increase burliness rate in hybridization. In order to earlier ... methods is greatly limited due to a long juvenile phase of ... Plant materials .... This fragment may be linked ... reduce high temperature which affected pollen germi-.

Investigating the conformational stability of prion strains through a kinetic replication model.

Directory of Open Access Journals (Sweden)

Mattia Zampieri

2009-07-01

Full Text Available Prion proteins are known to misfold into a range of different aggregated forms, showing different phenotypic and pathological states. Understanding strain specificities is an important problem in the field of prion disease. Little is known about which PrP(Sc structural properties and molecular mechanisms determine prion replication, disease progression and strain phenotype. The aim of this work is to investigate, through a mathematical model, how the structural stability of different aggregated forms can influence the kinetics of prion replication. The model-based results suggest that prion strains with different conformational stability undergoing in vivo replication are characterizable in primis by means of different rates of breakage. A further role seems to be played by the aggregation rate (i.e. the rate at which a prion fibril grows. The kinetic variability introduced in the model by these two parameters allows us to reproduce the different characteristic features of the various strains (e.g., fibrils' mean length and is coherent with all experimental observations concerning strain-specific behavior.

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.

Infection and Replication of Influenza Virus at the Ocular Surface.

Science.gov (United States)

Creager, Hannah M; Kumar, Amrita; Zeng, Hui; Maines, Taronna R; Tumpey, Terrence M; Belser, Jessica A

2018-04-01

Although influenza viruses typically cause respiratory tract disease, some viruses, particularly those with an H7 hemagglutinin, have been isolated from the eyes of conjunctivitis cases. Previous work has shown that isolates of multiple subtypes from both ocular and respiratory infections are capable of replication in human ex vivo ocular tissues and corneal or conjunctival cell monolayers, leaving the determinants of ocular tropism unclear. Here, we evaluated the effect of several variables on tropism for ocular cells cultured in vitro and examined the potential effect of the tear film on viral infectivity. All viruses tested were able to replicate in primary human corneal epithelial cell monolayers subjected to aerosol inoculation. The temperature at which cells were cultured postinoculation minimally affected infectivity. Replication efficiency, in contrast, was reduced at 33°C relative to that at 37°C, and this effect was slightly greater for the conjunctivitis isolates than for the respiratory ones. With the exception of a seasonal H3N2 virus, the subset of viruses studied in multilayer corneal tissue constructs also replicated productively after either aerosol or liquid inoculation. Human tears significantly inhibited the hemagglutination of both ocular and nonocular isolates, but the effect on viral infectivity was more variable, with tears reducing the infectivity of nonocular isolates more than ocular isolates. These data suggest that most influenza viruses may be capable of establishing infection if they reach the surface of ocular cells but that this is more likely for ocular-tropic viruses, as they are better able to maintain their infectivity during passage through the tear film. IMPORTANCE The potential spread of zoonotic influenza viruses to humans represents an important threat to public health. Unfortunately, despite the importance of cellular and tissue tropism to pathogenesis, determinants of influenza virus tropism have yet to be fully

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....

Knowledge work and work-related stress

DEFF Research Database (Denmark)

Ipsen, Christine

2006-01-01

Work-related stress is an increasing problem in Europe. Earlier studies have stated that knowledge-work comprises working conditions which reflect a good psychosocial environment. Recent Danish studies, however, point at stress being an increasing problem in knowledge-intensive companies...... with informally, individu-ally and incidentally. It is only when problems exist that enhanced support is offered in order to help an employee to cope or recover. As most workplace initiatives work at this tertiary level, the sources of work-related and organiza-tional stress are not reduced or eliminated...... as good and stimulating, but has on the other hand sides to it which can cause frustration and stress. The implication of organisational characteristics of the knowledge-intensive companies studied is a transfer of the responsibility for ones own working-life. Consequently, issues are dealt...

Modeling DNA Replication.

Science.gov (United States)

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.

Science.gov (United States)

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

Directory of Open Access Journals (Sweden)

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

Directory of Open Access Journals (Sweden)

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.

Science.gov (United States)

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.

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

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.

Directory of Open Access Journals (Sweden)

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.

DNA replication after mutagenic treatment in Hordeum vulgare.

Science.gov (United States)

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.

New paradigms in the repair of oxidative damage in human genome: mechanisms ensuring repair of mutagenic base lesions during replication and involvement of accessory proteins.

Science.gov (United States)

Dutta, Arijit; Yang, Chunying; Sengupta, Shiladitya; Mitra, Sankar; Hegde, Muralidhar L

2015-05-01

Oxidized bases in the mammalian genome, which are invariably mutagenic due to their mispairing property, are continuously induced by endogenous reactive oxygen species and more abundantly after oxidative stress. Unlike bulky base adducts induced by UV and other environmental mutagens in the genome that block replicative DNA polymerases, oxidatively damaged bases such as 5-hydroxyuracil, produced by oxidative deamination of cytosine in the template strand, do not block replicative polymerases and thus need to be repaired prior to replication to prevent mutation. Following up our earlier studies, which showed that the Nei endonuclease VIII like 1 (NEIL1) DNA glycosylase, one of the five base excision repair (BER)-initiating enzymes in mammalian cells, has enhanced expression during the S-phase and higher affinity for replication fork-mimicking single-stranded (ss) DNA substrates, we recently provided direct experimental evidence for NEIL1's role in replicating template strand repair. The key requirement for this event, which we named as the 'cow-catcher' mechanism of pre-replicative BER, is NEIL1's non-productive binding (substrate binding without product formation) to the lesion base in ss DNA template to stall DNA synthesis, causing fork regression. Repair of the lesion in reannealed duplex is then carried out by NEIL1 in association with the DNA replication proteins. NEIL1 (and other BER-initiating enzymes) also interact with several accessory and non-canonical proteins including the heterogeneous nuclear ribonucleoprotein U and Y-box-binding protein 1 as well as high mobility group box 1 protein, whose precise roles in BER are still obscure. In this review, we have discussed the recent advances in our understanding of oxidative genome damage repair pathways with particular focus on the pre-replicative template strand repair and the role of scaffold factors like X-ray repairs cross-complementing protein 1 and poly (ADP-ribose) polymerase 1 and other accessory

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.

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.

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.

Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis.

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Cai, Hui; Zhang, Yu; Lu, Mijia; Liang, Xueya; Jennings, Ryan; Niewiesk, Stefan; Li, Jianrong

2016-08-15

Human metapneumovirus (hMPV) is a major causative agent of upper- and lower-respiratory-tract infections in infants, the elderly, and immunocompromised individuals worldwide. Like all pneumoviruses, hMPV encodes the zinc binding protein M2-1, which plays important regulatory roles in RNA synthesis. The M2-1 protein is phosphorylated, but the specific role(s) of the phosphorylation in viral replication and pathogenesis remains unknown. In this study, we found that hMPV M2-1 is phosphorylated at amino acid residues S57 and S60. Subsequent mutagenesis found that phosphorylation is not essential for zinc binding activity and oligomerization, whereas inhibition of zinc binding activity abolished the phosphorylation and oligomerization of the M2-1 protein. Using a reverse genetics system, recombinant hMPVs (rhMPVs) lacking either one or both phosphorylation sites in the M2-1 protein were recovered. These recombinant viruses had a significant decrease in both genomic RNA replication and mRNA transcription. In addition, these recombinant viruses were highly attenuated in cell culture and cotton rats. Importantly, rhMPVs lacking phosphorylation in the M2-1 protein triggered high levels of neutralizing antibody and provided complete protection against challenge with wild-type hMPV. Collectively, these data demonstrated that phosphorylation of the M2-1 protein upregulates hMPV RNA synthesis, replication, and pathogenesis in vivo The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Currently, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that phosphorylation of M2-1 is essential for virus

Earlier vegetation green-up has reduced spring dust storms.

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Fan, Bihang; Guo, Li; Li, Ning; Chen, Jin; Lin, Henry; Zhang, Xiaoyang; Shen, Miaogen; Rao, Yuhan; Wang, Cong; Ma, Lei

2014-10-24

The observed decline of spring dust storms in Northeast Asia since the 1950s has been attributed to surface wind stilling. However, spring vegetation growth could also restrain dust storms through accumulating aboveground biomass and increasing surface roughness. To investigate the impacts of vegetation spring growth on dust storms, we examine the relationships between recorded spring dust storm outbreaks and satellite-derived vegetation green-up date in Inner Mongolia, Northern China from 1982 to 2008. We find a significant dampening effect of advanced vegetation growth on spring dust storms (r = 0.49, p = 0.01), with a one-day earlier green-up date corresponding to a decrease in annual spring dust storm outbreaks by 3%. Moreover, the higher correlation (r = 0.55, p storm outbreak ratio (the ratio of dust storm outbreaks to times of strong wind events) indicates that such effect is independent of changes in surface wind. Spatially, a negative correlation is detected between areas with advanced green-up dates and regional annual spring dust storms (r = -0.49, p = 0.01). This new insight is valuable for understanding dust storms dynamics under the changing climate. Our findings suggest that dust storms in Inner Mongolia will be further mitigated by the projected earlier vegetation green-up in the warming world.

Comprehensive methods for earlier detection and monitoring of forest decline

Science.gov (United States)

Jennifer Pontius; Richard Hallett

2014-01-01

Forested ecosystems are threatened by invasive pests, pathogens, and unusual climatic events brought about by climate change. Earlier detection of incipient forest health problems and a quantitatively rigorous assessment method is increasingly important. Here, we describe a method that is adaptable across tree species and stress agents and practical for use in the...

Chaotic interactions of self-replicating RNA.

Science.gov (United States)

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.

Replicating empirical research in behavioral ecology: how and why it should be done but rarely ever is.

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Kelly, Clint D

2006-09-01

That empirical evidence is replicable is the foundation of science. Ronald Fisher a founding father of biostatistics, recommended that a null hypothesis be rejected more than once because "no isolated experiment, however significant in itself can suffice for the experimental demonstration of any natural phenomenon" (Fisher 1974:14). Despite this demand, animal behaviorists and behavioral ecologists seldom replicate studies. This practice is not part of our scientific culture, as it is in chemistry or physics, due to a number of factors, including a general disdain by journal editors and thesis committees for unoriginal work. I outline why and how we should replicate empirical studies, which studies should be given priority, and then elaborate on why we do not engage in this necessary endeavor. I also explain how to employ various statistics to test the replicability of a series of studies and illustrate these using published studies from the literature.

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.

Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.

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Lance D Eckerle

2010-05-01

Full Text Available Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN in nonstructural protein 14 (nsp14 of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication

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

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

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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

Replication of Brucella abortus and Brucella melitensis in fibroblasts does not require Atg5-dependent macroautophagy.

Science.gov (United States)

Hamer, Isabelle; Goffin, Emeline; De Bolle, Xavier; Letesson, Jean-Jacques; Jadot, Michel

2014-09-02

Several intracellular bacterial pathogens have evolved subtle strategies to subvert vesicular trafficking pathways of their host cells to avoid killing and to replicate inside the cells. Brucellae are Gram-negative facultative intracellular bacteria that are responsible for brucellosis, a worldwide extended chronic zoonosis. Following invasion, Brucella abortus is found in a vacuole that interacts first with various endosomal compartments and then with endoplasmic reticulum sub-compartments. Brucella establishes its replication niche in ER-derived vesicles. In the past, it has been proposed that B. abortus passed through the macroautophagy pathway before reaching its niche of replication. However, recent experiments provided evidence that the classical macroautophagy pathway was not involved in the intracellular trafficking and the replication of B. abortus in bone marrow-derived macrophages and in HeLa cells. In contrast, another study showed that macroautophagy favoured the survival and the replication of Brucella melitensis in infected RAW264.7 macrophages. This raises the possibility that B. abortus and B. melitensis followed different intracellular pathways before replicating. In the present work, we have addressed this issue by comparing the replication rate of B. abortus and B. melitensis in embryonic fibroblasts derived from wild-type and Atg5-/- mice, Atg5 being a core component of the canonical macroautophagic pathway. Our results indicate that both B. abortus S2308 and B. melitensis 16M strains are able to invade and replicate in Atg5-deficient fibroblasts, suggesting that the canonical Atg5-dependent macroautophagic pathway is dispensable for Brucella replication. The number of viable bacteria was even slightly higher in Atg5-/- fibroblasts than in wild-type fibroblasts. This increase could be due to a more efficient uptake or to a better survival rate of bacteria before the beginning of the replication in Atg5-deficient cells as compared to wild

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

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

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

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

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...

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

2017-06-01

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.

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.

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

Directory of Open Access Journals (Sweden)

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.

MITA/STING and Its Alternative Splicing Isoform MRP Restrict Hepatitis B Virus Replication.

Science.gov (United States)

Liu, Shuhui; Zhao, Kaitao; Su, Xi; Lu, Lu; Zhao, He; Zhang, Xianwen; Wang, Yun; Wu, Chunchen; Chen, Jizheng; Zhou, Yuan; Hu, Xue; Wang, Yanyi; Lu, Mengji; Chen, Xinwen; Pei, Rongjuan

2017-01-01

An efficient clearance of hepatitis B virus (HBV) requires the coordinated work of both the innate and adaptive immune responses. MITA/STING, an adapter protein of the innate immune signaling pathways, plays a key role in regulating innate and adaptive immune responses to DNA virus infection. Previously, we identified an alternatively spliced isoform of MITA/STING, called MITA-related protein (MRP), and found that MRP could specifically block MITA-mediated interferon (IFN) induction while retaining the ability to activate NF-κB. Here, we asked whether MITA/STING and MRP were able to control the HBV replication. Both MITA/STING and MRP significantly inhibited HBV replication in vitro. MITA overexpression stimulated IRF3-IFN pathway; while MRP overexpression activated NF-κB pathway, suggesting these two isoforms may inhibit HBV replication through different ways. Using a hydrodynamic injection (HI) mouse model, we found that HBV replication was reduced following MITA/STING and MRP expression vectors in mice and was enhanced by the knockout of MITA/STING (MITA/STING-/-). The HBV specific humoral and CD8+ T cell responses were impaired in MITA/STING deficient mice, suggesting the participation of MITA/STING in the initiation of host adaptive immune responses. In summary, our data suggest that MITA/STING and MRP contribute to HBV control via modulation of the innate and adaptive responses.

Regulated eukaryotic DNA replication origin firing with purified proteins.

Science.gov (United States)

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.

Science.gov (United States)

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.

An Enduring Health Risk of Childhood Adversity: Earlier, More Severe, and Longer Lasting Work Disability in Adult Life.

Science.gov (United States)

Laditka, Sarah B; Laditka, James N

2018-02-08

Childhood adversity has been linked with adult health problems. We hypothesized that childhood adversity would also be associated with work limitations due to physical or nervous health problems, known as work disability. With data from the Panel Study of Income Dynamics (PSID) (1968-2013; n=6,045; 82,374 transitions; 129,107 person-years) and the 2014 PSID Childhood Retrospective Circumstances Study, we estimated work disability transition probabilities with multinomial logistic Markov models. Four or more adversities defined a high level. Microsimulations quantified adult work disability patterns for African American and non-Hispanic white women and men, accounting for age, education, race, sex, diabetes, heart disease, obesity, and sedentary behavior. Childhood adversity was significantly associated with work disability. Of African American women with high adversity, 10.2% had moderate work disability at age 30 versus 4.1% with no reported adversities; comparable results for severe work disability were 5.6% versus 1.9% (both pwork disability remained significant after adjusting for diabetes, heart disease, obesity, and sedentary behavior (pwork disability throughout adult life. © The Author(s) 2018. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Termination of DNA replication forks: "Breaking up is hard to do".

Science.gov (United States)

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.

RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

Energy Technology Data Exchange (ETDEWEB)

Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); James, John; Prescott, Alan [Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Haga, Ismar R. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); Beard, Philippa M., E-mail: pip.beard@roslin.ed.ac.uk [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom)

2015-01-15

Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.

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

Chromatin Constrains the Initiation and Elongation of DNA Replication.

Science.gov (United States)

Devbhandari, Sujan; Jiang, Jieqing; Kumar, Charanya; Whitehouse, Iestyn; Remus, Dirk

2017-01-05

Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Replication, falsification, and the crisis of confidence in social psychology.

Science.gov (United States)

Earp, Brian D; Trafimow, David

2015-01-01

The (latest) crisis in confidence in social psychology has generated much heated discussion about the importance of replication, including how it should be carried out as well as interpreted by scholars in the field. For example, what does it mean if a replication attempt "fails"-does it mean that the original results, or the theory that predicted them, have been falsified? And how should "failed" replications affect our belief in the validity of the original research? In this paper, we consider the replication debate from a historical and philosophical perspective, and provide a conceptual analysis of both replication and falsification as they pertain to this important discussion. Along the way, we highlight the importance of auxiliary assumptions (for both testing theories and attempting replications), and introduce a Bayesian framework for assessing "failed" replications in terms of how they should affect our confidence in original findings.

Replication, falsification, and the crisis of confidence in social psychology

Science.gov (United States)

Earp, Brian D.; Trafimow, David

2015-01-01

The (latest) crisis in confidence in social psychology has generated much heated discussion about the importance of replication, including how it should be carried out as well as interpreted by scholars in the field. For example, what does it mean if a replication attempt “fails”—does it mean that the original results, or the theory that predicted them, have been falsified? And how should “failed” replications affect our belief in the validity of the original research? In this paper, we consider the replication debate from a historical and philosophical perspective, and provide a conceptual analysis of both replication and falsification as they pertain to this important discussion. Along the way, we highlight the importance of auxiliary assumptions (for both testing theories and attempting replications), and introduce a Bayesian framework for assessing “failed” replications in terms of how they should affect our confidence in original findings. PMID:26042061

SCF^{Cyclin F}-dependent degradation of CDC6 suppresses DNA re-replication

DEFF Research Database (Denmark)

Walter, David; Hoffmann, Saskia; Komseli, Eirini-Stavroula

2016-01-01

interact through defined sequence motifs that promote CDC6 ubiquitylation and degradation. Absence of Cyclin F or expression of a stable mutant of CDC6 promotes re-replication and genome instability in cells lacking the CDT1 inhibitor Geminin. Together, our work reveals a novel SCF(Cyclin F...

Universal Temporal Profile of Replication Origin Activation in Eukaryotes

Science.gov (United States)

Goldar, Arach

2011-03-01

The complete and faithful transmission of eukaryotic genome to daughter cells involves the timely duplication of mother cell's DNA. DNA replication starts at multiple chromosomal positions called replication origin. From each activated replication origin two replication forks progress in opposite direction and duplicate the mother cell's DNA. While it is widely accepted that in eukaryotic organisms replication origins are activated in a stochastic manner, little is known on the sources of the observed stochasticity. It is often associated to the population variability to enter S phase. We extract from a growing Saccharomyces cerevisiae population the average rate of origin activation in a single cell by combining single molecule measurements and a numerical deconvolution technique. We show that the temporal profile of the rate of origin activation in a single cell is similar to the one extracted from a replicating cell population. Taking into account this observation we exclude the population variability as the origin of observed stochasticity in origin activation. We confirm that the rate of origin activation increases in the early stage of S phase and decreases at the latter stage. The population average activation rate extracted from single molecule analysis is in prefect accordance with the activation rate extracted from published micro-array data, confirming therefore the homogeneity and genome scale invariance of dynamic of replication process. All these observations point toward a possible role of replication fork to control the rate of origin activation.

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…

Polymorphism in ficolin-1 ( FCN1 ) gene is associated with an earlier ...

Indian Academy of Sciences (India)

-1 (FCN1) gene is associated with an earlier onset of type 1 diabetes mellitus in children and adolescents from northeast Brazil. ZILMA PEREIRA DOS ANJOSA MANUELLA MARIA SILVA SANTOS NATASSIA JAVORSKI RODRIGUES ...

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.

RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress

DEFF Research Database (Denmark)

Bhowmick, Rahul; Minocherhomji, Sheroy; Hickson, Ian D

2016-01-01

Homologous recombination (HR) is necessary to counteract DNA replication stress. Common fragile site (CFS) loci are particularly sensitive to replication stress and undergo pathological rearrangements in tumors. At these loci, replication stress frequently activates DNA repair synthesis in mitosis...... replication stress at CFS loci during S-phase. In contrast, MiDAS is RAD52 dependent, and RAD52 is required for the timely recruitment of MUS81 and POLD3 to CFSs in early mitosis. Our results provide further mechanistic insight into MiDAS and define a specific function for human RAD52. Furthermore, selective...

Astronomical discoveries you can make, too! replicating the work of the great observers

CERN Document Server

Buchheim, Robert K

2015-01-01

You too can follow in the steps of the great astronomers such as Hipparchus, Galileo, Kepler and Hubble, who all contributed so much to our modern understanding of the cosmos. This book gives the student or amateur astronomer the following tools to replicate some of these seminal observations from their own homes:   With your own eyes: Use your own observations and measurements to discover and confirm the phenomena of the seasons, the analemma and the equation of time, the logic behind celestial coordinates, and even the precession of the equinoxes.   With a consumer-grade digital camera: Record the changing brightness of an eclipsing binary star and show that a pulsating star changes color as it brightens and dims. Add an inexpensive diffraction grating to your camera and see the variety of spectral features in the stars, and demonstrate that the Sun’s spectrum is similar to one particular type of stellar spectrum.   With a backyard telescope: Add a CCD imager and you can measure the scale of the Solar ...

Laying a Solid Foundation: Strategies for Effective Program Replication

Science.gov (United States)

Summerville, Geri

2009-01-01

The replication of proven social programs is a cost-effective and efficient way to achieve large-scale, positive social change. Yet there has been little guidance available about how to approach program replication and limited development of systems--at local, state or federal levels--to support replication efforts. "Laying a Solid Foundation:…

Mapping autonomously replicating sequence elements in a 73-kb ...

Indian Academy of Sciences (India)

Autonomously replicating sequence (ARS) elements are the genetic determinants of replication origin function in yeasts. They can be easily identified as the plasmids containing them transform yeast cells at a high frequency. As the first step towards identifying all potential replication origins in a 73-kb region of the long arm ...

Checkpoint-dependent RNR induction promotes fork restart after replicative stress.

Science.gov (United States)

Morafraile, Esther C; Diffley, John F X; Tercero, José Antonio; Segurado, Mónica

2015-01-20

The checkpoint kinase Rad53 is crucial to regulate DNA replication in the presence of replicative stress. Under conditions that interfere with the progression of replication forks, Rad53 prevents Exo1-dependent fork degradation. However, although EXO1 deletion avoids fork degradation in rad53 mutants, it does not suppress their sensitivity to the ribonucleotide reductase (RNR) inhibitor hydroxyurea (HU). In this case, the inability to restart stalled forks is likely to account for the lethality of rad53 mutant cells after replication blocks. Here we show that Rad53 regulates replication restart through the checkpoint-dependent transcriptional response, and more specifically, through RNR induction. Thus, in addition to preventing fork degradation, Rad53 prevents cell death in the presence of HU by regulating RNR-expression and localization. When RNR is induced in the absence of Exo1 and RNR negative regulators, cell viability of rad53 mutants treated with HU is increased and the ability of replication forks to restart after replicative stress is restored.

Selective visual working memory in fear of spiders: the role of automaticity and material-specificity.

Science.gov (United States)

Reinecke, Andrea; Becker, Eni S; Rinck, Mike

2009-12-01

Following cognitive models of anxiety, biases occur if threat processing is automatic versus strategic. Therefore, most of these models predict attentional bias, but not explicit memory bias. We suggest dividing memory into the highly automatic working memory (WM) component versus long-term memory when investigating bias in anxiety. WM for threat has rarely been investigated although its main function is stimulus monitoring, particularly important in anxiety. We investigated WM for spiders in spider fearfuls (SFs) versus non-anxious controls (NACs). In Experiment 1 (23 SFs/24 NACs), we replicated an earlier WM study, reducing strategic processing options. This led to stronger group differences and, thus, clearer WM threat biases. There were no group differences in Experiment 2 (18 SFs/19 NACs), using snakes instead of spiders to test whether WM biases are material-specific. This article supports cognitive models of anxiety in that biases are more likely to occur when reducing strategic processing. However, it contradicts the assumption that explicit memory biases are not characteristic of anxiety.

Identification of the determinants of efficient Pestivirus replication

OpenAIRE

Risager, Peter Christian; Belsham, Graham; Rasmussen, Thomas Bruun

2013-01-01

The key for the survival of a virus is to copy its own genome into progeny genomes that allows continued reproduction. The mechanism behind this "copy function" or "replication" is a wellorganized process that involves the formation of a replication complex in the cell and interactions between the viral proteins. The replication process in single-stranded RNA viruses of positive polarity requires a particular enzyme, an RNA dependent RNA polymerase, that has no direct counterpart elsewhere in...

Nuclear mitochondrial DNA activates replication in Saccharomyces cerevisiae.

Directory of Open Access Journals (Sweden)

Laurent Chatre

Full Text Available The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in.

Using Replication Projects in Teaching Research Methods

Science.gov (United States)

Standing, Lionel G.; Grenier, Manuel; Lane, Erica A.; Roberts, Meigan S.; Sykes, Sarah J.

2014-01-01

It is suggested that replication projects may be valuable in teaching research methods, and also address the current need in psychology for more independent verification of published studies. Their use in an undergraduate methods course is described, involving student teams who performed direct replications of four well-known experiments, yielding…

Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription.

Science.gov (United States)

Cifuentes-Muñoz, Nicolás; Branttie, Jean; Slaughter, Kerri Beth; Dutch, Rebecca Ellis

2017-12-15

Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications. IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to

Regulation of replication fork progression through histone supply and demand

DEFF Research Database (Denmark)

Groth, Anja; Corpet, Armelle; Cook, Adam J L

2007-01-01

DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. Here, we characterize a complex in which the human histone c...... progression and histone supply and demand.......1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. We propose that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and thus provides a means to fine-tune replication fork...

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

Science.gov (United States)

Larsen, Nicolai B; Sass, Ehud; Suski, Catherine; Mankouri, Hocine W; Hickson, Ian D

2014-04-07

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, to specific DNA sequences called Ter. Here, we demonstrate that Tus-Ter modules also induce polar RF pausing when engineered into the Saccharomyces cerevisiae genome. This heterologous RF barrier is distinct from a number of previously characterized, protein-mediated, RF pause sites in yeast, as it is neither Tof1-dependent nor counteracted by the Rrm3 helicase. Although the yeast replisome can overcome RF pausing at Tus-Ter modules, this event triggers site-specific homologous recombination that requires the RecQ helicase, Sgs1, for its timely resolution. We propose that Tus-Ter can be utilized as a versatile, site-specific, heterologous DNA replication-perturbing system, with a variety of potential applications.

Chromosome biology: conflict management for replication and transcription.

Science.gov (United States)

Dewar, James M; Walter, Johannes C

2013-03-04

A recent study has uncovered a new mechanism that attenuates DNA replication during periods of heightened gene expression to avoid collisions between replication and transcription. Copyright © 2013 Elsevier Ltd. All rights reserved.

Distribution of DNA replication proteins in Drosophila cells

Science.gov (United States)

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

2007-01-01

Background DNA replication in higher eukaryotic cells is organized in discrete subnuclear sites called replication foci (RF). During the S phase, most replication proteins assemble at the RF by interacting with PCNA via a PCNA binding domain (PBD). This has been shown to occur for many mammalian replication proteins, but it is not known whether this mechanism is conserved in evolution. Results Fluorescent fusions of mammalian replication proteins, Dnmt1, HsDNA Lig I and HsPCNA were analyzed for their ability to target to RF in Drosophila cells. Except for HsPCNA, none of the other proteins and their deletions showed any accumulation at RF in Drosophila cells. We hypothesized that in Drosophila cells there might be some other peptide sequence responsible for targeting proteins to RF. To test this, we identified the DmDNA Lig I and compared the protein sequence with HsDNA Lig I. The two orthologs shared the PBD suggesting a functionally conserved role for this domain in the Drosophila counterpart. A series of deletions of DmDNA Lig I were analyzed for their ability to accumulate at RF in Drosophila and mammalian cells. Surprisingly, no accumulation at RF was observed in Drosophila cells, while in mammalian cells DmDNA Lig I accumulated at RF via its PBD. Further, GFP fusions with the PBD domains from Dnmt1, HsDNA Lig I and DmDNA Lig I, were able to target to RF only in mammalian cells but not in Drosophila cells. Conclusion We show that S phase in Drosophila cells is characterized by formation of RF marked by PCNA like in mammalian cells. However, other than PCNA none of the replication proteins and their deletions tested here showed accumulation at RF in Drosophila cells while the same proteins and deletions are capable of accumulating at RF in mammalian cells. We hypothesize that unlike mammalian cells, in Drosophila cells, replication proteins do not form long-lasting interactions with the replication machinery, and rather perform their functions via very

Parasites Sustain and Enhance RNA-Like Replicators through Spatial Self-Organisation.

Directory of Open Access Journals (Sweden)

Enrico Sandro Colizzi

2016-04-01

Full Text Available In a prebiotic RNA world, parasitic behaviour may be favoured because template dependent replication happens in trans, thus being altruistic. Spatially extended systems are known to reduce harmful effects of parasites. Here we present a spatial system to show that evolution of replication is (indirectly enhanced by strong parasites, and we characterise the phase transition that leads to this mode of evolution. Building on the insights of this analysis, we identify two scenarios, namely periodic disruptions and longer replication time-span, in which speciation occurs and an evolved parasite-like lineage enables the evolutionary increase of replication rates in replicators. Finally, we show that parasites co-evolving with replicators are selected to become weaker, i.e. worse templates for replication when the duration of replication is increased. We conclude that parasites may not be considered a problem for evolution in a prebiotic system, but a degree of freedom that can be exploited by evolution to enhance the evolvability of replicators, by means of emergent levels of selection.

Replication stress activates DNA repair synthesis in mitosis

DEFF Research Database (Denmark)

Minocherhomji, Sheroy; Ying, Songmin; Bjerregaard, Victoria A

2015-01-01

Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps...... into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early...... mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest...

Specificity and function of Archaeal DNA replication initiator proteins

DEFF Research Database (Denmark)

Samson, Rachel Y.; Xu, Yanqun; Gadelha, Catarina

2013-01-01

Chromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC) that recruits the replicative helicase MCM(2-7) via Cdc6 and Cdt1. We find that the three origins...... to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels...

Structure of replicating intermediates of human herpesvirus type 6

International Nuclear Information System (INIS)

Severini, Alberto; Sevenhuysen, Claire; Garbutt, Michael; Tipples, Graham A.

2003-01-01

We have studied the structure of the replicative intermediates of human herpesvirus 6 (HHV-6) using pulsed-field gel electrophoresis, partial digestion, two-dimensional gel electrophoresis, and sedimentation centrifugation. The results show that DNA replication of HHV-6 produces head-to-tail concatemeric intermediates as well as approximately equal amounts of circular monomers or oligomers. Unlike the situation in herpes simplex virus, the intermediates of human herpesvirus 6 replication are not highly branched, suggesting a difference in the mechanism of replication or a lower frequency of homologous recombination in human herpesvirus 6 compared to herpes simplex virus

Transgene Expression and Host Cell Responses to Replication-Defective, Single-Cycle, and Replication-Competent Adenovirus Vectors

Directory of Open Access Journals (Sweden)

Catherine M. Crosby

2017-02-01

Full Text Available Most adenovirus (Ad vectors are E1 gene deleted replication defective (RD-Ad vectors that deliver one transgene to the cell and all expression is based on that one gene. In contrast, E1-intact replication-competent Ad (RC-Ad vectors replicate their DNA and their transgenes up to 10,000-fold, amplifying transgene expression markedly higher than RD-Ad vectors. While RC-Ad are more potent, they run the real risk of causing adenovirus infections in vector recipients and those that administer them. To gain the benefits of transgene amplification, but avoid the risk of Ad infections, we developed “single cycle” Ad (SC-Ad vectors. SC-Ads amplify transgene expression and generated markedly stronger and more persistent immune responses than RD-Ad as expected. However, they also unexpectedly generated stronger immune responses than RC-Ad vectors. To explore the basis of this potency here, we compared gene expression and the cellular responses to infection to these vectors in vitro and in vivo. In vitro, in primary human lung epithelial cells, SC- and RC-Ad amplified their genomes more than 400-fold relative to RD-Ad with higher replication by SC-Ad. This replication translated into higher green fluorescent protein (GFP expression for 48 h by SC- and RC-Ad than by RD-Ad. In vitro, in the absence of an immune system, RD-Ad expression became higher by 72 h coincident with cell death mediated by SC- and RC-Ad and release of transgene product from the dying cells. When the vectors were compared in human THP-1 Lucia- interferon-stimulated gene (ISG cells, which are a human monocyte cell line that have been modified to quantify ISG activity, RC-Ad6 provoked significantly stronger ISG responses than RD- or SC-Ad. In mice, intravenous or intranasal injection produced up to 100-fold genome replication. Under these in vivo conditions in the presence of the immune system, luciferase expression by RC and SC-Ad was markedly higher than that by RD-Ad. In

Are Psychology Journals Anti-replication? A Snapshot of Editorial Practices

OpenAIRE

Martin, G. N.; Clarke, Richard M.

2017-01-01

Recent research in psychology has highlighted a number of replication problems in the discipline, with publication bias – the preference for publishing original and positive results, and a resistance to publishing negative results and replications- identified as one reason for replication failure. However, little empirical research exists to demonstrate that journals explicitly refuse to publish replications. We reviewed the instructions to authors and the published aims of 1151 psychology jo...

Prescription Stimulant Use is Associated with Earlier Onset of Psychosis

OpenAIRE

Moran, Lauren V.; Masters, Grace A.; Pingali, Samira; Cohen, Bruce M.; Liebson, Elizabeth; Rajarethinam, R.P.; Ongur, Dost

2015-01-01

A childhood history of attention deficit hyperactivity disorder (ADHD) is common in psychotic disorders, yet prescription stimulants may interact adversely with the physiology of these disorders. Specifically, exposure to stimulants leads to long-term increases in dopamine release. We therefore hypothesized that individuals with psychotic disorders previously exposed to prescription stimulants will have an earlier onset of psychosis. Age of onset of psychosis (AOP) was compared in individuals...

On Scalability and Replicability of Smart Grid Projects—A Case Study

Directory of Open Access Journals (Sweden)

Lukas Sigrist

2016-03-01

Full Text Available This paper studies the scalability and replicability of smart grid projects. Currently, most smart grid projects are still in the R&D or demonstration phases. The full roll-out of the tested solutions requires a suitable degree of scalability and replicability to prevent project demonstrators from remaining local experimental exercises. Scalability and replicability are the preliminary requisites to perform scaling-up and replication successfully; therefore, scalability and replicability allow for or at least reduce barriers for the growth and reuse of the results of project demonstrators. The paper proposes factors that influence and condition a project’s scalability and replicability. These factors involve technical, economic, regulatory and stakeholder acceptance related aspects, and they describe requirements for scalability and replicability. In order to assess and evaluate the identified scalability and replicability factors, data has been collected from European and national smart grid projects by means of a survey, reflecting the projects’ view and results. The evaluation of the factors allows quantifying the status quo of on-going projects with respect to the scalability and replicability, i.e., they provide a feedback on to what extent projects take into account these factors and on whether the projects’ results and solutions are actually scalable and replicable.

CRISPR-mediated control of the bacterial initiation of replication.

Science.gov (United States)

Wiktor, Jakub; Lesterlin, Christian; Sherratt, David J; Dekker, Cees

2016-05-05

Programmable control of the cell cycle has been shown to be a powerful tool in cell-biology studies. Here, we develop a novel system for controlling the bacterial cell cycle, based on binding of CRISPR/dCas9 to the origin-of-replication locus. Initiation of replication of bacterial chromosomes is accurately regulated by the DnaA protein, which promotes the unwinding of DNA at oriC We demonstrate that the binding of CRISPR/dCas9 to any position within origin or replication blocks the initiation of replication. Serial-dilution plating, single-cell fluorescence microscopy, and flow-cytometry experiments show that ongoing rounds of chromosome replication are finished upon CRISPR/dCas9 binding, but no new rounds are initiated. Upon arrest, cells stay metabolically active and accumulate cell mass. We find that elevating the temperature from 37 to 42°C releases the CRISR/dCas9 replication inhibition, and we use this feature to recover cells from the arrest. Our simple and robust method of controlling the bacterial cell cycle is a useful asset for synthetic biology and DNA-replication studies in particular. The inactivation of CRISPR/dCas9 binding at elevated temperatures may furthermore be of wide interest for CRISPR/Cas9 applications in genomic engineering. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Interplay between Selenium Levels and Replicative Senescence in WI-38 Human Fibroblasts: A Proteomic Approach.

Science.gov (United States)

Hammad, Ghania; Legrain, Yona; Touat-Hamici, Zahia; Duhieu, Stéphane; Cornu, David; Bulteau, Anne-Laure; Chavatte, Laurent

2018-01-20

Selenoproteins are essential components of antioxidant defense, redox homeostasis, and cell signaling in mammals, where selenium is found in the form of a rare amino acid, selenocysteine. Selenium, which is often limited both in food intake and cell culture media, is a strong regulator of selenoprotein expression and selenoenzyme activity. Aging is a slow, complex, and multifactorial process, resulting in a gradual and irreversible decline of various functions of the body. Several cellular aspects of organismal aging are recapitulated in the replicative senescence of cultured human diploid fibroblasts, such as embryonic lung fibroblast WI-38 cells. We previously reported that the long-term growth of young WI-38 cells with high (supplemented), moderate (control), or low (depleted) concentrations of selenium in the culture medium impacts their replicative lifespan, due to rapid changes in replicative senescence-associated markers and signaling pathways. In order to gain insight into the molecular link between selenium levels and replicative senescence, in the present work, we have applied a quantitative proteomic approach based on 2-Dimensional Differential in-Gel Electrophoresis (2D-DIGE) to the study of young and presenescent cells grown in selenium-supplemented, control, or depleted media. Applying a restrictive cut-off (spot intensity ±50% and a p value iii) spots varying in response to selenium concentration in presenescent cells. Interestingly, a 72% overlap between the impact of senescence and selenium was observed in our proteomic results, demonstrating a strong interplay between selenium, selenoproteins, and replicative senescence.

Replication and Inhibitors of Enteroviruses and Parechoviruses

Directory of Open Access Journals (Sweden)

Lonneke van der Linden

2015-08-01

Full Text Available The Enterovirus (EV and Parechovirus genera of the picornavirus family include many important human pathogens, including poliovirus, rhinovirus, EV-A71, EV-D68, and human parechoviruses (HPeV. They cause a wide variety of diseases, ranging from a simple common cold to life-threatening diseases such as encephalitis and myocarditis. At the moment, no antiviral therapy is available against these viruses and it is not feasible to develop vaccines against all EVs and HPeVs due to the great number of serotypes. Therefore, a lot of effort is being invested in the development of antiviral drugs. Both viral proteins and host proteins essential for virus replication can be used as targets for virus inhibitors. As such, a good understanding of the complex process of virus replication is pivotal in the design of antiviral strategies goes hand in hand with a good understanding of the complex process of virus replication. In this review, we will give an overview of the current state of knowledge of EV and HPeV replication and how this can be inhibited by small-molecule inhibitors.

Attenuation of Replication-Competent Adenovirus Serotype 26 Vaccines by Vectorization.

Science.gov (United States)

Maxfield, Lori F; Abbink, Peter; Stephenson, Kathryn E; Borducchi, Erica N; Ng'ang'a, David; Kirilova, Marinela M; Paulino, Noelix; Boyd, Michael; Shabram, Paul; Ruan, Qian; Patel, Mayank; Barouch, Dan H

2015-11-01

Replication-competent adenovirus (rcAd)-based vaccine vectors may theoretically provide immunological advantages over replication-incompetent Ad vectors, but they also raise additional potential clinical and regulatory issues. We produced replication-competent Ad serotype 26 (rcAd26) vectors by adding the E1 region back into a replication-incompetent Ad26 vector backbone with the E3 or E3/E4 regions deleted. We assessed the effect of vectorization on the replicative capacity of the rcAd26 vaccines. Attenuation occurred in a stepwise fashion, with E3 deletion, E4 deletion, and human immunodeficiency virus type 1 (HIV-1) envelope (Env) gene insertion all contributing to reduced replicative capacity compared to that with the wild-type Ad26 vector. The rcAd26 vector with E3 and E4 deleted and containing the Env transgene exhibited 2.7- to 4.4-log-lower replicative capacity than that of the wild-type Ad26 in vitro. This rcAd26 vector is currently being evaluated in a phase 1 clinical trial. Attenuation as a result of vectorization and transgene insertion has implications for the clinical development of replication-competent vaccine vectors. Copyright © 2015, Maxfield et al.

Using Replicates in Information Retrieval Evaluation.

Science.gov (United States)

Voorhees, Ellen M; Samarov, Daniel; Soboroff, Ian

2017-09-01

This article explores a method for more accurately estimating the main effect of the system in a typical test-collection-based evaluation of information retrieval systems, thus increasing the sensitivity of system comparisons. Randomly partitioning the test document collection allows for multiple tests of a given system and topic (replicates). Bootstrap ANOVA can use these replicates to extract system-topic interactions-something not possible without replicates-yielding a more precise value for the system effect and a narrower confidence interval around that value. Experiments using multiple TREC collections demonstrate that removing the topic-system interactions substantially reduces the confidence intervals around the system effect as well as increases the number of significant pairwise differences found. Further, the method is robust against small changes in the number of partitions used, against variability in the documents that constitute the partitions, and the measure of effectiveness used to quantify system effectiveness.

Experimenter gender and replicability in science.

Science.gov (United States)

Chapman, Colin D; Benedict, Christian; Schiöth, Helgi B

2018-01-01

There is a replication crisis spreading through the annals of scientific inquiry. Although some work has been carried out to uncover the roots of this issue, much remains unanswered. With this in mind, this paper investigates how the gender of the experimenter may affect experimental findings. Clinical trials are regularly carried out without any report of the experimenter's gender and with dubious knowledge of its influence. Consequently, significant biases caused by the experimenter's gender may lead researchers to conclude that therapeutics or other interventions are either overtreating or undertreating a variety of conditions. Bearing this in mind, this policy paper emphasizes the importance of reporting and controlling for experimenter gender in future research. As backdrop, it explores what we know about the role of experimenter gender in influencing laboratory results, suggests possible mechanisms, and suggests future areas of inquiry.

Are Psychology Journals Anti-replication? A Snapshot of Editorial Practices.

Science.gov (United States)

Martin, G N; Clarke, Richard M

2017-01-01

Recent research in psychology has highlighted a number of replication problems in the discipline, with publication bias - the preference for publishing original and positive results, and a resistance to publishing negative results and replications- identified as one reason for replication failure. However, little empirical research exists to demonstrate that journals explicitly refuse to publish replications. We reviewed the instructions to authors and the published aims of 1151 psychology journals and examined whether they indicated that replications were permitted and accepted. We also examined whether journal practices differed across branches of the discipline, and whether editorial practices differed between low and high impact journals. Thirty three journals (3%) stated in their aims or instructions to authors that they accepted replications. There was no difference between high and low impact journals. The implications of these findings for psychology are discussed.

The rolling-circle melting-pot model for porcine circovirus DNA replication

Science.gov (United States)

A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication (Ori) among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. Porcine circo...

Inhibition of spring viraemia of carp virus replication in an Epithelioma papulosum cyprini cell line by RNAi

Science.gov (United States)

Gotesman, M; Soliman, H; Besch, R; El-Matbouli, M

2015-01-01

Spring viraemia of carp virus (SVCV) is an aetiological agent of a serious disease affecting carp farms in Europe and is a member of the Rhabdoviridae family of viruses. The genome of SVCV codes for five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). RNA-mediated interference (RNAi) by small interfering RNAs (siRNAs) is a powerful tool to inhibit gene transcription and is used to study genes important for viral replication. In previous studies regarding another member of Rhabdoviridae, siRNA inhibition of the rabies virus nucleoprotein gene provided in vitro and in vivo protection against rabies. In this study, synthetic siRNA molecules were designed to target SVCV-N and SVCV-P transcripts to inhibit SVCV replication and were tested in an epithelioma papulosum cyprini (EPC) cell line. Inhibition of gene transcription was measured by real-time quantitative reverse-transcription PCR (RT-qPCR). The efficacy of using siRNA for inhibition of viral replication was analysed by RT-qPCR measurement of a reporter gene (glycoprotein) expression and by virus endpoint titration. Inhibition of nucleoprotein and phosphoprotein gene expression by siRNA reduced SVCV replication. However, use of tandem siRNAs that target phosphoprotein and nucleoprotein worked best at reducing SVCV replication. PMID:24460815

Molecular analysis of the replication program in unicellular model organisms.

Science.gov (United States)

Raghuraman, M K; Brewer, Bonita J

2010-01-01

Eukaryotes have long been reported to show temporal programs of replication, different portions of the genome being replicated at different times in S phase, with the added possibility of developmentally regulated changes in this pattern depending on species and cell type. Unicellular model organisms, primarily the budding yeast Saccharomyces cerevisiae, have been central to our current understanding of the mechanisms underlying the regulation of replication origins and the temporal program of replication in particular. But what exactly is a temporal program of replication, and how might it arise? In this article, we explore this question, drawing again on the wealth of experimental information in unicellular model organisms.

Regulation of DNA replication in irradiated cells by trans-acting factors

International Nuclear Information System (INIS)

Wang, Y.; Huq, M.S.; Cheng, X.; Iliakis, G.

1995-01-01

We compared DNA replication activity in cytoplasmic extracts prepared from irradiated and nonirradiated HeLa cells using a simian virus 40 (SV40)-based in vitro replication assay. The assay measures semi-conservative DNA replication in a plasmid carrying the SV40 origin of replication and requires SV40 T antigen as the sole noncellular protein. The plasmid DNA used in the replication reaction is never exposed to radiation. We find that replication of plasmid DNA is significantly reduced when cytoplasmic extracts from irradiated cells are used. Since plasmid replication proceeds to completion in extracts from irradiated cells, the observed reduction in the overall replication activity is probably due to a reduction in the efficiency of initiation events. The degree of inhibition of DNA replication after exposure to 10, 30 and 50 Gy X rays as measured in vitro using this assay is similar to that measured in intact cells immediately before processing for extract preparation. These observations are compatible with the induction or activation by ionizing radiation of a factor(s) that inhibits in trans DNA replication. The results contribute to our understanding of the mechanism(s) developed by the cells to regulate DNA replication when exposed to clastogenic agents. Such processes may be of significance in the restoration of DNA integrity, and may define yet another checkpoint operating during S at the level of clusters of replicons. 26 refs., 4 figs

Genome-wide alterations of the DNA replication program during tumor progression

Science.gov (United States)

Arneodo, A.; Goldar, A.; Argoul, F.; Hyrien, O.; Audit, B.

2016-08-01

Oncogenic stress is a major driving force in the early stages of cancer development. Recent experimental findings reveal that, in precancerous lesions and cancers, activated oncogenes may induce stalling and dissociation of DNA replication forks resulting in DNA damage. Replication timing is emerging as an important epigenetic feature that recapitulates several genomic, epigenetic and functional specificities of even closely related cell types. There is increasing evidence that chromosome rearrangements, the hallmark of many cancer genomes, are intimately associated with the DNA replication program and that epigenetic replication timing changes often precede chromosomic rearrangements. The recent development of a novel methodology to map replication fork polarity using deep sequencing of Okazaki fragments has provided new and complementary genome-wide replication profiling data. We review the results of a wavelet-based multi-scale analysis of genomic and epigenetic data including replication profiles along human chromosomes. These results provide new insight into the spatio-temporal replication program and its dynamics during differentiation. Here our goal is to bring to cancer research, the experimental protocols and computational methodologies for replication program profiling, and also the modeling of the spatio-temporal replication program. To illustrate our purpose, we report very preliminary results obtained for the chronic myelogeneous leukemia, the archetype model of cancer. Finally, we discuss promising perspectives on using genome-wide DNA replication profiling as a novel efficient tool for cancer diagnosis, prognosis and personalized treatment.

Checkpoint independence of most DNA replication origins in fission yeast

OpenAIRE

Mickle, Katie L; Ramanathan, Sunita; Rosebrock, Adam; Oliva, Anna; Chaudari, Amna; Yompakdee, Chulee; Scott, Donna; Leatherwood, Janet; Huberman, Joel A

2007-01-01

Abstract Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleo...

Replicative intermediates in UV-irradiated Simian virus 40

International Nuclear Information System (INIS)

Clark, J.M.; Hanawalt, P.C.

1984-01-01

The authors have used Simian virus 40 (SV40) as a probe to study the replication of UV-damaged DNA in mammalian cells. Viral DNA replication in infected monkey kidney cells was synchronized by incubating a mutant of SV40 (tsA58) temperature-sensitive for the initiation of DNA synthesis at the restrictive temperature and then adding aphidicolin to temporarily inhibit DNA synthesis at the permissive temperature while permitting pre-replicative events to occur. After removal of the drug, the infected cells were irradiated at 100 J/m 2 (254 nm) to produce 6-7 pyrimidine dimers per SV40 genome, and returned to the restrictive temperature to prevent reinitiation of replication from the SV40 origin. Replicative intermediates (RI) were labeled with [ 3 H]thymidine. The size distribution of daughter DNA strands in RI isolated shortly after irradiation was skewed towards lengths less than the interdimer spacing in parental DNA; this bias persisted for at least 1 h after irradiation, but disappeared within 3 h by which time the size of the newly-synthesized DNA exceeded the interdimer distance. Evidence was obtained for the generation at late times after irradiation, of Form I molecules in which the daughter DNA strand contain dimers. Thus DNA strand exchange as well as trans-dimer synthesis may be involved in the generation of supercoiled Form I DNA from 0V-damaged SV40 replicative intermediates. (Auth.)

Cross-catalytic peptide nucleic acid (PNA) replication based on templated ligation

DEFF Research Database (Denmark)

Singhal, Abhishek; Nielsen, Peter E

2014-01-01

We report the first PNA self-replicating system based on template directed cross-catalytic ligation, a process analogous to biological replication. Using two template PNAs and four pentameric precursor PNAs, all four possible carbodiimide assisted amide ligation products were detected...... precursors. Cross-catalytic product formation followed product inhibited kinetics, but approximately two replication rounds were observed. Analogous but less efficient replication was found for a similar tetrameric system. These results demonstrate that simpler nucleobase replication systems than natural...

X-ray repair replication in L1210 leukemia cells

International Nuclear Information System (INIS)

Lee, Y.C.; Byfield, J.E.; Bennett, L.R.; Chan, P.Y.M.

1974-01-01

Repair replication has been studied in detail in mouse L1210 leukemia cells. A method of identifying and quantitating repair replication using a pre- and postradiation block of normal replication with cytosine arabinoside is illustrated. The method derived does not require isolation of DNA per se and appears to be satisfactory for screening for inhibitors of repair replication. Repair replication can be demonstrated at doses in the 1000-rad range in bromouridine deoxyriboside-substituted cells and at slightly higher doses in nonsubstituted cells. Drugs that are known to bind to DNA inhibit this x-ray-induced repair replication. Drugs with these properties may be identified by the methods described and compared quantitatively in their ability to inhibit this type of x-ray damage. Since these phenomena can be demonstrated for low radiation doses and at drug concentrations attainable in vivo during human cancer chemotherapy this class of anticancer agent may be worthy of closer study. Application to the L1210 leukemia system should permit comparison of in vitro and in vivo drug effects in the context of the extensive in vivo pharmacological data already available for L1210 cells. (U.S.)

A Gossip-Based Optimistic Replication for Efficient Delay-Sensitive Streaming Using an Interactive Middleware Support System

Science.gov (United States)

Mavromoustakis, Constandinos X.; Karatza, Helen D.

2010-06-01

While sharing resources the efficiency is substantially degraded as a result of the scarceness of availability of the requested resources in a multiclient support manner. These resources are often aggravated by many factors like the temporal constraints for availability or node flooding by the requested replicated file chunks. Thus replicated file chunks should be efficiently disseminated in order to enable resource availability on-demand by the mobile users. This work considers a cross layered middleware support system for efficient delay-sensitive streaming by using each device's connectivity and social interactions in a cross layered manner. The collaborative streaming is achieved through the epidemically replicated file chunk policy which uses a transition-based approach of a chained model of an infectious disease with susceptible, infected, recovered and death states. The Gossip-based stateful model enforces the mobile nodes whether to host a file chunk or not or, when no longer a chunk is needed, to purge it. The proposed model is thoroughly evaluated through experimental simulation taking measures for the effective throughput Eff as a function of the packet loss parameter in contrast with the effectiveness of the replication Gossip-based policy.

Definition of herpes simplex virus type 1 helper activities for adeno-associated virus early replication events.

Directory of Open Access Journals (Sweden)

Nathalie Alazard-Dany

2009-03-01

Full Text Available The human parvovirus Adeno-Associated Virus (AAV type 2 can only replicate in cells co-infected with a helper virus, such as Adenovirus or Herpes Simplex Virus type 1 (HSV-1; whereas, in the absence of a helper virus, it establishes a latent infection. Previous studies demonstrated that the ternary HSV-1 helicase/primase (HP complex (UL5/8/52 and the single-stranded DNA-Binding Protein (ICP8 were sufficient to induce AAV-2 replication in transfected cells. We independently showed that, in the context of a latent AAV-2 infection, the HSV-1 ICP0 protein was able to activate rep gene expression. The present study was conducted to integrate these observations and to further explore the requirement of other HSV-1 proteins during early AAV replication steps, i.e. rep gene expression and AAV DNA replication. Using a cellular model that mimics AAV latency and composite constructs coding for various sets of HSV-1 genes, we first confirmed the role of ICP0 for rep gene expression and demonstrated a synergistic effect of ICP4 and, to a lesser extent, ICP22. Conversely, ICP27 displayed an inhibitory effect. Second, our analyses showed that the effect of ICP0, ICP4, and ICP22 on rep gene expression was essential for the onset of AAV DNA replication in conjunction with the HP complex and ICP8. Third, and most importantly, we demonstrated that the HSV-1 DNA polymerase complex (UL30/UL42 was critical to enhance AAV DNA replication to a significant level in transfected cells and that its catalytic activity was involved in this process. Altogether, this work represents the first comprehensive study recapitulating the series of early events taking place during HSV-1-induced AAV replication.

Homologous recombination in mammalian cells: effect of p53 and Bcl-2 proteins, replication inhibition and ionizing radiations

International Nuclear Information System (INIS)

Saintigny, Yannick

1999-01-01

The control of cell cycle, associated with the mechanisms of replication, DNA repair/recombination allows the cells to maintain their genetic integrity. The p53 protein ensures the control of G1/S transition. Its inactivation would allow to initial replication on damaged matrix and lead to the block of replication forks followed by DNA strand breaks, good substrates for recombination. This work shows that the expression of mutant p53 protein stimulates both spontaneous and radio-induced homologous recombination, independently of the control of cell cycle. Moreover, the use of a set of replication inhibitors show that inhibition of the replication elongation stimulates recombination more strongly than the initiation inhibition. Replication arrest by these inhibitors also significantly increases the number of DNA strand breaks. These results highlighted a point of action of p53 protein on the ultimate stages of the homologous recombination mechanism. Lastly, the expression of Bcl-2 protein inhibits apoptosis and increases survival, but specifically inhibits conservative recombination, after radiation as well as in absence of apoptotic stress. The extinction of this mechanism of DNA repair is associated with an increase of mutagenesis. Taken together, these results allow ta consider the maintenance of the genetic stability as a cellular network involving different pathways. A multiple stages model for tumoral progression can be deduced. (author) [fr

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'.

BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication.

Science.gov (United States)

Morosky, Stefanie; Lennemann, Nicholas J; Coyne, Carolyn B

2016-05-15

Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of BPIFB6 expression

BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication

Science.gov (United States)

Morosky, Stefanie; Lennemann, Nicholas J.

2016-01-01

ABSTRACT Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. IMPORTANCE Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of

DNA replication machinery is required for development in Drosophila.

Science.gov (United States)

Kohzaki, Hidetsugu; Asano, Maki; Murakami, Yota

2018-01-01

 In Drosophila , some factors involved in chromosome replication seem to be involved in gene amplification and endoreplication, which are actively utilized in particular tissue development, but direct evidence has not been shown. Therefore, we examined the effect of depletion of replication factors on these processes. First, we confirmed RNAi knockdown can be used for the depletion of replication factors by comparing the phenotypes of RNAi knockdown and deletion or point mutants of the components of DNA licensing factor, MCM2, MCM4 and Cdt1. Next, we found that tissue-specific RNAi knockdown of replication factors caused tissue-specific defects, probably due to defects in DNA replication. In particular, we found that depletion inhibited gene amplification of the chorion gene in follicle cells and endoreplication in salivary glands, showing that chromosomal DNA replication factors are required for these processes. Finally, using RNAi, we screened the genes for chromosomal DNA replication that affected tissue development. Interestingly, wing specific knockdown of Mcm10 induced wing formation defects. These results suggest that some components of chromosomal replication machinery are directly involved in tissue development.

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism.

Science.gov (United States)

Reynolds, John J; Bicknell, Louise S; Carroll, Paula; Higgs, Martin R; Shaheen, Ranad; Murray, Jennie E; Papadopoulos, Dimitrios K; Leitch, Andrea; Murina, Olga; Tarnauskaitė, Žygimantė; Wessel, Sarah R; Zlatanou, Anastasia; Vernet, Audrey; von Kriegsheim, Alex; Mottram, Rachel M A; Logan, Clare V; Bye, Hannah; Li, Yun; Brean, Alexander; Maddirevula, Sateesh; Challis, Rachel C; Skouloudaki, Kassiani; Almoisheer, Agaadir; Alsaif, Hessa S; Amar, Ariella; Prescott, Natalie J; Bober, Michael B; Duker, Angela; Faqeih, Eissa; Seidahmed, Mohammed Zain; Al Tala, Saeed; Alswaid, Abdulrahman; Ahmed, Saleem; Al-Aama, Jumana Yousuf; Altmüller, Janine; Al Balwi, Mohammed; Brady, Angela F; Chessa, Luciana; Cox, Helen; Fischetto, Rita; Heller, Raoul; Henderson, Bertram D; Hobson, Emma; Nürnberg, Peter; Percin, E Ferda; Peron, Angela; Spaccini, Luigina; Quigley, Alan J; Thakur, Seema; Wise, Carol A; Yoon, Grace; Alnemer, Maha; Tomancak, Pavel; Yigit, Gökhan; Taylor, A Malcolm R; Reijns, Martin A M; Simpson, Michael A; Cortez, David; Alkuraya, Fowzan S; Mathew, Christopher G; Jackson, Andrew P; Stewart, Grant S

2017-04-01

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

Anaphase onset before complete DNA replication with intact checkpoint responses

DEFF Research Database (Denmark)

Torres-Rosell, Jordi; De Piccoli, Giacomo; Cordon-Preciado, Violeta

2007-01-01

Cellular checkpoints prevent mitosis in the presence of stalled replication forks. Whether checkpoints also ensure the completion of DNA replication before mitosis is unknown. Here, we show that in yeast smc5-smc6 mutants, which are related to cohesin and condensin, replication is delayed, most...

Late replication domains are evolutionary conserved in the Drosophila genome.

Science.gov (United States)

Andreyenkova, Natalya G; Kolesnikova, Tatyana D; Makunin, Igor V; Pokholkova, Galina V; Boldyreva, Lidiya V; Zykova, Tatyana Yu; Zhimulev, Igor F; Belyaeva, Elena S

2013-01-01

Drosophila chromosomes are organized into distinct domains differing in their predominant chromatin composition, replication timing and evolutionary conservation. We show on a genome-wide level that genes whose order has remained unaltered across 9 Drosophila species display late replication timing and frequently map to the regions of repressive chromatin. This observation is consistent with the existence of extensive domains of repressive chromatin that replicate extremely late and have conserved gene order in the Drosophila genome. We suggest that such repressive chromatin domains correspond to a handful of regions that complete replication at the very end of S phase. We further demonstrate that the order of genes in these regions is rarely altered in evolution. Substantial proportion of such regions significantly coincide with large synteny blocks. This indicates that there are evolutionary mechanisms maintaining the integrity of these late-replicating chromatin domains. The synteny blocks corresponding to the extremely late-replicating regions in the D. melanogaster genome consistently display two-fold lower gene density across different Drosophila species.

Calcein represses human papillomavirus 16 E1-E2 mediated DNA replication via blocking their binding to the viral origin of replication.

Science.gov (United States)

Das, Dipon; Smith, Nathan W; Wang, Xu; Richardson, Stacie L; Hartman, Matthew C T; Morgan, Iain M

2017-08-01

Human papillomaviruses are causative agents in several human diseases ranging from genital warts to ano-genital and oropharyngeal cancers. Currently only symptoms of HPV induced disease are treated; there are no antivirals available that directly target the viral life cycle. Previously, we determined that the cellular protein TopBP1 interacts with the HPV16 replication/transcription factor E2. This E2-TopBP1 interaction is essential for optimal E1-E2 DNA replication and for the viral life cycle. The drug calcein disrupts the interaction of TopBP1 with itself and other host proteins to promote cell death. Here we demonstrate that calcein blocks HPV16 E1-E2 DNA replication via blocking the viral replication complex forming at the origin of replication. This occurs at non-toxic levels of calcein and demonstrates specificity as it does not block the ability of E2 to regulate transcription. We propose that calcein or derivatives could be developed as an anti-HPV therapeutic. Copyright © 2017 Elsevier Inc. All rights reserved.

The Song Remains the Same: A Replication and Extension of the MUSIC Model

Science.gov (United States)

Rentfrow, Peter J.; Goldberg, Lewis R.; Stillwell, David J.; Kosinski, Michal; Gosling, Samuel D.; Levitin, Daniel J.

2012-01-01

There is overwhelming anecdotal and empirical evidence for individual differences in musical preferences. However, little is known about what drives those preferences. Are people drawn to particular musical genres (e.g., rap, jazz) or to certain musical properties (e.g., lively, loud)? Recent findings suggest that musical preferences can be conceptualized in terms of five orthogonal dimensions: Mellow, Unpretentious, Sophisticated, Intense, and Contemporary (conveniently, MUSIC). The aim of the present research is to replicate and extend that work by empirically examining the hypothesis that musical preferences are based on preferences for particular musical properties and psychological attributes as opposed to musical genres. Findings from Study 1 replicated the five-factor MUSIC structure using musical excerpts from a variety of genres and subgenres and revealed musical attributes that differentiate each factor. Results from Studies 2 and 3 show that the MUSIC structure is recoverable using musical pieces from only the jazz and rock genres, respectively. Taken together, the current work provides strong evidence that preferences for music are determined by specific musical attributes and that the MUSIC model is a robust framework for conceptualizing and measuring such preferences. PMID:24825945

The Song Remains the Same: A Replication and Extension of the MUSIC Model.

Science.gov (United States)

Rentfrow, Peter J; Goldberg, Lewis R; Stillwell, David J; Kosinski, Michal; Gosling, Samuel D; Levitin, Daniel J

2012-12-01

There is overwhelming anecdotal and empirical evidence for individual differences in musical preferences. However, little is known about what drives those preferences. Are people drawn to particular musical genres (e.g., rap, jazz) or to certain musical properties (e.g., lively, loud)? Recent findings suggest that musical preferences can be conceptualized in terms of five orthogonal dimensions: Mellow, Unpretentious, Sophisticated, Intense, and Contemporary (conveniently, MUSIC). The aim of the present research is to replicate and extend that work by empirically examining the hypothesis that musical preferences are based on preferences for particular musical properties and psychological attributes as opposed to musical genres. Findings from Study 1 replicated the five-factor MUSIC structure using musical excerpts from a variety of genres and subgenres and revealed musical attributes that differentiate each factor. Results from Studies 2 and 3 show that the MUSIC structure is recoverable using musical pieces from only the jazz and rock genres, respectively. Taken together, the current work provides strong evidence that preferences for music are determined by specific musical attributes and that the MUSIC model is a robust framework for conceptualizing and measuring such preferences.

Working memory capacity in social anxiety disorder: Revisiting prior conclusions.

Science.gov (United States)

Waechter, Stephanie; Moscovitch, David A; Vidovic, Vanja; Bielak, Tatiana; Rowa, Karen; McCabe, Randi E

2018-04-01

In one of the few studies examining working memory processes in social anxiety disorder (SAD), Amir and Bomyea (2011) recruited participants with and without SAD to complete a working memory span task with neutral and social threat words. Those with SAD showed better working memory performance for social threat words compared to neutral words, suggesting an enhancement in processing efficiency for socially threatening information in SAD. The current study sought to replicate and extend these findings. In this study, 25 participants with a principal diagnosis of SAD, 24 anxious control (AC) participants with anxiety disorders other than SAD, and 27 healthy control (HC) participants with no anxiety disorder completed a working memory task with social threat, general threat, and neutral stimuli. The groups in the current study demonstrated similar working memory performance within each of the word type conditions, thus failing to replicate the principal findings of Amir and Bomyea (2011). Post hoc analyses revealed a significant association between higher levels of anxiety symptomatology and poorer overall WM performance. These results inform our understanding of working memory in the anxiety disorders and support the importance of replication in psychological research. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Oxytocin promotes intuitive rather than deliberated cooperation with the in-group

NARCIS (Netherlands)

Ten Velden, F.S.; Daughters, K.; De Dreu, C.K.W.

A contribution to a special issue on Hormones and Human Competition. In intergroup settings, individuals prefer cooperating with their in-group, and sometimes derogate and punish out-groups. Here we replicate earlier work showing that such in-group bounded cooperation is conditioned by oxytocin and

Replication stress, a source of epigenetic aberrations in cancer?

DEFF Research Database (Denmark)

Jasencakova, Zusana; Groth, Anja

2010-01-01

. Chromatin organization is transiently disrupted during DNA replication and maintenance of epigenetic information thus relies on faithful restoration of chromatin on the new daughter strands. Acute replication stress challenges proper chromatin restoration by deregulating histone H3 lysine 9 mono......-methylation on new histones and impairing parental histone recycling. This could facilitate stochastic epigenetic silencing by laying down repressive histone marks at sites of fork stalling. Deregulation of replication in response to oncogenes and other tumor-promoting insults is recognized as a significant source...... of genome instability in cancer. We propose that replication stress not only presents a threat to genome stability, but also jeopardizes chromatin integrity and increases epigenetic plasticity during tumorigenesis....

SMC1-Mediated Intra-S-Phase Arrest Facilitates Bocavirus DNA Replication

Science.gov (United States)

Luo, Yong; Deng, Xuefeng; Cheng, Fang; Li, Yi

2013-01-01

Activation of a host DNA damage response (DDR) is essential for DNA replication of minute virus of canines (MVC), a member of the genus Bocavirus of the Parvoviridae family; however, the mechanism by which DDR contributes to viral DNA replication is unknown. In the current study, we demonstrate that MVC infection triggers the intra-S-phase arrest to slow down host cellular DNA replication and to recruit cellular DNA replication factors for viral DNA replication. The intra-S-phase arrest is regulated by ATM (ataxia telangiectasia-mutated kinase) signaling in a p53-independent manner. Moreover, we demonstrate that SMC1 (structural maintenance of chromosomes 1) is the key regulator of the intra-S-phase arrest induced during infection. Either knockdown of SMC1 or complementation with a dominant negative SMC1 mutant blocks both the intra-S-phase arrest and viral DNA replication. Finally, we show that the intra-S-phase arrest induced during MVC infection was caused neither by damaged host cellular DNA nor by viral proteins but by replicating viral genomes physically associated with the DNA damage sensor, the Mre11-Rad50-Nbs1 (MRN) complex. In conclusion, the feedback loop between MVC DNA replication and the intra-S-phase arrest is mediated by ATM-SMC1 signaling and plays a critical role in MVC DNA replication. Thus, our findings unravel the mechanism underlying DDR signaling-facilitated MVC DNA replication and demonstrate a novel strategy of DNA virus-host interaction. PMID:23365434

Inhibition of DNA replication by ultraviolet light

International Nuclear Information System (INIS)

Edenberg, H.J.

1976-01-01

DNA replication in ultraviolet-irradiated HeLa cells was studied by two different techniques: measurements of the kinetics of semiconservative DNA synthesis, and DNA fiber autoradiography. In examining the kinetics of semiconservative DNA synthesis, density label was used to avoid measuring the incorporation due to repair replication. The extent of inhibition varied with time. After doses of less than 10 J/m 2 the rate was initially depressed but later showed some recovery. After higher doses, a constant, low rate of synthesis was seen for at least the initial 6 h. An analysis of these data indicated that the inhibition of DNA synthesis could be explained by replication forks halting at pyrimidine dimers. DNA fiber autoradiography was used to further characterize replication after ultraviolet irradiation. The average length of labeled segments in irradiated cells increased in the time immediately after irradiation, and then leveled off. This is the predicted pattern if DNA synthesis in each replicon continued at its previous rate until a lesion is reached, and then halted. The frequency of lesions that block synthesis is approximately the same as the frequency of pyrimidine dimers

Replication fidelity assessment of polymer large area sub-μm structured surfaces using fast angular intensity distribution measurements

DEFF Research Database (Denmark)

Calaon, M.; Hansen, H. N.; Tosello, G.

The present investigation addresses one of the key challenges in the product quality control of transparent polymer substrates, identified in the replication fidelity of sub-μm structures over large area. Additionally the work contributes to the development of new techniques focused on in......-line characterization of large nanostructured surfaces. In particular the aim of the present paper is to introduce initial development of a metrology approach to quantify the replication fidelity of produced 500 nm diameter semi-spheres via anodizing of aluminum (Al) and subsequent nickel electroforming to COC...

Culture in the cockpit: do Hofstede's dimensions replicate?

Science.gov (United States)

Merritt, A.; Helmreich, R. L. (Principal Investigator)

2000-01-01

Survey data collected from 9,400 male commercial airline pilots in 19 countries were used in a replication study of Hofstede's indexes of national culture. The analysis that removed the constraint of item equivalence proved superior, both conceptually and empirically, to the analysis using Hofstede's items and formulae as prescribed, and rendered significant replication correlations for all indexes (Individualism-Collectivism .96, Power Distance .87, Masculinity-Femininity .75, and Uncertainty Avoidance .68). The successful replication confirms that national culture exerts an influence on cockpit behavior over and above the professional culture of pilots, and that "one size fits all" training is inappropriate.

Oncolytic Replication of E1b-Deleted Adenoviruses

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Pei-Hsin Cheng

2015-11-01

Full Text Available Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viralmRNAexport, and cell cycle disruption.

Electron microscopic analysis of rotavirus assembly-replication intermediates

International Nuclear Information System (INIS)

Boudreaux, Crystal E.; Kelly, Deborah F.; McDonald, Sarah M.

2015-01-01

Rotaviruses (RVs) replicate their segmented, double-stranded RNA genomes in tandem with early virion assembly. In this study, we sought to gain insight into the ultrastructure of RV assembly-replication intermediates (RIs) using transmission electron microscopy (EM). Specifically, we examined a replicase-competent, subcellular fraction that contains all known RV RIs. Three never-before-seen complexes were visualized in this fraction. Using in vitro reconstitution, we showed that ~15-nm doughnut-shaped proteins in strings were nonstructural protein 2 (NSP2) bound to viral RNA transcripts. Moreover, using immunoaffinity-capture EM, we revealed that ~20-nm pebble-shaped complexes contain the viral RNA polymerase (VP1) and RNA capping enzyme (VP3). Finally, using a gel purification method, we demonstrated that ~30–70-nm electron-dense, particle-shaped complexes represent replicase-competent core RIs, containing VP1, VP3, and NSP2 as well as capsid proteins VP2 and VP6. The results of this study raise new questions about the interactions among viral proteins and RNA during the concerted assembly–replicase process. - Highlights: • Rotaviruses replicate their genomes in tandem with early virion assembly. • Little is known about rotavirus assembly-replication intermediates. • Assembly-replication intermediates were imaged using electron microscopy

Electron microscopic analysis of rotavirus assembly-replication intermediates

Energy Technology Data Exchange (ETDEWEB)

Boudreaux, Crystal E.; Kelly, Deborah F. [Virginia Tech Carilion School of Medicine and Research Institute, Roanoke, VA (United States); McDonald, Sarah M., E-mail: mcdonaldsa@vtc.vt.edu [Virginia Tech Carilion School of Medicine and Research Institute, Roanoke, VA (United States); Department of Biomedical Sciences and Pathobiology, Virginia—Maryland Regional College of Veterinary Medicine, Blacksburg, VA (United States)

2015-03-15

Rotaviruses (RVs) replicate their segmented, double-stranded RNA genomes in tandem with early virion assembly. In this study, we sought to gain insight into the ultrastructure of RV assembly-replication intermediates (RIs) using transmission electron microscopy (EM). Specifically, we examined a replicase-competent, subcellular fraction that contains all known RV RIs. Three never-before-seen complexes were visualized in this fraction. Using in vitro reconstitution, we showed that ~15-nm doughnut-shaped proteins in strings were nonstructural protein 2 (NSP2) bound to viral RNA transcripts. Moreover, using immunoaffinity-capture EM, we revealed that ~20-nm pebble-shaped complexes contain the viral RNA polymerase (VP1) and RNA capping enzyme (VP3). Finally, using a gel purification method, we demonstrated that ~30–70-nm electron-dense, particle-shaped complexes represent replicase-competent core RIs, containing VP1, VP3, and NSP2 as well as capsid proteins VP2 and VP6. The results of this study raise new questions about the interactions among viral proteins and RNA during the concerted assembly–replicase process. - Highlights: • Rotaviruses replicate their genomes in tandem with early virion assembly. • Little is known about rotavirus assembly-replication intermediates. • Assembly-replication intermediates were imaged using electron microscopy.

Managing Single-Stranded DNA during Replication Stress in Fission Yeast

Directory of Open Access Journals (Sweden)

Sarah A. Sabatinos

2015-09-01

Full Text Available Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

Replication termination and chromosome dimer resolution in the archaeon Sulfolobus solfataricus.

Science.gov (United States)

Duggin, Iain G; Dubarry, Nelly; Bell, Stephen D

2011-01-05

Archaea of the genus Sulfolobus have a single-circular chromosome with three replication origins. All three origins fire in every cell in every cell cycle. Thus, three pairs of replication forks converge and terminate in each replication cycle. Here, we report 2D gel analyses of the replication fork fusion zones located between origins. These indicate that replication termination involves stochastic fork collision. In bacteria, replication termination is linked to chromosome dimer resolution, a process that requires the XerC and D recombinases, FtsK and the chromosomal dif site. Sulfolobus encodes a single-Xer homologue and its deletion gave rise to cells with aberrant DNA contents and increased volumes. Identification of the chromosomal dif site that binds Xer in vivo, and biochemical characterization of Xer/dif recombination revealed that, in contrast to bacteria, dif is located outside the fork fusion zones. Therefore, it appears that replication termination and dimer resolution are temporally and spatially distinct processes in Sulfolobus.

Growth and replication of red rain cells at 121°C and their red fluorescence

Science.gov (United States)

Gangappa, Rajkumar; Wickramasinghe, Chandra; Wainwright, Milton; Kumar, A. Santhosh; Louis, Godfrey

2010-09-01

We have shown that the red cells found in the Red Rain (which fell on Kerala, India, in 2001) survive and grow after incubation for periods of up to two hours at 121°C . Under these conditions daughter cells appear within the original mother cells and the number of cells in the samples increases with length of exposure to 121°C. No such increase in cells occurs at room temperature, suggesting that the increase in daughter cells is brought about by exposure of the Red Rain cells to high temperatures. This is an independent confirmation of results reported earlier by two of the present authors, claiming that the cells can replicate under high pressure at temperatures upto 300°C. The flourescence behaviour of the red cells is shown to be in remarkable correspondence with the extended red emission observed in the Red Rectagle planetary nebula and other galactic and extragalactic dust clouds, suggesting, though not proving an extraterrestrial origin.

The replication of expansive production knowledge

DEFF Research Database (Denmark)

Wæhrens, Brian Vejrum; Yang, Cheng; Madsen, Erik Skov

2012-01-01

Purpose – With the aim to support offshore production line replication, this paper specifically aims to explore the use of templates and principles to transfer expansive productive knowledge embedded in a production line and understand the contingencies that influence the mix of these approaches......; and (2) rather than being viewed as alternative approaches, templates and principles should be seen as complementary once the transfer motive moves beyond pure replication. Research limitations – The concepts introduced in this paper were derived from two Danish cases. While acceptable for theory...

Evolution of Database Replication Technologies for WLCG

CERN Document Server

Baranowski, Zbigniew; Blaszczyk, Marcin; Dimitrov, Gancho; Canali, Luca

2015-01-01

In this article we summarize several years of experience on database replication technologies used at WLCG and we provide a short review of the available Oracle technologies and their key characteristics. One of the notable changes and improvement in this area in recent past has been the introduction of Oracle GoldenGate as a replacement of Oracle Streams. We report in this article on the preparation and later upgrades for remote replication done in collaboration with ATLAS and Tier 1 database administrators, including the experience from running Oracle GoldenGate in production. Moreover, we report on another key technology in this area: Oracle Active Data Guard which has been adopted in several of the mission critical use cases for database replication between online and offline databases for the LHC experiments.

Non-Equilibrium Thermodynamics of Self-Replicating Protocells

DEFF Research Database (Denmark)

Fellermann, Harold; Corominas-Murtra, Bernat; Hansen, Per Lyngs

2018-01-01

We provide a non-equilibrium thermodynamic description of the life-cycle of a droplet based, chemically feasible, system of protocells. By coupling the protocells metabolic kinetics with its thermodynamics, we demonstrate how the system can be driven out of equilibrium to ensure protocell growth...... and replication. This coupling allows us to derive the equations of evolution and to rigorously demonstrate how growth and replication life-cycle can be understood as a non-equilibrium thermodynamic cycle. The process does not appeal to genetic information or inheritance, and is based only on non......-equilibrium physics considerations. Our non-equilibrium thermodynamic description of simple, yet realistic, processes of protocell growth and replication, represents an advance in our physical understanding of a central biological phenomenon both in connection to the origin of life and for modern biology....

Class I ADP-ribosylation factors are involved in enterovirus 71 replication.

Directory of Open Access Journals (Sweden)

Jianmin Wang

Full Text Available Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies.

Class I ADP-ribosylation factors are involved in enterovirus 71 replication.

Science.gov (United States)

Wang, Jianmin; Du, Jiang; Jin, Qi

2014-01-01

Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies.

FBH1 Catalyzes Regression of Stalled Replication Forks

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Kasper Fugger

2015-03-01

Full Text Available DNA replication fork perturbation is a major challenge to the maintenance of genome integrity. It has been suggested that processing of stalled forks might involve fork regression, in which the fork reverses and the two nascent DNA strands anneal. Here, we show that FBH1 catalyzes regression of a model replication fork in vitro and promotes fork regression in vivo in response to replication perturbation. Cells respond to fork stalling by activating checkpoint responses requiring signaling through stress-activated protein kinases. Importantly, we show that FBH1, through its helicase activity, is required for early phosphorylation of ATM substrates such as CHK2 and CtIP as well as hyperphosphorylation of RPA. These phosphorylations occur prior to apparent DNA double-strand break formation. Furthermore, FBH1-dependent signaling promotes checkpoint control and preserves genome integrity. We propose a model whereby FBH1 promotes early checkpoint signaling by remodeling of stalled DNA replication forks.

Chromosomal Replication Complexity: A Novel DNA Metrics and Genome Instability Factor.

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Andrei Kuzminov

2016-10-01

Full Text Available As the ratio of the copy number of the most replicated to the unreplicated regions in the same chromosome, the definition of chromosomal replication complexity (CRC appears to leave little room for variation, being either two during S-phase or one otherwise. However, bacteria dividing faster than they replicate their chromosome spike CRC to four and even eight. A recent experimental inquiry about the limits of CRC in Escherichia coli revealed two major reasons to avoid elevating it further: (i increased chromosomal fragmentation and (ii complications with subsequent double-strand break repair. Remarkably, examples of stable elevated CRC in eukaryotic chromosomes are well known under various terms like "differential replication," "underreplication," "DNA puffs," "onion-skin replication," or "re-replication" and highlight the phenomenon of static replication fork (sRF. To accurately describe the resulting "amplification by overinitiation," I propose a new term: "replification" (subchromosomal overreplication. In both prokaryotes and eukaryotes, replification, via sRF processing, causes double-strand DNA breaks and, with their repair elevating chromosomal rearrangements, represents a novel genome instability factor. I suggest how static replication bubbles could be stabilized and speculate that some tandem duplications represent such persistent static bubbles. Moreover, I propose how static replication bubbles could be transformed into tandem duplications, double minutes, or inverted triplications. Possible experimental tests of these models are discussed.

Inhibition of spring viraemia of carp virus replication in an Epithelioma papulosum cyprini cell line by RNAi.

Science.gov (United States)

Gotesman, M; Soliman, H; Besch, R; El-Matbouli, M

2015-02-01

Spring viraemia of carp virus (SVCV) is an aetiological agent of a serious disease affecting carp farms in Europe and is a member of the Rhabdoviridae family of viruses. The genome of SVCV codes for five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). RNA-mediated interference (RNAi) by small interfering RNAs (siRNAs) is a powerful tool to inhibit gene transcription and is used to study genes important for viral replication. In previous studies regarding another member of Rhabdoviridae, siRNA inhibition of the rabies virus nucleoprotein gene provided in vitro and in vivo protection against rabies. In this study, synthetic siRNA molecules were designed to target SVCV-N and SVCV-P transcripts to inhibit SVCV replication and were tested in an epithelioma papulosum cyprini (EPC) cell line. Inhibition of gene transcription was measured by real-time quantitative reverse-transcription PCR (RT-qPCR). The efficacy of using siRNA for inhibition of viral replication was analysed by RT-qPCR measurement of a reporter gene (glycoprotein) expression and by virus endpoint titration. Inhibition of nucleoprotein and phosphoprotein gene expression by siRNA reduced SVCV replication. However, use of tandem siRNAs that target phosphoprotein and nucleoprotein worked best at reducing SVCV replication. © 2014 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.

Dynamic and nucleolin-dependent localization of human cytomegalovirus UL84 to the periphery of viral replication compartments and nucleoli.

Science.gov (United States)

Bender, Brian J; Coen, Donald M; Strang, Blair L

2014-10-01

proteins required for HCMV genome replication and replicating viral DNA. We found that UL84 localizes with viral proteins, viral DNA, and the cellular nucleolar protein nucleolin in the subnuclear replication compartments in which viral DNA replication occurs. Unexpectedly, we also found localization of UL84 with nucleolin in nucleoli and showed that the presence of nucleolin is involved in localization of UL84 to the nucleus. These results add to previous work showing the importance of nucleolin in replication compartment architecture and viral DNA synthesis and are relevant to understanding UL84 function. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Replicating Health Economic Models: Firm Foundations or a House of Cards?

Science.gov (United States)

Bermejo, Inigo; Tappenden, Paul; Youn, Ji-Hee

2017-11-01

Health economic evaluation is a framework for the comparative analysis of the incremental health gains and costs associated with competing decision alternatives. The process of developing health economic models is usually complex, financially expensive and time-consuming. For these reasons, model development is sometimes based on previous model-based analyses; this endeavour is usually referred to as model replication. Such model replication activity may involve the comprehensive reproduction of an existing model or 'borrowing' all or part of a previously developed model structure. Generally speaking, the replication of an existing model may require substantially less effort than developing a new de novo model by bypassing, or undertaking in only a perfunctory manner, certain aspects of model development such as the development of a complete conceptual model and/or comprehensive literature searching for model parameters. A further motivation for model replication may be to draw on the credibility or prestige of previous analyses that have been published and/or used to inform decision making. The acceptability and appropriateness of replicating models depends on the decision-making context: there exists a trade-off between the 'savings' afforded by model replication and the potential 'costs' associated with reduced model credibility due to the omission of certain stages of model development. This paper provides an overview of the different levels of, and motivations for, replicating health economic models, and discusses the advantages, disadvantages and caveats associated with this type of modelling activity. Irrespective of whether replicated models should be considered appropriate or not, complete replicability is generally accepted as a desirable property of health economic models, as reflected in critical appraisal checklists and good practice guidelines. To this end, the feasibility of comprehensive model replication is explored empirically across a small

A Transactional Asynchronous Replication Scheme for Mobile Database Systems

Institute of Scientific and Technical Information of China (English)

丁治明; 孟小峰; 王珊

2002-01-01

In mobile database systems, mobility of users has a significant impact on data replication. As a result, the various replica control protocols that exist today in traditional distributed and multidatabase environments are no longer suitable. To solve this problem, a new mobile database replication scheme, the Transaction-Level Result-Set Propagation (TLRSP)model, is put forward in this paper. The conflict detection and resolution strategy based on TLRSP is discussed in detail, and the implementation algorithm is proposed. In order to compare the performance of the TLRSP model with that of other mobile replication schemes, we have developed a detailed simulation model. Experimental results show that the TLRSP model provides an efficient support for replicated mobile database systems by reducing reprocessing overhead and maintaining database consistency.

Diversity of the DNA Replication System in the Archaea Domain

Directory of Open Access Journals (Sweden)

Felipe Sarmiento

2014-01-01

Full Text Available The precise and timely duplication of the genome is essential for cellular life. It is achieved by DNA replication, a complex process that is conserved among the three domains of life. Even though the cellular structure of archaea closely resembles that of bacteria, the information processing machinery of archaea is evolutionarily more closely related to the eukaryotic system, especially for the proteins involved in the DNA replication process. While the general DNA replication mechanism is conserved among the different domains of life, modifications in functionality and in some of the specialized replication proteins are observed. Indeed, Archaea possess specific features unique to this domain. Moreover, even though the general pattern of the replicative system is the same in all archaea, a great deal of variation exists between specific groups.

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.

Diverse Effects of Cyclosporine on Hepatitis C Virus Strain Replication

Science.gov (United States)

Ishii, Naoto; Watashi, Koichi; Hishiki, Takayuki; Goto, Kaku; Inoue, Daisuke; Hijikata, Makoto; Wakita, Takaji; Kato, Nobuyuki; Shimotohno, Kunitada

2006-01-01

Recently, a production system for infectious particles of hepatitis C virus (HCV) utilizing the genotype 2a JFH1 strain has been developed. This strain has a high capacity for replication in the cells. Cyclosporine (CsA) has a suppressive effect on HCV replication. In this report, we characterize the anti-HCV effect of CsA. We observe that the presence of viral structural proteins does not influence the anti-HCV activity of CsA. Among HCV strains, the replication of genotype 1b replicons was strongly suppressed by treatment with CsA. In contrast, JFH1 replication was less sensitive to CsA and its analog, NIM811. Replication of JFH1 did not require the cellular replication cofactor, cyclophilin B (CyPB). CyPB stimulated the RNA binding activity of NS5B in the genotype 1b replicon but not the genotype 2a JFH1 strain. These findings provide an insight into the mechanisms of diversity governing virus-cell interactions and in the sensitivity of these strains to antiviral agents. PMID:16611911

Complex Dynamic Development of Poliovirus Membranous Replication Complexes

Science.gov (United States)

Nair, Vinod; Hansen, Bryan T.; Hoyt, Forrest H.; Fischer, Elizabeth R.; Ehrenfeld, Ellie

2012-01-01

Replication of all positive-strand RNA viruses is intimately associated with membranes. Here we utilize electron tomography and other methods to investigate the remodeling of membranes in poliovirus-infected cells. We found that the viral replication structures previously described as “vesicles” are in fact convoluted, branching chambers with complex and dynamic morphology. They are likely to originate from cis-Golgi membranes and are represented during the early stages of infection by single-walled connecting and branching tubular compartments. These early viral organelles gradually transform into double-membrane structures by extension of membranous walls and/or collapsing of the luminal cavity of the single-membrane structures. As the double-membrane regions develop, they enclose cytoplasmic material. At this stage, a continuous membranous structure may have double- and single-walled membrane morphology at adjacent cross-sections. In the late stages of the replication cycle, the structures are represented mostly by double-membrane vesicles. Viral replication proteins, double-stranded RNA species, and actively replicating RNA are associated with both double- and single-membrane structures. However, the exponential phase of viral RNA synthesis occurs when single-membrane formations are predominant in the cell. It has been shown previously that replication complexes of some other positive-strand RNA viruses form on membrane invaginations, which result from negative membrane curvature. Our data show that the remodeling of cellular membranes in poliovirus-infected cells produces structures with positive curvature of membranes. Thus, it is likely that there is a fundamental divergence in the requirements for the supporting cellular membrane-shaping machinery among different groups of positive-strand RNA viruses. PMID:22072780

Repair of DNA in replicated and unreplicated portions of the human genome

International Nuclear Information System (INIS)

Waters, R.

1979-01-01

Portions of the human genome that have replicated after ultraviolet light irradiation and those that remain unreplicated have both been examined for the distribution of pyrimidine dimers and the extent of repair replication following their removal. The data indicate that the number of unrepaired dimers and the extent of repair replication seen after their excision are equal in the replicated and unreplicated DNA. Furthermore, the daughter strand of replicated DNA is larger than the average interdimer distance found in the parental strand. Hence, DNA replication in normal human fibroblasts is clearly capable of getting past pyrimidine dimers, and a preferential repair of such lesions in DNA that is about to be or has been replicated does not operate to any visible extent in these cells. (author)

Interferon lambda inhibits dengue virus replication in epithelial cells.

Science.gov (United States)

Palma-Ocampo, Helen K; Flores-Alonso, Juan C; Vallejo-Ruiz, Verónica; Reyes-Leyva, Julio; Flores-Mendoza, Lilian; Herrera-Camacho, Irma; Rosas-Murrieta, Nora H; Santos-López, Gerardo

2015-09-28

In viral disease, infection is controlled at the cellular level by type I interferon (IFN-I), but dengue virus (DENV) has the ability to inhibit this response. Type III interferon, also known as lambda IFN (IFN-III or IFN-λ), is a complementary pathway to the antiviral response by IFN-I. This work analyzed the IFN-λ (IFN-III) mediated antiviral response against DENV serotype 2 (DENV-2) infection. Dengue fever patients were sampled to determine their IFN-λ levels by ELISA. To study the IFN-λ response during DENV infection we selected the epithelial cell line C33-A, and we demonstrated that it is permissive to DENV-2 infection. The effect of IFN-λ on virus replication was determined in these cells, in parallel to the expression of IFN-stimulated genes (ISGs), and Suppressor of Cytokine Signaling (SOCS), genes measured by RT-qPCR. We found increased (~1.8 times) serological IFN-λ in dengue fever patients compared to healthy blood donors. IFN-λ inhibited DENV-2 replication in a dose-dependent manner in vitro. The reduction of viral titer corresponded with increased ISG mRNA levels (MX1 and OAS1), with the highest inhibition occurring at ISG's peak expression. Presence of IFN-negative regulators, SOCS1 and SOCS3, during DENV-2 infection was associated with reduced IFN-λ1 expression. Evidence described here suggests that IFN-λ is a good candidate inhibitor of viral replication in dengue infection. Mechanisms for the cellular and organismal interplay between DENV and IFN- λ need to be further studied as they could provide insights into strategies to treat this disease. Furthermore, we report a novel epithelial model to study dengue infection in vitro.

Replication studies in longevity

DEFF Research Database (Denmark)

Varcasia, O; Garasto, S; Rizza, T

2001-01-01

In Danes we replicated the 3'APOB-VNTR gene/longevity association study previously carried out in Italians, by which the Small alleles (less than 35 repeats) had been identified as frailty alleles for longevity. In Danes, neither genotype nor allele frequencies differed between centenarians and 20...

Non-Replicating Adenovirus-Vectored Anthrax Vaccine

International Nuclear Information System (INIS)

Van Kampen, K. R.; Zhang, J.; Jex, E.; Tang, D. C.

2007-01-01

As bioterrorism is emerging as a national threat, it is urgent to develop a new generation of anthrax vaccines that can be rapidly produced and mass administered in an emergency setting. We have demonstrated that protective immunity against anthrax spores could be elicited in mice by intranasal administration of a non-replicating human adenovirus serotype 5 (Ad5)-derived vector encoding Bacillus anthracis protective antigen (PA) in a single-dose regimen. The potency of an Ad5 vector encoding PA was remarkably enhanced by codon optimization of the PA gene to match the tRNA pool found in human cells. This nasal vaccine can be mass-administered by non-medical personnel during a bioterrorist attack. In addition, replication-competent adenovirus (RCA)-free Ad5-vectored anthrax vaccines can be mass produced in PER.C6 cells in serum-free wave bioreactors and purified by column chromatography to meet a surge in demand. The non-replicating nature of this new generation of anthrax vaccine ensures an excellent safety profile for vaccines and the environment.(author)

The Anisotropy of Replicated Aluminum Foams

Directory of Open Access Journals (Sweden)

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.

Origin Licensing Requires ATP Binding and Hydrolysis by the MCM Replicative Helicase

Science.gov (United States)

Coster, Gideon; Frigola, Jordi; Beuron, Fabienne; Morris, Edward P.; Diffley, John F.X.

2014-01-01

Summary Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs. PMID:25087873

Chromatin replication and histone dynamics

DEFF Research Database (Denmark)

Alabert, Constance; Jasencakova, Zuzana; Groth, Anja

2017-01-01

Inheritance of the DNA sequence and its proper organization into chromatin is fundamental for genome stability and function. Therefore, how specific chromatin structures are restored on newly synthesized DNA and transmitted through cell division remains a central question to understand cell fate...... choices and self-renewal. Propagation of genetic information and chromatin-based information in cycling cells entails genome-wide disruption and restoration of chromatin, coupled with faithful replication of DNA. In this chapter, we describe how cells duplicate the genome while maintaining its proper...... organization into chromatin. We reveal how specialized replication-coupled mechanisms rapidly assemble newly synthesized DNA into nucleosomes, while the complete restoration of chromatin organization including histone marks is a continuous process taking place throughout the cell cycle. Because failure...

Association of RANTES with the replication of severe acute respiratory syndrome coronavirus in THP-1 cells.

Science.gov (United States)

Li, D; Wu, N; Yao, H; Bader, A; Brockmeyer, Norbert H; Altmeyer, P

2005-03-29

Severe acute respiratory syndrome (SARS) is a novel infectious disease which is characterized by an overaggressive immune response. Chemokines are important inflammatory mediators and regulate disease due to viral infection. In previous study, we found that SARS-CoV has the ability to replicate in mononuclear cells. In present work, we sought to characterize the replication of SARS-CoV at the presence of RANTES in THP-1 cells. To determine whether RANTES play an role in the process of SARS, THP-1 cells were incubated with heat-inactivated SARS-CoV and ELISA was used to test RANTES levels in the supernatants; Then the effect of dexamethasone on the induced secretion was evaluated. Real-time PCR was used to investigate the effort of RANTES on the replication of SARS-CoV in vitro. Macrophages, induced by THP-1 cells, were used as cell model. Inactive SARS-CoV could induce THP-1 cells secret RANTES and this increase effect could not be suppressed by DXM. RANTES itself could inhibit the replication of SARS-CoV in THP-1 cells when it was added into the culture before or at the same time with the virus; No inhibition effect was shown when RANTES were added into the culture after SARS-CoV infected the cells.

Electrochemically replicated smooth aluminum foils for anodic alumina nanochannel arrays

International Nuclear Information System (INIS)

Biring, Sajal; Tsai, K-T; Sur, Ujjal Kumar; Wang, Y-L

2008-01-01

A fast electrochemical replication technique has been developed to fabricate large-scale ultra-smooth aluminum foils by exploiting readily available large-scale smooth silicon wafers as the masters. Since the adhesion of aluminum on silicon depends on the time of surface pretreatment in water, it is possible to either detach the replicated aluminum from the silicon master without damaging the replicated aluminum and master or integrate the aluminum film to the silicon substrate. Replicated ultra-smooth aluminum foils are used for the growth of both self-organized and lithographically guided long-range ordered arrays of anodic alumina nanochannels without any polishing pretreatment

Suppression of Coronavirus Replication by Cyclophilin Inhibitors

Directory of Open Access Journals (Sweden)

Takashi Sasaki

2013-05-01

Full Text Available Coronaviruses infect a variety of mammalian and avian species and cause serious diseases in humans, cats, mice, and birds in the form of severe acute respiratory syndrome (SARS, feline infectious peritonitis (FIP, mouse hepatitis, and avian infectious bronchitis, respectively. No effective vaccine or treatment has been developed for SARS-coronavirus or FIP virus, both of which cause lethal diseases. It has been reported that a cyclophilin inhibitor, cyclosporin A (CsA, could inhibit the replication of coronaviruses. CsA is a well-known immunosuppressive drug that binds to cellular cyclophilins to inhibit calcineurin, a calcium-calmodulin-activated serine/threonine-specific phosphatase. The inhibition of calcineurin blocks the translocation of nuclear factor of activated T cells from the cytosol into the nucleus, thus preventing the transcription of genes encoding cytokines such as interleukin-2. Cyclophilins are peptidyl-prolyl isomerases with physiological functions that have been described for many years to include chaperone and foldase activities. Also, many viruses require cyclophilins for replication; these include human immunodeficiency virus, vesicular stomatitis virus, and hepatitis C virus. However, the molecular mechanisms leading to the suppression of viral replication differ for different viruses. This review describes the suppressive effects of CsA on coronavirus replication.

Reduction of Glucose Metabolism in Olfactory Bulb is an Earlier Alzheimer's Disease-related Biomarker in 5XFAD Mice

Directory of Open Access Journals (Sweden)

Nai-An Xiao

2015-01-01

Conclusions: The decline of (18F-FDG uptake in the olfactory bulb occurs earlier than other incidents, serving as an earlier in vivo biological marker of AD in 5XFAD mice and making early diagnosis of AD possibly.

Cell lethality after selective irradiation of the DNA replication fork

International Nuclear Information System (INIS)

Hofer, K.G.; Warters, R.L.

1985-01-01

It has been suggested that nascent DNA located at the DNA replication fork may exhibit enhanced sensitivity to radiation damage. To evaluate this hypothesis, Chinese hamster ovary cells (CHO) were labeled with 125 I-iododeoxyuridine ( 125 IUdR) either in the presence or absence of aphidicolin. Aphidicolin (5 μg/ml) reduced cellular 125 IUdR incorporation to 3-5% of the control value. The residual 125 I incorporation appeared to be restricted to low molecular weight (sub-replicon sized) fragments of DNA which were more sensitive to micrococcal nuclease attack and less sensitive to high salt DNase I digestion than randomly labeled DNA. These findings suggest that DNA replicated in the presence of aphidicolin remains localized at the replication fork adjacent to the nuclear matrix. Based on these observations an attempt was made to compare the lethal consequences of 125 I decays at the replication fork to that of 125 I decays randomly distributed over the entire genome. Regardless of the distribution of decay events, all treatment groups exhibited identical dose-response curves (D 0 : 101 125 I decays/cell). Since differential irradiation of the replication complex did not result in enhanced cell lethality, it can be concluded that neither the nascent DNA nor the protein components (replicative enzymes, nuclear protein matrix) associated with the DNA replication site constitute key radiosensitive targets within the cellular genome. (orig.)

An Inadvertent Concurrent Replication: Same Roadmap, Different Journey

Science.gov (United States)

Lemons, Christopher J.; King, Seth A.; Davidson, Kimberly A.; Berryessa, Teresa L.; Gajjar, Shimul A.; Sacks, Lia H.

2016-01-01

Replication is a critical aspect of scientific inquiry that presents a variety of challenges to researchers, even under the best of conditions. We conducted a review of replication rates in special education journals similar to the review conducted by Makel et al. in this issue. Unknowingly conducting independent reviews allowed for an unexpected…

Perfectionism in Gifted Adolescents: A Replication and Extension

Science.gov (United States)

Margot, Kelly C.; Rinn, Anne N.

2016-01-01

To provide further generalizability for the results garnered by two previous studies, the authors conducted a methodological replication. In addition to adding to the body of replication research done with gifted students, the purpose of this study was to examine perfectionism differences among gifted adolescents in regards to gender, birth order,…

The adoption of sustainable innovations: The role of instrumental, environmental, and symbolic attributes for earlier and later adopters

NARCIS (Netherlands)

H. Noppers, E.; Keizer, K.; Bockarjova, M.; Steg, L.

2015-01-01

We investigated motivations of potential earlier and later adopters for adopting sustainable innovations. A large questionnaire study revealed that potential earlier adopters of innovative cars evaluated the symbolic attributes of electric cars, but not the instrumental and environmental attributes,

Darwinian Evolution of Mutualistic RNA Replicators with Different Genes

Science.gov (United States)

Mizuuchi, R.; Ichihashi, N.

2017-07-01

We report a sustainable long-term replication and evolution of two distinct cooperative RNA replicators encoding different genes. One of the RNAs evolved to maintain or increase the cooperativity, despite selective advantage of selfish mutations.

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

High-affinity DNA-binding Domains of Replication Protein A (RPA) Direct SMARCAL1-dependent Replication Fork Remodeling*

Science.gov (United States)

Bhat, Kamakoti P.; Bétous, Rémy; Cortez, David

2015-01-01

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. PMID:25552480

High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.

Science.gov (United States)

Bhat, Kamakoti P; Bétous, Rémy; Cortez, David

2015-02-13

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Replication-mediated disassociation of replication protein A-XPA complex upon DNA damage: implications for RPA handing off.

Science.gov (United States)

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2012-08-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.

Replication-mediated disassociation of replication protein A–XPA complex upon DNA damage: implications for RPA handing off

Science.gov (United States)

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2013-01-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA–XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA–XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA–XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed. PMID:22578086

Signal replication in a DNA nanostructure

Science.gov (United States)

Mendoza, Oscar; Houmadi, Said; Aimé, Jean-Pierre; Elezgaray, Juan

2017-01-01

Logic circuits based on DNA strand displacement reaction are the basic building blocks of future nanorobotic systems. The circuits tethered to DNA origami platforms present several advantages over solution-phase versions where couplings are always diffusion-limited. Here we consider a possible implementation of one of the basic operations needed in the design of these circuits, namely, signal replication. We show that with an appropriate preparation of the initial state, signal replication performs in a reproducible way. We also show the existence of side effects concomitant to the high effective concentrations in tethered circuits, such as slow leaky reactions and cross-activation.

In vitro replication of poliovirus

International Nuclear Information System (INIS)

Lubinski, J.M.

1986-01-01

Poliovirus is a member of the Picornaviridae whose genome is a single stranded RNA molecule of positive polarity surrounded by a proteinaceous capsid. Replication of poliovirus occurs via negative strand intermediates in infected cells using a virally encoded RNA-dependent RNA polymerase and host cell proteins. The authors have exploited the fact that complete cDNA copies of the viral genome when transfected onto susceptible cells generate virus. Utilizing the bacteriophage SP6 DNA dependent RNA polymerase system to synthesize negative strands in vitro and using these in an in vitro reaction the authors have generated full length infectious plus strands. Mutagenesis of the 5' and 3' ends of the negative and positive strands demonstrated that replication could occur either de novo or be extensions of the templates from their 3' ends or from nicks occurring during replication. The appearance of dimeric RNA molecules generated in these reactions was not dependent upon the same protein required for de novo initiation. Full length dimeric RNA molecules using a 5' 32 P end-labelled oligo uridylic acid primer and positive strand template were demonstrated in vitro containing only the 35,000 Mr host protein and the viral RNA-dependent RNA polymerase. A model for generating positive strands without protein priming by cleavage of dimeric RNA molecules was proposed

Replication of Merkel cell polyomavirus induces reorganization of promyelocytic leukemia nuclear bodies.

Science.gov (United States)

Neumann, Friederike; Czech-Sioli, Manja; Dobner, Thomas; Grundhoff, Adam; Schreiner, Sabrina; Fischer, Nicole

2016-11-01

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma (MCC), a rare but aggressive skin cancer. The virus is highly prevalent: 60-80 % of adults are seropositive; however, cells permissive for MCPyV infection are unknown. Consequently, very little information about the MCPyV life cycle is available. Until recently, MCPyV replication could only be studied using a semi-permissive in vitro replication system (Neumann et al., 2011; Feng et al., 2011, Schowalter et al., 2011). MCPyV replication most likely depends on subnuclear structures such as promyelocytic leukemia protein nuclear bodies (PML-NBs), which are known to play regulatory roles in the infection of many DNA viruses. Here, we investigated PML-NB components as candidate host factors to control MCPyV DNA replication. We showed that PML-NBs change in number and size in cells actively replicating MCPyV proviral DNA. We observed a significant increase in PML-NBs in cells positive for MCPyV viral DNA replication. Interestingly, a significant amount of cells actively replicating MCPyV did not show any Sp100 expression. While PML and Daxx had no effect on MCPyV DNA replication, MCPyV replication was increased in cells depleted for Sp100, strongly suggesting that Sp100 is a negative regulator of MCPyV DNA replication.

Initiation of DNA replication: functional and evolutionary aspects

Science.gov (United States)

Bryant, John A.; Aves, Stephen J.

2011-01-01

Background The initiation of DNA replication is a very important and highly regulated step in the cell division cycle. It is of interest to compare different groups of eukaryotic organisms (a) to identify the essential molecular events that occur in all eukaryotes, (b) to start to identify higher-level regulatory mechanisms that are specific to particular groups and (c) to gain insights into the evolution of initiation mechanisms. Scope This review features a wide-ranging literature survey covering replication origins, origin recognition and usage, modification of origin usage (especially in response to plant hormones), assembly of the pre-replication complex, loading of the replisome, genomics, and the likely origin of these mechanisms and proteins in Archaea. Conclusions In all eukaryotes, chromatin is organized for DNA replication as multiple replicons. In each replicon, replication is initiated at an origin. With the exception of those in budding yeast, replication origins, including the only one to be isolated so far from a plant, do not appear to embody a specific sequence; rather, they are AT-rich, with short tracts of locally bent DNA. The proteins involved in initiation are remarkably similar across the range of eukaryotes. Nevertheless, their activity may be modified by plant-specific mechanisms, including regulation by plant hormones. The molecular features of initiation are seen in a much simpler form in the Archaea. In particular, where eukaryotes possess a number of closely related proteins that form ‘hetero-complexes’ (such as the origin recognition complex and the MCM complex), archaeans typically possess one type of protein (e.g. one MCM) that forms a homo-complex. This suggests that several eukaryotic initiation proteins have evolved from archaeal ancestors by gene duplication and divergence. PMID:21508040

Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus

DEFF Research Database (Denmark)

Jensen, Rasmus Bugge

2006-01-01

Progression through the Caulobacter crescentus cell cycle is coupled to a cellular differentiation program. The swarmer cell is replicationally quiescent, and DNA replication initiates at the swarmer-to-stalked cell transition. There is a very short delay between initiation of DNA replication...

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

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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

Two subunits of human ORC are dispensable for DNA replication and proliferation.

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Shibata, Etsuko; Kiran, Manjari; Shibata, Yoshiyuki; Singh, Samarendra; Kiran, Shashi; Dutta, Anindya

2016-12-01

The six-subunit Origin Recognition Complex (ORC) is believed to be an essential eukaryotic ATPase that binds to origins of replication as a ring-shaped heterohexamer to load MCM2-7 and initiate DNA replication. We have discovered that human cell lines in culture proliferate with intact chromosomal origins of replication after disruption of both alleles of ORC2 or of the ATPase subunit, ORC1 . The ORC1 or ORC2 -depleted cells replicate with decreased chromatin loading of MCM2-7 and become critically dependent on another ATPase, CDC6, for survival and DNA replication. Thus, either the ORC ring lacking a subunit, even its ATPase subunit, can load enough MCM2-7 in partnership with CDC6 to initiate DNA replication, or cells have an ORC-independent, CDC6-dependent mechanism to load MCM2-7 on origins of replication.

USP7 is a SUMO deubiquitinase essential for DNA replication

DEFF Research Database (Denmark)

Lecona, Emilio; Rodriguez-Acebes, Sara; Specks, Julia

2016-01-01

Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates DNA replication. We have previously shown that chromatin around replisomes is rich in SUMO and poor in Ub, whereas mature chromatin exhibits an opposite pattern. How this SUMO-rich, Ub-poor environment...... is maintained at sites of DNA replication in mammalian cells remains unexplored. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Inhibition or genetic deletion of USP7 leads...... to the accumulation of Ub on SUMOylated proteins, which are displaced away from replisomes. Our findings provide a model explaining the differential accumulation of SUMO and Ub at replication forks and identify an essential role of USP7 in DNA replication that should be considered in the development of USP7...

Maintaining replication origins in the face of genomic change.

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Di Rienzi, Sara C; Lindstrom, Kimberly C; Mann, Tobias; Noble, William S; Raghuraman, M K; Brewer, Bonita J

2012-10-01

Origins of replication present a paradox to evolutionary biologists. As a collection, they are absolutely essential genomic features, but individually are highly redundant and nonessential. It is therefore difficult to predict to what extent and in what regard origins are conserved over evolutionary time. Here, through a comparative genomic analysis of replication origins and chromosomal replication patterns in the budding yeasts Saccharomyces cerevisiae and Lachancea waltii, we assess to what extent replication origins survived genomic change produced from 150 million years of evolution. We find that L. waltii origins exhibit a core consensus sequence and nucleosome occupancy pattern highly similar to those of S. cerevisiae origins. We further observe that the overall progression of chromosomal replication is similar between L. waltii and S. cerevisiae. Nevertheless, few origins show evidence of being conserved in location between the two species. Among the conserved origins are those surrounding centromeres and adjacent to histone genes, suggesting that proximity to an origin may be important for their regulation. We conclude that, over evolutionary time, origins maintain sequence, structure, and regulation, but are continually being created and destroyed, with the result that their locations are generally not conserved.

Compulsive Buying: Earlier Illicit Drug Use, Impulse Buying, Depression, and Adult ADHD Symptoms

Science.gov (United States)

Brook, Judith S.; Zhang, Chenshu; Brook, David W.; Leukefeld, Carl G.

2015-01-01

This longitudinal study examined the association between psychosocial antecedents, including illicit drug use, and adult compulsive buying (CB) across a 29-year time period from mean age 14 to mean age 43. Participants originally came from a community-based random sample of residents in two upstate New York counties. Multivariate linear regression analysis was used to study the relationship between the participant’s earlier psychosocial antecedents and adult CB in the fifth decade of life. The results of the multivariate linear regression analyses showed that gender (female), earlier adult impulse buying (IB), depressive mood, illicit drug use, and concurrent ADHD symptoms were all significantly associated with adult CB at mean age 43. It is important that clinicians treating CB in adults should consider the role of drug use, symptoms of ADHD, IB, depression, and family factors in CB. PMID:26165963

Intracellular Detection of Viral Transcription and Replication Using RNA FISH

Science.gov (United States)

2016-05-26

Chapter 14. Intracellular detection of viral transcription and replication using RNA FISH i. Summary/Abstract Many hemorrhagic fever viruses...only allow entirely new investigations into the replication of these viruses, but also how this method can be applied to any virus with a known...localization, TurboFISH, hemorrhagic fever virus replication 1. Introduction RNA FISH was developed as a method to visualize cellular RNA by binding a

The Legal Road To Replicating Silicon Valley

OpenAIRE

John Armour; Douglas Cumming

2004-01-01

Must policymakers seeking to replicate the success of Silicon Valley’s venture capital market first replicate other US institutions, such as deep and liquid stock markets? Or can legal reforms alone make a significant difference? In this paper, we compare the economic and legal determinants of venture capital investment, fundraising and exits. We introduce a cross-sectional and time series empirical analysis across 15 countries and 13 years of data spanning an entire business cycle. We show t...

Feline coronavirus replication is affected by both cyclophilin A and cyclophilin B.

Science.gov (United States)

Tanaka, Yoshikazu; Sato, Yuka; Sasaki, Takashi

2017-02-01

Feline coronavirus (FCoV) causes the fatal disease feline infectious peritonitis, which is currently incurable by drug treatment, and no effective vaccines are available. Cyclosporin A (CsA), a cyclophilin (Cyp) inhibitor, inhibits the replication of FCoV in vitro and in vivo as well as the replication of human and animal coronaviruses. However, the mechanism underlying the regulation of coronavirus replication by CsA is unknown. In this study, we analysed the role of Cyps in FCoV replication using knockdown and knockout cells specific to Cyps. Inhibition of CypA and CypB reduced FCoV replication, with replication in knockout cells being much less than that in knockdown cells. Furthermore, the proteins expressed by CypA and CypB harbouring mutations in their respective predicted peptidyl-prolyl cis-transisomerase active sites, which also alter the affinities between Cyps and CsA, inhibited FCoV replication. These findings indicate that the peptidyl-prolyl cis-transisomerase active sites of Cyps might be required for FCoV replication.

Replication of innovation in professional service firms: options for leveraging knowledge

NARCIS (Netherlands)

Tuominen, T.; van der Aa, W.

2013-01-01

In this conceptual paper we elaborate on the replication of innovation in professional service firms (PSFs). We do this by integrating literature streams on replication, knowledge sharing, productization of services and management of PSF’s. First we discuss the object of replication in order to

An oncolytic adenovirus enhances antiangiogenic and antitumoral effects of a replication-deficient adenovirus encoding endostatin by rescuing its selective replication in nasopharyngeal carcinoma cells

International Nuclear Information System (INIS)

Liu, Ran-yi; Zhou, Ling; Zhang, Yan-ling; Huang, Bi-jun; Ke, Miao-la; Chen, Jie-min; Li, Li-xia; Fu, Xiang; Wu, Jiang-xue; Huang, Wenlin

2013-01-01

Highlights: •H101 promotes endostatin expression by Ad-Endo via rescuing Ad-Endo replication. •H101 rescued Ad-Endo replication by supplying E1A and E1B19k proteins. •Ad-Endo enhanced the cytotoxicity of H101 in NPC cells. •Ad-Endo and oncolytic Ad H101 have synergistic antitumor effects on NPC. -- Abstract: A replication-deficient adenovirus (Ad) encoding secreted human endostatin (Ad-Endo) has been demonstrated to have promising antiangiogenic and antitumoral effects. The E1B55k-deleted Ad H101 can selectively lyse cancer cells. In this study, we explored the antitumor effects and cross-interactions of Ad-Endo and H101 on nasopharyngeal carcinoma (NPC). The results showed that H101 dramatically promoted endostatin expression by Ad-Endo via rescuing Ad-Endo replication in NPC cells, and the expressed endostatin proteins significantly inhibited the proliferation of human umbilical vein endothelial cells. E1A and E1B19k products are required for the rescuing of H101 to Ad-Endo replication in CNE-1 and CNE-2 cells, but not in C666-1 cells. On the other hand, Ad-Endo enhanced the cytotoxicity of H101 by enhancing Ad replication in NPC cells. The combination of H101 and Ad-Endo significantly inhibited CNE-2 xenografts growth through the increased endostatin expression and Ad replication. These findings indicate that the combination of Ad-Endo gene therapy and oncolytic Ad therapeutics could be promising in comprehensive treatment of NPC

An oncolytic adenovirus enhances antiangiogenic and antitumoral effects of a replication-deficient adenovirus encoding endostatin by rescuing its selective replication in nasopharyngeal carcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Liu, Ran-yi, E-mail: liuranyi@mail.sysu.edu.cn [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Zhou, Ling [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Zhang, Yan-ling [School of Biotechnology, Southern Medical University, Guangzhou 510515 (China); Huang, Bi-jun; Ke, Miao-la; Chen, Jie-min [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Li, Li-xia [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); General Hospital of Guangzhou Military Command of PLA, Guangzhou 510010 (China); Fu, Xiang; Wu, Jiang-xue [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Huang, Wenlin, E-mail: hwenl@mail.sysu.edu.cn [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Guangdong Provincial Key Laboratory of Tumor-Targeted Drug, Doublle Bioproducts Inc., Guangzhou 510663 (China)

2013-12-13

Highlights: •H101 promotes endostatin expression by Ad-Endo via rescuing Ad-Endo replication. •H101 rescued Ad-Endo replication by supplying E1A and E1B19k proteins. •Ad-Endo enhanced the cytotoxicity of H101 in NPC cells. •Ad-Endo and oncolytic Ad H101 have synergistic antitumor effects on NPC. -- Abstract: A replication-deficient adenovirus (Ad) encoding secreted human endostatin (Ad-Endo) has been demonstrated to have promising antiangiogenic and antitumoral effects. The E1B55k-deleted Ad H101 can selectively lyse cancer cells. In this study, we explored the antitumor effects and cross-interactions of Ad-Endo and H101 on nasopharyngeal carcinoma (NPC). The results showed that H101 dramatically promoted endostatin expression by Ad-Endo via rescuing Ad-Endo replication in NPC cells, and the expressed endostatin proteins significantly inhibited the proliferation of human umbilical vein endothelial cells. E1A and E1B19k products are required for the rescuing of H101 to Ad-Endo replication in CNE-1 and CNE-2 cells, but not in C666-1 cells. On the other hand, Ad-Endo enhanced the cytotoxicity of H101 by enhancing Ad replication in NPC cells. The combination of H101 and Ad-Endo significantly inhibited CNE-2 xenografts growth through the increased endostatin expression and Ad replication. These findings indicate that the combination of Ad-Endo gene therapy and oncolytic Ad therapeutics could be promising in comprehensive treatment of NPC.

Focus Article: Replication in Second Language Writing Research

Science.gov (United States)

Porte, Graeme; Richards, Keith

2012-01-01

This paper discusses the meaning and range of replication in L2 research from both quantitative and qualitative perspectives. In the first half of the paper, it will be argued that key quantitative studies need to be replicated to have their robustness and generalizability tested and that this is a requirement of scientific inquiry. Such research…

The Replication Mechanism in a Romanian ERP System Environment

Directory of Open Access Journals (Sweden)

Iuliana SCORTA

2008-01-01

Full Text Available Modern Relational Database Management Systems have Replication technology. Large enterprises are often spread around the country, and although WANs can be very fast and reliable these days, it is often better for each location to have a local copy of data rather than have a single database at a central location. This usually means that replication is a requirement in order for each location to have the most up-to-date data. This paper reveals a replication mechanism implemented in a Romanian ERP system environment.

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.

Science.gov (United States)

Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

2016-06-01

Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.

Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.

Science.gov (United States)

Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F Peter; Zhang, Huidong

2016-01-01

Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, Escherichia coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection.

Science.gov (United States)

Cui, Hongguang; Wang, Aiming

2016-05-15

The potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein and induces the endoplasmic reticulum (ER)-originated replication vesicles that target the chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a Canadian Plum pox virus (PPV) isolate. We found that deletion of any of the short motifs of 6K1 (each of which ranged from 5 to 13 amino acids), most of the 6K1 sequence (but with the conserved sequence of the cleavage sites being retained), or all of the 6K1 sequence in the PPV infectious clone abolished viral replication. The trans expression of 6K1 or the cis expression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 for viral replication. Disruption of the N- or C-terminal cleavage site of 6K1, which prevented the release of 6K1 from the polyprotein, either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that green fluorescent protein-tagged 6K1 formed punctate inclusions at the viral early infection stage and colocalized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication and is an important viral element of the viral replication complex at the early infection stage. Potyviruses account for more than 30% of known plant viruses and consist of many agriculturally important viruses. The genomes of potyviruses encode two polyproteins that are proteolytically processed into 11 mature

New Methods to Address Old Challenges: The Use of Administrative Data for Longitudinal Replication Studies of Child Maltreatment.

Science.gov (United States)

Hurren, Emily; Stewart, Anna; Dennison, Susan

2017-09-15

Administrative data are crucial to the "big data" revolution of social science and have played an important role in the development of child maltreatment research. These data are also of value to administrators, policy makers, and clinicians. The focus of this paper is the use of administrative data to produce and replicate longitudinal studies of child maltreatment. Child protection administrative data have several advantages. They are often population-based, and allow longitudinal examination of child maltreatment and complex multi-level analyses. They also allow comparison across subgroups and minority groups, remove burden from individuals to disclose traumatic experiences, and can be less biased than retrospective recall. Finally, they can be linked to data from other agencies to explore comorbidity and outcomes, and are comparatively cost and time effective. The benefits and challenges associated with the use of administrative data for longitudinal child maltreatment research become magnified when these data are used to produce replications. Techniques to address challenges and support future replication efforts include developing a biographical understanding of the systems from which the data are drawn, using multiple data sources to contextualize the data and research results, recognizing and adopting various approaches to replication, and documenting all data coding and manipulation processes. These techniques are illustrated in this paper via a case study of previous replication work.