Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

Directory of Open Access Journals (Sweden)

Natascha Krömmelbein

2016-02-01

Full Text Available The human cytomegalovirus (HCMV replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production.

A Role of hIPI3 in DNA Replication Licensing in Human Cells.

Science.gov (United States)

Huang, Yining; Amin, Aftab; Qin, Yan; Wang, Ziyi; Jiang, Huadong; Liang, Lu; Shi, Linjing; Liang, Chun

2016-01-01

The yeast Ipi3p is required for DNA replication and cell viability in Sacharomyces cerevisiae. It is an essential component of the Rix1 complex (Rix1p/Ipi2p-Ipi1p-Ipi3p) that is required for the processing of 35S pre-rRNA in pre-60S ribosomal particles and for the initiation of DNA replication. The human IPI3 homolog is WDR18 (WD repeat domain 18), which shares significant homology with yIpi3p. Here we report that knockdown of hIPI3 resulted in substantial defects in the chromatin association of the MCM complex, DNA replication, cell cycle progression and cell proliferation. Importantly, hIPI3 silencing did not result in a reduction of the protein level of hCDC6, hMCM7, or the ectopically expressed GFP protein, indicating that protein synthesis was not defective in the same time frame of the DNA replication and cell cycle defects. Furthermore, the mRNA and protein levels of hIPI3 fluctuate in the cell cycle, with the highest levels from M phase to early G1 phase, similar to other pre-replicative (pre-RC) proteins. Moreover, hIPI3 interacts with other replication-initiation proteins, co-localizes with hMCM7 in the nucleus, and is important for the nuclear localization of hMCM7. We also found that hIPI3 preferentially binds to the origins of DNA replication including those at the c-Myc, Lamin-B2 and β-Globin loci. These results indicate that hIPI3 is involved in human DNA replication licensing independent of its role in ribosome biogenesis.

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

Radiation-induced inhibition of human endothelial cells replicating in culture

International Nuclear Information System (INIS)

DeGowin, R.L.; Lewis, L.J.; Mason, R.E.; Borke, M.K.; Hoak, J.C.

1976-01-01

The radiosensitivity of some tumors may depend upon the sensitivity of their microvasculature to radiation. Heretofore, the dose-response of human endothelial cells replicating in tissue culture has not been published. In studies reported here, we exposed flasks containing 4 to 7 x 10 4 genetically identical human endothelial cells to doses of x irradiation from 125 to 1000 rad. During the phase of logarithmic growth, cell counts were compared to those of an unirradiated control to construct a dose--response curve. Similar studies were performed with normal fibroblasts. We found that 160 rad suppressed endothelial cell replication by 37 percent. Although recovery was evident with doses of 500 rad, no net increase in cell number occurred in 3 weeks in flasks of endothelial cells that received 750 or 1000 rad. Fibroblasts were slightly less sensitive under these conditions. To our knowledge, this is the first report of a radiation dose--response curve for human endothelial cells replicating in culture

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.

Megabase replication domains along the human genome: relation to chromatin structure and genome organisation.

Science.gov (United States)

Audit, Benjamin; Zaghloul, Lamia; Baker, Antoine; Arneodo, Alain; Chen, Chun-Long; d'Aubenton-Carafa, Yves; Thermes, Claude

2013-01-01

In higher eukaryotes, the absence of specific sequence motifs, marking the origins of replication has been a serious hindrance to the understanding of (i) the mechanisms that regulate the spatio-temporal replication program, and (ii) the links between origins activation, chromatin structure and transcription. In this chapter, we review the partitioning of the human genome into megabased-size replication domains delineated as N-shaped motifs in the strand compositional asymmetry profiles. They collectively span 28.3% of the genome and are bordered by more than 1,000 putative replication origins. We recapitulate the comparison of this partition of the human genome with high-resolution experimental data that confirms that replication domain borders are likely to be preferential replication initiation zones in the germline. In addition, we highlight the specific distribution of experimental and numerical chromatin marks along replication domains. Domain borders correspond to particular open chromatin regions, possibly encoded in the DNA sequence, and around which replication and transcription are highly coordinated. These regions also present a high evolutionary breakpoint density, suggesting that susceptibility to breakage might be linked to local open chromatin fiber state. Altogether, this chapter presents a compartmentalization of the human genome into replication domains that are landmarks of the human genome organization and are likely to play a key role in genome dynamics during evolution and in pathological situations.

A serine palmitoyltransferase inhibitor blocks hepatitis C virus replication in human hepatocytes.

Science.gov (United States)

Katsume, Asao; Tokunaga, Yuko; Hirata, Yuichi; Munakata, Tsubasa; Saito, Makoto; Hayashi, Hitohisa; Okamoto, Koichi; Ohmori, Yusuke; Kusanagi, Isamu; Fujiwara, Shinya; Tsukuda, Takuo; Aoki, Yuko; Klumpp, Klaus; Tsukiyama-Kohara, Kyoko; El-Gohary, Ahmed; Sudoh, Masayuki; Kohara, Michinori

2013-10-01

Host cell lipid rafts form a scaffold required for replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs) produce sphingolipids, which are essential components of the lipid rafts that associate with HCV nonstructural proteins. Prevention of the de novo synthesis of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and thereby inhibits HCV replication. We investigated the ability of the SPT inhibitor NA808 to prevent HCV replication in cells and mice. We tested the ability of NA808 to inhibit SPT's enzymatic activity in FLR3-1 replicon cells. We used a replicon system to select for HCV variants that became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory concentration, after 14 rounds of cell passage. We assessed the ability of NA808 or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in mice with humanized livers (transplanted with human hepatocytes). NA808 was injected intravenously, with or without pegylated interferon alfa-2a and HCV polymerase and/or protease inhibitors. NA808 prevented HCV replication via noncompetitive inhibition of SPT; no resistance mutations developed. NA808 prevented replication of all HCV genotypes tested in mice with humanized livers. Intravenous NA808 significantly reduced viral load in the mice and had synergistic effects with pegylated interferon alfa-2a and HCV polymerase and protease inhibitors. The SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

From the chromatin interaction network to the organization of the human genome into replication N/U-domains

International Nuclear Information System (INIS)

Boulos, Rasha E; Julienne, Hanna; Baker, Antoine; Jensen, Pablo; Arneodo, Alain; Audit, Benjamin; Chen, Chun-Long; D'Aubenton-Carafa, Yves; Thermes, Claude; Petryk, Nataliya; Kahli, Malik; Hyrien, Olivier; Goldar, Arach

2014-01-01

The three-dimensional (3D) architecture of the mammalian nucleus is now being unraveled thanks to the recent development of chromatin conformation capture (3C) technologies. Here we report the results of a combined multiscale analysis of genome-wide mean replication timing and chromatin conformation data that reveal some intimate relationships between chromatin folding and human DNA replication. We previously described megabase replication N/U-domains as mammalian multiorigin replication units, and showed that their borders are ‘master’ replication initiation zones that likely initiate cascades of origin firing responsible for the stereotypic replication of these domains. Here, we demonstrate that replication N/U-domains correspond to the structural domains of self-interacting chromatin, and that their borders act as insulating regions both in high-throughput 3C (Hi-C) data and high-resolution 3C (4C) experiments. Further analyses of Hi-C data using a graph-theoretical approach reveal that N/U-domain borders are long-distance, interconnected hubs of the chromatin interaction network. Overall, these results and the observation that a well-defined ordering of chromatin states exists from N/U-domain borders to centers suggest that ‘master’ replication initiation zones are at the heart of a high-order, epigenetically controlled 3D organization of the human genome. (paper)

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

Directory of Open Access Journals (Sweden)

Majid Eshaghi

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

Repurposing Lesogaberan to Promote Human Islet Cell Survival and β-Cell Replication

Directory of Open Access Journals (Sweden)

Jide Tian

2017-01-01

Full Text Available The activation of β-cell’s A- and B-type gamma-aminobutyric acid receptors (GABAA-Rs and GABAB-Rs can promote their survival and replication, and the activation of α-cell GABAA-Rs promotes their conversion into β-cells. However, GABA and the most clinically applicable GABA-R ligands may be suboptimal for the long-term treatment of diabetes due to their pharmacological properties or potential side-effects on the central nervous system (CNS. Lesogaberan (AZD3355 is a peripherally restricted high-affinity GABAB-R-specific agonist, originally developed for the treatment of gastroesophageal reflux disease (GERD that appears to be safe for human use. This study tested the hypothesis that lesogaberan could be repurposed to promote human islet cell survival and β-cell replication. Treatment with lesogaberan significantly enhanced replication of human islet cells in vitro, which was abrogated by a GABAB-R antagonist. Immunohistochemical analysis of human islets that were grafted into immune-deficient mice revealed that oral treatment with lesogaberan promoted human β-cell replication and islet cell survival in vivo as effectively as GABA (which activates both GABAA-Rs and GABAB-Rs, perhaps because of its more favorable pharmacokinetics. Lesogaberan may be a promising drug candidate for clinical studies of diabetes intervention and islet transplantation.

Enhanced replication of herpes simplex virus type 1 in human cells

International Nuclear Information System (INIS)

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

1991-01-01

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

Human cytomegalovirus renders cells non-permissive for replication of herpes simplex viruses

International Nuclear Information System (INIS)

Cockley, K.D.

1988-01-01

The herpes simplex virus (HSV) genome during production infection in vitro may be subject to negative regulation which results in modification of the cascade of expression of herpes virus macromolecular synthesis leading to establishment of HSV latency. In the present study, human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of HSV type-1 (HSV-1). A delay in HSV replication of 15 hr as well as a consistent, almost 1000-fold inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 hr after superinfection were observed compared with controls infected with HSV alone. HSV type-2 (HSV-2) replication was similarly inhibited in HCMV-infected HEL cells. Prior ultraviolet-irradiation (UV) of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HCMV deoxyribonucleic acid (DNA) negative temperature-sensitive (ts) mutants inhibited HSV replications as efficiently as wild-type (wt) HCMV at the non-permissive temperature. Evidence for penetration and replication of superinfecting HSV into HCMV-infected cells was provided by blot hybridization of HSV DNA synthesized in HSV-superinfected cell cultures and by cesium chloride density gradient analysis of [ 3 H]-labeled HSV-1-superinfected cells

Histone hypoacetylation is required to maintain late replication timing of constitutive heterochromatin.

Science.gov (United States)

Casas-Delucchi, Corella S; van Bemmel, Joke G; Haase, Sebastian; Herce, Henry D; Nowak, Danny; Meilinger, Daniela; Stear, Jeffrey H; Leonhardt, Heinrich; Cardoso, M Cristina

2012-01-01

The replication of the genome is a spatio-temporally highly organized process. Yet, its flexibility throughout development suggests that this process is not genetically regulated. However, the mechanisms and chromatin modifications controlling replication timing are still unclear. We made use of the prominent structure and defined heterochromatic landscape of pericentric regions as an example of late replicating constitutive heterochromatin. We manipulated the major chromatin markers of these regions, namely histone acetylation, DNA and histone methylation, as well as chromatin condensation and determined the effects of these altered chromatin states on replication timing. Here, we show that manipulation of DNA and histone methylation as well as acetylation levels caused large-scale heterochromatin decondensation. Histone demethylation and the concomitant decondensation, however, did not affect replication timing. In contrast, immuno-FISH and time-lapse analyses showed that lowering DNA methylation, as well as increasing histone acetylation, advanced the onset of heterochromatin replication. While dnmt1(-)(/)(-) cells showed increased histone acetylation at chromocenters, histone hyperacetylation did not induce DNA demethylation. Hence, we propose that histone hypoacetylation is required to maintain normal heterochromatin duplication dynamics. We speculate that a high histone acetylation level might increase the firing efficiency of origins and, concomitantly, advances the replication timing of distinct genomic regions.

Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq).

Science.gov (United States)

Langley, Alexander R; Gräf, Stefan; Smith, James C; Krude, Torsten

2016-12-01

Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Linker Histone Phosphorylation Regulates Global Timing of Replication Origin Firing*S⃞

Science.gov (United States)

Thiriet, Christophe; Hayes, Jeffrey J.

2009-01-01

Despite the presence of linker histone in all eukaryotes, the primary function(s) of this histone have been difficult to clarify. Knock-out experiments indicate that H1s play a role in regulation of only a small subset of genes but are an essential component in mouse development. Here, we show that linker histone (H1) is involved in the global regulation of DNA replication in Physarum polycephalum. We find that genomic DNA of H1 knock-down cells is more rapidly replicated, an effect due at least in part to disruption of the native timing of replication fork firing. Immunoprecipitation experiments demonstrate that H1 is transiently lost from replicating chromatin via a process facilitated by phosphorylation. Our results suggest that linker histones generate a chromatin environment refractory to replication and that their transient removal via protein phosphorylation during S phase is a critical step in the epigenetic regulation of replication timing. PMID:19015270

Silencing of the pentose phosphate pathway genes influences DNA replication in human fibroblasts.

Science.gov (United States)

Fornalewicz, Karolina; Wieczorek, Aneta; Węgrzyn, Grzegorz; Łyżeń, Robert

2017-11-30

Previous reports and our recently published data indicated that some enzymes of glycolysis and the tricarboxylic acid cycle can affect the genome replication process by changing either the efficiency or timing of DNA synthesis in human normal cells. Both these pathways are connected with the pentose phosphate pathway (PPP pathway). The PPP pathway supports cell growth by generating energy and precursors for nucleotides and amino acids. Therefore, we asked if silencing of genes coding for enzymes involved in the pentose phosphate pathway may also affect the control of DNA replication in human fibroblasts. Particular genes coding for PPP pathway enzymes were partially silenced with specific siRNAs. Such cells remained viable. We found that silencing of the H6PD, PRPS1, RPE genes caused less efficient enterance to the S phase and decrease in efficiency of DNA synthesis. On the other hand, in cells treated with siRNA against G6PD, RBKS and TALDO genes, the fraction of cells entering the S phase was increased. However, only in the case of G6PD and TALDO, the ratio of BrdU incorporation to DNA was significantly changed. The presented results together with our previously published studies illustrate the complexity of the influence of genes coding for central carbon metabolism on the control of DNA replication in human fibroblasts, and indicate which of them are especially important in this process. Copyright © 2017 Elsevier B.V. All rights reserved.

R-loops and initiation of DNA replication in human cells: a missing link?

Directory of Open Access Journals (Sweden)

Rodrigo eLombraña

2015-04-01

Full Text Available The unanticipated widespread occurrence of stable hybrid DNA/RNA structures (R-loops in human cells and the increasing evidence of their involvement in several human malignancies have invigorated the research on R-loop biology in recent years. Here we propose that physiological R-loop formation at CpG island promoters can contribute to DNA replication origin specification at these regions, the most efficient replication initiation sites in mammalian cells. Quite likely, this occurs by the strand-displacement reaction activating the formation of G-quadruplex structures that target the Origin Recognition Complex (ORC in the single-stranded conformation. In agreement with this, we found that R-loops co-localize with the ORC within the same CpG island region in a significant fraction of these efficient replication origins, precisely at the position displaying the highest density of G4 motifs. This scenario builds on the connection between transcription and replication in human cells and suggests that R-loop dysregulation at CpG island promoter-origins might contribute to the phenotype of DNA replication abnormalities and loss of genome integrity detected in cancer cells.

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.

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

Science.gov (United States)

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.

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.

Human keratinocytes restrict chikungunya virus replication at a post-fusion step

Energy Technology Data Exchange (ETDEWEB)

Bernard, Eric [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Hamel, Rodolphe [Laboratoire Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution, Contrôle, UMR 5290 CNRS/IRD/UM1, Montpellier (France); Neyret, Aymeric [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Ekchariyawat, Peeraya [Laboratoire Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution, Contrôle, UMR 5290 CNRS/IRD/UM1, Montpellier (France); Molès, Jean-Pierre [INSERM U1058, UM1, CHU Montpellier (France); Simmons, Graham [Blood Systems Research Institute, San Francisco, CA 94118 (United States); Chazal, Nathalie [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Desprès, Philippe [Unité Interactions Moléculaires Flavivirus-Hôtes, Institut Pasteur, Paris (France); and others

2015-02-15

Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV.

Human keratinocytes restrict chikungunya virus replication at a post-fusion step

International Nuclear Information System (INIS)

Bernard, Eric; Hamel, Rodolphe; Neyret, Aymeric; Ekchariyawat, Peeraya; Molès, Jean-Pierre; Simmons, Graham; Chazal, Nathalie; Desprès, Philippe

2015-01-01

Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV

The pathological consequences of impaired genome integrity in humans; disorders of the DNA replication machinery.

Science.gov (United States)

O'Driscoll, Mark

2017-01-01

Accurate and efficient replication of the human genome occurs in the context of an array of constitutional barriers, including regional topological constraints imposed by chromatin architecture and processes such as transcription, catenation of the helical polymer and spontaneously generated DNA lesions, including base modifications and strand breaks. DNA replication is fundamentally important for tissue development and homeostasis; differentiation programmes are intimately linked with stem cell division. Unsurprisingly, impairments of the DNA replication machinery can have catastrophic consequences for genome stability and cell division. Functional impacts on DNA replication and genome stability have long been known to play roles in malignant transformation through a variety of complex mechanisms, and significant further insights have been gained from studying model organisms in this context. Congenital hypomorphic defects in components of the DNA replication machinery have been and continue to be identified in humans. These disorders present with a wide range of clinical features. Indeed, in some instances, different mutations in the same gene underlie different clinical presentations. Understanding the origin and molecular basis of these features opens a window onto the range of developmental impacts of suboptimal DNA replication and genome instability in humans. Here, I will briefly overview the basic steps involved in DNA replication and the key concepts that have emerged from this area of research, before switching emphasis to the pathological consequences of defects within the DNA replication network; the human disorders. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major.

Science.gov (United States)

Lombraña, Rodrigo; Álvarez, Alba; Fernández-Justel, José Miguel; Almeida, Ricardo; Poza-Carrión, César; Gomes, Fábia; Calzada, Arturo; Requena, José María; Gómez, María

2016-08-09

Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs). Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major

Directory of Open Access Journals (Sweden)

Rodrigo Lombraña

2016-08-01

Full Text Available Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs. Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance.

Repair of human DNA in molecules that replicate or remain unreplicated following ultraviolet irradiation

International Nuclear Information System (INIS)

Waters, R.

1980-01-01

The extent of DNA replication, the incidence of uv induced pyrimidine dimers and the repair replication observed after their excision was monitored in human fibroblasts uv irradiated with single or split uv doses. The excision repair processes were measured in molecules that remained unreplicated or in those that replicated after the latter uv irradiation. Less DNA replication was observed after a split as opposed to single uv irradiation. Furthermore, a split dose did not modify the excision parameters measured after a single irradiation, regardless of whether the DNA had replicated or not

Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork.

Science.gov (United States)

Parajuli, Shankar; Teasley, Daniel C; Murali, Bhavna; Jackson, Jessica; Vindigni, Alessandro; Stewart, Sheila A

2017-09-15

Faithful DNA replication is essential for genome stability. To ensure accurate replication, numerous complex and redundant replication and repair mechanisms function in tandem with the core replication proteins to ensure DNA replication continues even when replication challenges are present that could impede progression of the replication fork. A unique topological challenge to the replication machinery is posed by RNA-DNA hybrids, commonly referred to as R-loops. Although R-loops play important roles in gene expression and recombination at immunoglobulin sites, their persistence is thought to interfere with DNA replication by slowing or impeding replication fork progression. Therefore, it is of interest to identify DNA-associated enzymes that help resolve replication-impeding R-loops. Here, using DNA fiber analysis, we demonstrate that human ribonuclease H1 (RNH1) plays an important role in replication fork movement in the mammalian nucleus by resolving R-loops. We found that RNH1 depletion results in accumulation of RNA-DNA hybrids, slowing of replication forks, and increased DNA damage. Our data uncovered a role for RNH1 in global DNA replication in the mammalian nucleus. Because accumulation of RNA-DNA hybrids is linked to various human cancers and neurodegenerative disorders, our study raises the possibility that replication fork progression might be impeded, adding to increased genomic instability and contributing to disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage.

Science.gov (United States)

Wang, Yan; Mao, Lili; Kankanala, Jayakanth; Wang, Zhengqiang; Geraghty, Robert J

2017-02-01

The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease. Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus

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.

Human Papilloma Viral DNA Replicates as a Stable Episome in Cultured Epidermal Keratinocytes

Science.gov (United States)

Laporta, Robert F.; Taichman, Lorne B.

1982-06-01

Human papilloma virus (HPV) is poorly understood because systems for its growth in tissue culture have not been developed. We report here that cultured human epidermal keratinocytes could be infected with HPV from plantar warts and that the viral DNA persisted and replicated as a stable episome. There were 50-200 copies of viral DNA per cell and there was no evidence to indicate integration of viral DNA into the cellular genome. There was also no evidence to suggest that viral DNA underwent productive replication. We conclude that cultured human epidermal keratinocytes may be a model for the study of certain aspects of HPV biology.

Replication Banding Patterns in Human Chromosomes Detected Using 5-ethynyl-2'-deoxyuridine Incorporation

International Nuclear Information System (INIS)

Hoshi, Osamu; Ushiki, Tatsuo

2011-01-01

A novel technique using the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into replicating DNA is described for the analysis of replicating banding patterns of human metaphase chromosomes. Human lymphocytes were synchronized with excess thymidine and treated with EdU during the late S phase of the cell cycle. The incorporated EdU was then detected in metaphase chromosomes using Alexa Fluor® 488 azides, through the 1,3-dipolar cycloaddition reaction of organic azides with the terminal acetylene group of EdU. Chromosomes with incorporated EdU showed a banding pattern similar to G-banding of normal human chromosomes. Imaging by atomic force microscopy (AFM) in liquid conditions showed that the structure of the chromosomes was well preserved even after EdU treatment. Comparison between fluorescence microscopy and AFM images of the same chromosome 1 indicated the presence of ridges and grooves in the chromatid arm, features that have been previously reported in relation to G-banding. These results suggest an intimate relationship between EdU-induced replication bands and G- or R-bands in human chromosomes. This technique is thus useful for analyzing the structure of chromosomes in relation to their banding patterns following DNA replication in the S phase

Difference-based clustering of short time-course microarray data with replicates

Directory of Open Access Journals (Sweden)

Kim Jihoon

2007-07-01

Full Text Available Abstract Background There are some limitations associated with conventional clustering methods for short time-course gene expression data. The current algorithms require prior domain knowledge and do not incorporate information from replicates. Moreover, the results are not always easy to interpret biologically. Results We propose a novel algorithm for identifying a subset of genes sharing a significant temporal expression pattern when replicates are used. Our algorithm requires no prior knowledge, instead relying on an observed statistic which is based on the first and second order differences between adjacent time-points. Here, a pattern is predefined as the sequence of symbols indicating direction and the rate of change between time-points, and each gene is assigned to a cluster whose members share a similar pattern. We evaluated the performance of our algorithm to those of K-means, Self-Organizing Map and the Short Time-series Expression Miner methods. Conclusions Assessments using simulated and real data show that our method outperformed aforementioned algorithms. Our approach is an appropriate solution for clustering short time-course microarray data with replicates.

Enhanced replication of attenuated HSV-1 in irradiated human glioma xenografts

International Nuclear Information System (INIS)

Advani, Sunil J.; Kataoka, Yasushi; Sibley, Greg S.; Song, Paul Y.; Hallahan, Dennis E.; Roizman, Bernard; Weichselbaum, Ralph R.

1997-01-01

Purpose: Previously we had shown that combining ionizing radiation (IR) with attenuated replication competent HSV-1 (R3616) significantly increased glioma xenograft eradication compared to IR or virus alone. One hypothesis is that IR induces cell factors that contribute to augment viral replication thereby increasing the efficacy of attenuated HSV-1. The purpose of this study was to examine if IR altered viral replication of attenuated HSV-1 in glioma xenografts Material and Methods: Human U-87MG glioma cells were grown in the hindlimb of athymic mice and grown to >200 mm 3 . Tumors were infected with 2x10 7 plaque forming units (pfu) of R3616 ( γ1 34.5 - ) or R7020 (multimutated, γ1 34.5 + ) on day 0 and irradiated with 20 Gy on day 1 and 25 Gy on day 2. Tumors were harvested 3, 5, 7, and 14 days after viral injection. Tumors were homogenized and sonnicated. Serial dilutions of tumor extract were overlaid on Vero cells to determine the number of pfu. In addition, in-situ hybridization to HSV-1 DNA was performed on tumors harvested at day 7. Results: In-situ hybridization revealed larger numbers of glial cells infected with HSV along with a greater distribution in the irradiated tumors compared to non-irradiated tumors. We next quantified viral particles in infected tumors +/- IR: Conclusion: Herein we demonstrate radiation enhanced viral replication as one of the interactive effects of combining IR and attenuated HSV in treating glioma xenografts and a potential therapeutic motif in the treatment of gliomas. To reduce normal tissue toxicity of HSV in glioma therapy, viruses must be attenuated. However, attenuating the virus compromises its replication and thus its potential efficacy. Our results indicate that IR augments the amount of virus recovered from human glioma xenografts for up to 3 days post IR. The results do not appear to be related to a specific mutation in the herpes genome but rather to herpes viruses in general. Yields of R7020 were greater than R

Identifying significant temporal variation in time course microarray data without replicates

Directory of Open Access Journals (Sweden)

Porter Weston

2009-03-01

Full Text Available Abstract Background An important component of time course microarray studies is the identification of genes that demonstrate significant time-dependent variation in their expression levels. Until recently, available methods for performing such significance tests required replicates of individual time points. This paper describes a replicate-free method that was developed as part of a study of the estrous cycle in the rat mammary gland in which no replicate data was collected. Results A temporal test statistic is proposed that is based on the degree to which data are smoothed when fit by a spline function. An algorithm is presented that uses this test statistic together with a false discovery rate method to identify genes whose expression profiles exhibit significant temporal variation. The algorithm is tested on simulated data, and is compared with another recently published replicate-free method. The simulated data consists both of genes with known temporal dependencies, and genes from a null distribution. The proposed algorithm identifies a larger percentage of the time-dependent genes for a given false discovery rate. Use of the algorithm in a study of the estrous cycle in the rat mammary gland resulted in the identification of genes exhibiting distinct circadian variation. These results were confirmed in follow-up laboratory experiments. Conclusion The proposed algorithm provides a new approach for identifying expression profiles with significant temporal variation without relying on replicates. When compared with a recently published algorithm on simulated data, the proposed algorithm appears to identify a larger percentage of time-dependent genes for a given false discovery rate. The development of the algorithm was instrumental in revealing the presence of circadian variation in the virgin rat mammary gland during the estrous cycle.

From Hayflick to Walford: the role of T cell replicative senescence in human aging.

Science.gov (United States)

Effros, Rita B

2004-06-01

The immunologic theory of aging, proposed more than 40 years ago by Roy Walford, suggests that the normal process of aging in man and in animals is pathogenetically related to faulty immunological processes. Since that time, research on immunological aging has undergone extraordinary expansion, leading to new information in areas spanning from molecular biology and cell signaling to large-scale clinical studies. Investigation in this area has also provided unexpected insights into HIV disease, many aspects of which represent accelerated immunological aging. This article describes the initial insights and vision of Roy Walford into one particular facet of human immunological aging, namely, the potential relevance of the well-studied human fibroblast replicative senescence model, initially developed by Leonard Hayflick, to cells of the immune system. Extensive research on T cell senescence in cell culture has now documented changes in vitro that closely mirror alterations occurring during in vivo aging in humans, underscoring the biological significance of T cell replicative senescence. Moreover, the inclusion of high proportions of putatively senescent T cells in the 'immune risk phenotype' that is associated with early mortality in octogenarians provides initial clinical confirmation of both the immunologic theory of aging and the role of the T cell Hayflick Limit in human aging, two areas of gerontological research pioneered by Roy Walford.

The V protein of canine distemper virus is required for virus replication in human epithelial cells.

Directory of Open Access Journals (Sweden)

Noriyuki Otsuki

Full Text Available Canine distemper virus (CDV becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at position 267 of the V protein caused this growth defect. Analyses using H358 cells constitutively expressing the CDV V protein showed that the V protein with a cysteine, but not that with a tyrosine, at this position effectively blocked the interferon-stimulated signal transduction pathway, and supported virus replication of 007Lm in H358 cells. Thus, the V protein as well as the C protein appears to be functional and essential for CDV replication in human epithelial cells.

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

Science.gov (United States)

Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

2017-05-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

Activation of nucleotide oligomerization domain 2 (NOD2 by human cytomegalovirus initiates innate immune responses and restricts virus replication.

Directory of Open Access Journals (Sweden)

Arun Kapoor

Full Text Available Nucleotide-binding oligomerization domain 2 (NOD2 is an important innate immune sensor of bacterial pathogens. Its induction results in activation of the classic NF-κB pathway and alternative pathways including type I IFN and autophagy. Although the importance of NOD2 in recognizing RNA viruses has recently been identified, its role in sensing DNA viruses has not been studied. We report that infection with human cytomegalovirus (HCMV results in significant induction of NOD2 expression, beginning as early as 2 hours post infection and increasing steadily 24 hours post infection and afterwards. Infection with human herpesvirus 1 and 2 does not induce NOD2 expression. While the HCMV-encoded glycoprotein B is not required for NOD2 induction, a replication competent virion is necessary. Lentivirus-based NOD2 knockdown in human foreskin fibroblasts (HFFs and U373 glioma cells leads to enhanced HCMV replication along with decreased levels of interferon beta (IFN-β and the pro-inflammatory cytokine, IL8. NOD2 induction in HCMV-infected cells activates downstream NF-κB and interferon pathways supported by reduced nuclear localization of NF-κB and pIRF3 in NOD2 knockdown HFFs. Stable overexpression of NOD2 in HFFs restricts HCMV replication in association with increased levels of IFN-β and IL8. Similarly, transient overexpression of NOD2 in U373 cells or its downstream kinase, RIPK2, results in decreased HCMV replication and enhanced cytokine responses. However, overexpression of a mutant NOD2, 3020insC, associated with severe Crohn's disease, results in enhanced HCMV replication and decreased levels of IFN-β in U373 cells. These results show for the first time that NOD2 plays a significant role in HCMV replication and may provide a model for studies of HCMV recognition by the host cell and HCMV colitis in Crohn's disease.

Common RNA replication signals exist among group 2 coronaviruses: evidence for in vivo recombination between animal and human coronavius molecules

International Nuclear Information System (INIS)

Wu, H.-Y.; Guy, James S.; Yoo, Dongwan; Vlasak, Reinhard; Urbach, Ena; Brian, David A.

2003-01-01

5' and 3' UTR sequences on the coronavirus genome are known to carry cis-acting elements for DI RNA replication and presumably also virus genome replication. 5' UTR-adjacent coding sequences are also thought to harbor cis-acting elements. Here we have determined the 5' UTR and adjacent 289-nt sequences, and 3' UTR sequences, for six group 2 coronaviruses and have compared them to each other and to three previously reported group 2 members. Extensive regions of highly similar UTR sequences were found but small regions of divergence were also found indicating group 2 coronaviruses could be subdivided into those that are bovine coronavirus (BCoV)-like (BCoV, human respiratory coronavirus-OC43, human enteric coronavirus, porcine hemagglutinating encephalomyelitis virus, and equine coronavirus) and those that are murine hepatitis virus (MHV)-like (A59, 2, and JHM strains of MHV, puffinosis virus, and rat sialodacryoadenitis virus). The 3' UTRs of BCoV and MHV have been previously shown to be interchangeable. Here, a reporter-containing BCoV DI RNA was shown to be replicated by all five BCoV-like helper viruses and by MHV-H2 (a human cell-adapted MHV strain), a representative of the MHV-like subgroup, demonstrating group 2 common 5' and 3' replication signaling elements. BCoV DI RNA, furthermore, acquired the leader of HCoV-OC43 by leader switching, demonstrating for the first time in vivo recombination between animal and human coronavirus molecules. These results indicate that common replication signaling elements exist among group 2 coronaviruses despite a two-cluster pattern within the group and imply there could exist a high potential for recombination among group members

Replication of UV-irradiated DNA in human cell extracts: Evidence for mutagenic bypass of pyrimidine dimers

International Nuclear Information System (INIS)

Thomas, D.C.; Kunkel, T.A.

1993-01-01

The authors have examined the efficiency and fidelity of simian virus 40-origin-dependent replication of UV-irradiated double-stranded DNA in extracts of human cells. Using as a mutational target the α-complementation domain of the Escherichia coli lacZ gene in bacteriophage M13mp2DNA, replication of undamaged DNA in HeLa cell extracts was highly accurate, whereas replication of DNA irradiated with UV light (280-320 nm) was both less efficient and less accurate. Replication was inhibited by irradiation in a dose-dependent manner. Nonetheless, covalently closed, monomer-length circular products were generated that were resistant to digestion by Dpn I, showing that they resulted from semiconservative replication. These products were incised by T4 endonuclease V, whereas the undamaged replication products were not, suggesting that pyrimidine dimers were bypassed during replication. When replicated, UV-irradiated DNA was used to transfect an E. coli α-complementation host strain to score mutant M13mp2 plaques, the mutant plaque frequency was substantially higher than that obtained with either unirradiated, replicated DNA, or unreplicated, UV-irradiated DNA. Both the increased mutagenicity and the inhibition of replication associated with UV irradiation were reversed by treatment of the irradiated DNA with photolyase before replication. Sequence analysis of mutants resulting from replication of UV-irradiated DNA demonstrated that most mutants contained C → T transition errors at dipyrimidine sites. A few mutants contained 1-nt frameshift errors or tandem double CC → TT substitutions. The data are consistent with the interpretation that pyrimidine dimers are bypassed during replication by the multiprotein replication apparatus in human cell extracts and that this bypass is mutagenic primarily via misincorporation of dAMP opposite a cytosine (or uracil) in the dimer. 56 refs., 2 figs., 3 tabs

Histone acetylation regulates the time of replication origin firing.

Science.gov (United States)

Vogelauer, Maria; Rubbi, Liudmilla; Lucas, Isabelle; Brewer, Bonita J; Grunstein, Michael

2002-11-01

The temporal firing of replication origins throughout S phase in yeast depends on unknown determinants within the adjacent chromosomal environment. We demonstrate here that the state of histone acetylation of surrounding chromatin is an important regulator of temporal firing. Deletion of RPD3 histone deacetylase causes earlier origin firing and concurrent binding of the replication factor Cdc45p to origins. In addition, increased acetylation of histones in the vicinity of the late origin ARS1412 by recruitment of the histone acetyltransferase Gcn5p causes ARS1412 alone to fire earlier. These data indicate that histone acetylation is a direct determinant of the timing of origin firing.

Human parvovirus B19: a mechanistic overview of infection and DNA replication

Science.gov (United States)

Luo, Yong; Qiu, Jianming

2015-01-01

Human parvovirus B19 (B19V) is a human pathogen that belongs to genus Erythroparvovirus of the Parvoviridae family, which is composed of a group of small DNA viruses with a linear single-stranded DNA genome. B19V mainly infects human erythroid progenitor cells and causes mild to severe hematological disorders in patients. However, recent clinical studies indicate that B19V also infects nonerythroid lineage cells, such as myocardial endothelial cells, and may be associated with other disease outcomes. Several cell culture systems, including permissive and semipermissive erythroid lineage cells, nonpermissive human embryonic kidney 293 cells and recently reported myocardial endothelial cells, have been used to study the mechanisms underlying B19V infection and B19V DNA replication. This review aims to summarize recent advances in B19V studies with a focus on the mechanisms of B19V tropism specific to different cell types and the cellular pathways involved in B19V DNA replication including cellular signaling transduction and cell cycle arrest. PMID:26097496

MicroRNA regulation of human protease genes essential for influenza virus replication.

Directory of Open Access Journals (Sweden)

Victoria A Meliopoulos

Full Text Available Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB, cAMP/calcium signaling (CRE/CREB, and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.

MicroRNA regulation of human protease genes essential for influenza virus replication.

Science.gov (United States)

Meliopoulos, Victoria A; Andersen, Lauren E; Brooks, Paula; Yan, Xiuzhen; Bakre, Abhijeet; Coleman, J Keegan; Tompkins, S Mark; Tripp, Ralph A

2012-01-01

Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.

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

Protein-protein and protein-nucleic acid interactions within subcellular compartments are required for viral genome replication. To understand the localization of the human cytomegalovirus viral replication factor UL84 relative to other proteins involved in viral DNA synthesis and to replicating viral DNA in infected cells, we created a recombinant virus expressing a FLAG-tagged version of UL84 (UL84FLAG) and used this virus in immunofluorescence assays. UL84FLAG localization differed at early and late times of infection, transitioning from diffuse distribution throughout the nucleus to exclusion from the interior of replication compartments, with some concentration at the periphery of replication compartments with newly labeled DNA and the viral DNA polymerase subunit UL44. Early in infection, UL84FLAG colocalized with the viral single-stranded DNA binding protein UL57, but colocalization became less prominent as infection progressed. A portion of UL84FLAG also colocalized with the host nucleolar protein nucleolin at the peripheries of both replication compartments and nucleoli. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a dramatic elimination of UL84FLAG from replication compartments and other parts of the nucleus and its accumulation in the cytoplasm. Reciprocal coimmunoprecipitation of viral proteins from infected cell lysates revealed association of UL84, UL44, and nucleolin. These results indicate that UL84 localization during infection is dynamic, which is likely relevant to its functions, and suggest that its nuclear and subnuclear localization is highly dependent on direct or indirect interactions with nucleolin. Importance: The protein-protein interactions among viral and cellular proteins required for replication of the human cytomegalovirus (HCMV) DNA genome are poorly understood. We sought to understand how an enigmatic HCMV protein critical for virus replication, UL84, localizes relative to other viral and cellular

Human FEN1 Expression and Solubility Patterson in DNA Replication and Repair

National Research Council Canada - National Science Library

Carrier, Richard

1999-01-01

Flap endo-/exonuclease (FEN1) is a highly conserved protein shown to be one of 10 essential human proteins required for the production of form I DNA following DNA replication from the simian virus 40 (SV40...

Interaction of RECQ4 and MCM10 is important for efficient DNA replication origin firing in human cells

DEFF Research Database (Denmark)

Kliszczak, Maciej; Sedlackova, Hana; Pitchai, Ganesha P

2015-01-01

DNA replication is a highly coordinated process that is initiated at multiple replication origins in eukaryotes. These origins are bound by the origin recognition complex (ORC), which subsequently recruits the Mcm2-7 replicative helicase in a Cdt1/Cdc6-dependent manner. In budding yeast, two...... essential replication factors, Sld2 and Mcm10, are then important for the activation of replication origins. In humans, the putative Sld2 homolog, RECQ4, interacts with MCM10. Here, we have identified two mutants of human RECQ4 that are deficient in binding to MCM10. We show that these RECQ4 variants...... are able to complement the lethality of an avian cell RECQ4 deletion mutant, indicating that the essential function of RECQ4 in vertebrates is unlikely to require binding to MCM10. Nevertheless, we show that the RECQ4-MCM10 interaction is important for efficient replication origin firing....

Timing, coordination, and rhythm: Acrobatics at the DNA replication fork

KAUST Repository

Hamdan, Samir

2010-04-09

In DNA replication, the antiparallel nature of the parental duplex imposes certain constraints on the activity of the DNA polymerases that synthesize new DNA. The leading-strand polymerase advances in a continuous fashion, but the lagging-strand polymerase is forced to restart at short intervals. In several prokaryotic systems studied so far, this problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. The timing of Okazaki fragment synthesis and loop formation is determined by a subtle interplay of enzymatic activities at the fork. Recent developments in single-molecule techniques have enabled the direct observation of these processes and have greatly contributed to a better understanding of the dynamic nature of the replication fork. Here, we will review recent experimental advances, present the current models, and discuss some of the exciting developments in the field. 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Timing, coordination, and rhythm: Acrobatics at the DNA replication fork

KAUST Repository

Hamdan, Samir; van Oijen, Antoine M.

2010-01-01

In DNA replication, the antiparallel nature of the parental duplex imposes certain constraints on the activity of the DNA polymerases that synthesize new DNA. The leading-strand polymerase advances in a continuous fashion, but the lagging-strand polymerase is forced to restart at short intervals. In several prokaryotic systems studied so far, this problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. The timing of Okazaki fragment synthesis and loop formation is determined by a subtle interplay of enzymatic activities at the fork. Recent developments in single-molecule techniques have enabled the direct observation of these processes and have greatly contributed to a better understanding of the dynamic nature of the replication fork. Here, we will review recent experimental advances, present the current models, and discuss some of the exciting developments in the field. 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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...

Human FEN1 Expression and Solubility Patterson in DNA Replication and Repair

National Research Council Canada - National Science Library

Carrier, Richard

1999-01-01

.... I show that human FEN1 mRNA and protein levels increase in a cell cycle-dependent manner, with peak mRNA and protein levels attained coincident with S phase DNA replication in both primary and transformed cells...

The Tat protein of human immunodeficiency virus-1 enhances hepatitis C virus replication through interferon gamma-inducible protein-10

Directory of Open Access Journals (Sweden)

Qu Jing

2012-04-01

Full Text Available Abstract Background Co-infection with human immunodeficiency virus-1 (HIV-1 and hepatitis C virus (HCV is associated with faster progression of liver disease and an increase in HCV persistence. However, the mechanism by which HIV-1 accelerates the progression of HCV liver disease remains unknown. Results HIV-1/HCV co-infection is associated with increased expression of interferon gamma-induced protein-10 (IP-10 mRNA in peripheral blood mononuclear cells (PBMCs. HCV RNA levels were higher in PBMCs of patients with HIV-1/HCV co-infection than in patients with HCV mono-infection. HIV-1 Tat and IP-10 activated HCV replication in a time-dependent manner, and HIV-1 Tat induced IP-10 production. In addition, the effect of HIV-1 Tat on HCV replication was blocked by anti-IP-10 monoclonal antibody, demonstrating that the effect of HIV-1 Tat on HCV replication depends on IP-10. Taken together, these results suggest that HIV-1 Tat protein activates HCV replication by upregulating IP-10 production. Conclusions HIV-1/HCV co-infection is associated with increased expression of IP-10 mRNA and replication of HCV RNA. Furthermore, both HIV-1 Tat and IP-10 activate HCV replication. HIV-1 Tat activates HCV replication by upregulating IP-10 production. These results expand our understanding of HIV-1 in HCV replication and the mechanism involved in the regulation of HCV replication mediated by HIV-1 during co-infection.

Identification of a 450-bp region of human papillomavirus type 1 that promotes episomal replication in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Chattopadhyay, Anasuya; Schmidt, Martin C.; Khan, Saleem A.

2005-01-01

Human papillomaviruses (HPVs) replicate as nuclear plasmids in infected cells. Since the DNA replication machinery is generally conserved between humans and Saccharomyces cerevisiae, we studied whether HPV-1 DNA can replicate in yeast. Plasmids containing a selectable marker (with or without a yeast centromere) and either the full-length HPV-1 genome or various regions of the viral long control region (LCR) and the 3' end of the L1 gene were introduced into S. cerevisiae and their ability to replicate episomally was investigated. Our results show that HPV-1 sequences promote episomal replication of plasmids although the yeast centromere is required for plasmid retention. We have mapped the autonomously replicating sequence activity of HPV-1 DNA to a 450 base-pair sequence (HPV-1 nt 6783-7232) that includes 293 nucleotides from the 5' region of the viral LCR and 157 nucleotides from the 3' end of the L1 gene. The HPV-1 ARS does not include the binding sites for the viral E1 and E2 proteins, and these proteins are dispensable for replication in S. cerevisiae

Mechanism of Error-Free DNA Replication Past Lucidin-Derived DNA Damage by Human DNA Polymerase κ.

Science.gov (United States)

Yockey, Oliver P; Jha, Vikash; Ghodke, Pratibha P; Xu, Tianzuo; Xu, Wenyan; Ling, Hong; Pradeepkumar, P I; Zhao, Linlin

2017-11-20

DNA damage impinges on genetic information flow and has significant implications in human disease and aging. Lucidin-3-O-primeveroside (LuP) is an anthraquinone derivative present in madder root, which has been used as a coloring agent and food additive. LuP can be metabolically converted to genotoxic compound lucidin, which subsequently forms lucidin-specific N 2 -2'-deoxyguanosine (N 2 -dG) and N 6 -2'-deoxyadenosine (N 6 -dA) DNA adducts. Lucidin is mutagenic and carcinogenic in rodents but has low carcinogenic risks in humans. To understand the molecular mechanism of low carcinogenicity of lucidin in humans, we performed DNA replication assays using site-specifically modified oligodeoxynucleotides containing a structural analogue (LdG) of lucidin-N 2 -dG DNA adduct and determined the crystal structures of DNA polymerase (pol) κ in complex with LdG-bearing DNA and an incoming nucleotide. We examined four human pols (pol η, pol ι, pol κ, and Rev1) in their efficiency and accuracy during DNA replication with LdG; these pols are key players in translesion DNA synthesis. Our results demonstrate that pol κ efficiently and accurately replicates past the LdG adduct, whereas DNA replication by pol η, pol ι is compromised to different extents. Rev1 retains its ability to incorporate dCTP opposite the lesion albeit with decreased efficiency. Two ternary crystal structures of pol κ illustrate that the LdG adduct is accommodated by pol κ at the enzyme active site during insertion and postlesion-extension steps. The unique open active site of pol κ allows the adducted DNA to adopt a standard B-form for accurate DNA replication. Collectively, these biochemical and structural data provide mechanistic insights into the low carcinogenic risk of lucidin in humans.

In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

International Nuclear Information System (INIS)

Dresler, S.; Frattini, M.G.; Robinson-Hill, R.M.

1988-01-01

Using permeable diploid human fibroblasts, the authors have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent K m values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 μM. For UV-induced DNA repair synthesis, the apparent K m values were substantially lower, ranging from 0.11 to 0.44 μM for AG1518 cells and from 0.06 to 0.24 μM for IMR-90 cells. Recent data implicate DNA polymerase δ in UV-induced repair synthesis and suggest that DNA polymerases α and δ are both involved in semiconservative replication. They measured K m values for dGTP and dTTP for polymerases α and δ, for comparison with the values for replication and repair synthesis. The deoxyribonucleotide K m values for DNA polymerase δ are much greater than the K m values for UV-induced repair synthesis, suggesting that when polymerase δ functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases. The K m values for UV-induced repair synthesis are 5-80-fold lower than deoxyribonucleotide concentrations that have been reported for intact cultured diploid human fibroblasts. For replication, however, the K m for dGTP is only slightly lower than the average cellular dGTP concentration that has been reported for exponentially growing human fibroblasts. This finding is consistent with the concept that nucleotide compartmentation is required for the attainment of high rates of DNA replication in vivo

Proteome-wide analysis of SUMO2 targets in response to pathological DNA replication stress in human cells.

Science.gov (United States)

Bursomanno, Sara; Beli, Petra; Khan, Asif M; Minocherhomji, Sheroy; Wagner, Sebastian A; Bekker-Jensen, Simon; Mailand, Niels; Choudhary, Chunaram; Hickson, Ian D; Liu, Ying

2015-01-01

SUMOylation is a form of post-translational modification involving covalent attachment of SUMO (Small Ubiquitin-like Modifier) polypeptides to specific lysine residues in the target protein. In human cells, there are four SUMO proteins, SUMO1-4, with SUMO2 and SUMO3 forming a closely related subfamily. SUMO2/3, in contrast to SUMO1, are predominantly involved in the cellular response to certain stresses, including heat shock. Substantial evidence from studies in yeast has shown that SUMOylation plays an important role in the regulation of DNA replication and repair. Here, we report a proteomic analysis of proteins modified by SUMO2 in response to DNA replication stress in S phase in human cells. We have identified a panel of 22 SUMO2 targets with increased SUMOylation during DNA replication stress, many of which play key functions within the DNA replication machinery and/or in the cellular response to DNA damage. Interestingly, POLD3 was found modified most significantly in response to a low dose aphidicolin treatment protocol that promotes common fragile site (CFS) breakage. POLD3 is the human ortholog of POL32 in budding yeast, and has been shown to act during break-induced recombinational repair. We have also shown that deficiency of POLD3 leads to an increase in RPA-bound ssDNA when cells are under replication stress, suggesting that POLD3 plays a role in the cellular response to DNA replication stress. Considering that DNA replication stress is a source of genome instability, and that excessive replication stress is a hallmark of pre-neoplastic and tumor cells, our characterization of SUMO2 targets during a perturbed S-phase should provide a valuable resource for future functional studies in the fields of DNA metabolism and cancer biology. Copyright © 2014 Elsevier B.V. All rights reserved.

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.)

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....

Nicotinamide extends replicative lifespan of human cells.

Science.gov (United States)

Kang, Hyun Tae; Lee, Hyung Il; Hwang, Eun Seong

2006-10-01

We found that an ongoing application of nicotinamide to normal human fibroblasts not only attenuated expression of the aging phenotype but also increased their replicative lifespan, causing a greater than 1.6-fold increase in the number of population doublings. Although nicotinamide by itself does not act as an antioxidant, the cells cultured in the presence of nicotinamide exhibited reduced levels of reactive oxygen species (ROS) and oxidative damage products associated with cellular senescence, and a decelerated telomere shortening rate without a detectable increase in telomerase activity. Furthermore, in the treated cells growing beyond the original Hayflick limit, the levels of p53, p21WAF1, and phospho-Rb proteins were similar to those in actively proliferating cells. The nicotinamide treatment caused a decrease in ATP levels, which was stably maintained until the delayed senescence point. Nicotinamide-treated cells also maintained high mitochondrial membrane potential but a lower respiration rate and superoxide anion level. Taken together, in contrast to its demonstrated pro-aging effect in yeast, nicotinamide extends the lifespan of human fibroblasts, possibly through reduction in mitochondrial activity and ROS production.

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

Science.gov (United States)

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

Real-time single-molecule observation of rolling-circle DNA replication

NARCIS (Netherlands)

Tanner, Nathan A.; Loparo, Joseph J.; Hamdan, Samir M.; Jergic, Slobodan; Dixon, Nicholas E.; Oijen, Antoine M. van

2009-01-01

We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities

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.

Primary Human Placental Trophoblasts are Permissive for Zika Virus (ZIKV) Replication.

Science.gov (United States)

Aagaard, Kjersti M; Lahon, Anismrita; Suter, Melissa A; Arya, Ravi P; Seferovic, Maxim D; Vogt, Megan B; Hu, Min; Stossi, Fabio; Mancini, Michael A; Harris, R Alan; Kahr, Maike; Eppes, Catherine; Rac, Martha; Belfort, Michael A; Park, Chun Shik; Lacorazza, Daniel; Rico-Hesse, Rebecca

2017-01-27

Zika virus (ZIKV) is an emerging mosquito-borne (Aedes genus) arbovirus of the Flaviviridae family. Although ZIKV has been predominately associated with a mild or asymptomatic dengue-like disease, its appearance in the Americas has been accompanied by a multi-fold increase in reported incidence of fetal microcephaly and brain malformations. The source and mode of vertical transmission from mother to fetus is presumptively transplacental, although a causal link explaining the interval delay between maternal symptoms and observed fetal malformations following infection has been missing. In this study, we show that primary human placental trophoblasts from non-exposed donors (n = 20) can be infected by primary passage ZIKV-FLR isolate, and uniquely allowed for ZIKV viral RNA replication when compared to dengue virus (DENV). Consistent with their being permissive for ZIKV infection, primary trophoblasts expressed multiple putative ZIKV cell entry receptors, and cellular function and differentiation were preserved. These findings suggest that ZIKV-FLR strain can replicate in human placental trophoblasts without host cell destruction, thereby serving as a likely permissive reservoir and portal of fetal transmission with risk of latent microcephaly and malformations.

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.

The disparity between human cell senescence in vitro and lifelong replication in vivo.

Science.gov (United States)

Rubin, Harry

2002-07-01

Cultured human fibroblasts undergo senescence (a loss of replicative capacity) after a uniform, fixed number of approximately 50 population doublings, commonly termed the Hayflick limit. It has been long known from clonal and other quantitative studies, however, that cells decline in replicative capacity from the time of explantation and do so in a stochastic manner, with a half-life of only approximately 8 doublings. The apparent 50-cell doubling limit reflects the expansive propagation of the last surviving clone. The relevance of either figure to survival of cells in the body is questionable, given that stem cells in some renewing tissues undergo >1,000 divisions in a lifetime with no morphological sign of senescence. Oddly enough, these observations have had little if any effect on general acceptance of the Hayflick limit in its original form. The absence of telomerase in cultured human cells and the shortening of telomeres at each population doubling have suggested that telomere length acts as a mitotic clock that accounts for their limited lifespan. This concept assumed an iconic character with the report that ectopic expression of telomerase by a vector greatly extended the lifespan of human cells. That something similar might occur in vivo seemed consistent with initial reports that most human somatic tissues lack telomerase activity. More careful study, however, has revealed telomerase activity in stem cells and some dividing transit cells of many renewing tissues and even in dividing myocytes of repairing cardiac muscle. It now seems likely that telomerase is active in vivo where and when it is needed to maintain tissue integrity. Caution is recommended in applying telomerase inhibition to kill telomerase-expressing cancer cells, because it would probably damage stem cells in essential organs and even increase the likelihood of secondary cancers. The risk may be especially high in sun-exposed skin, where there are usually thousands of p53-mutant clones of

MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

Directory of Open Access Journals (Sweden)

Erkko Ylösmäki

Full Text Available MicroRNAs (miRNAs are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5 in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

Analysis of cis and trans Requirements for DNA Replication at the Right-End Hairpin of the Human Bocavirus 1 Genome.

Science.gov (United States)

Shen, Weiran; Deng, Xuefeng; Zou, Wei; Engelhardt, John F; Yan, Ziying; Qiu, Jianming

2016-09-01

Parvoviruses are single-stranded DNA viruses that use the palindromic structures at the ends of the viral genome for their replication. The mechanism of parvovirus replication has been studied mostly in the dependoparvovirus adeno-associated virus 2 (AAV2) and the protoparvovirus minute virus of mice (MVM). Here, we used human bocavirus 1 (HBoV1) to understand the replication mechanism of bocaparvovirus. HBoV1 is pathogenic to humans, causing acute respiratory tract infections, especially in young children under 2 years old. By using the duplex replicative form of the HBoV1 genome in human embryonic kidney 293 (HEK293) cells, we identified the HBoV1 minimal replication origin at the right-end hairpin (OriR). Mutagenesis analyses confirmed the putative NS1 binding and nicking sites within the OriR. Of note, unlike the large nonstructural protein (Rep78/68 or NS1) of other parvoviruses, HBoV1 NS1 did not specifically bind OriR in vitro, indicating that other viral and cellular components or the oligomerization of NS1 is required for NS1 binding to the OriR. In vivo studies demonstrated that residues responsible for NS1 binding and nicking are within the origin-binding domain. Further analysis identified that the small nonstructural protein NP1 is required for HBoV1 DNA replication at OriR. NP1 and other viral nonstructural proteins (NS1 to NS4) colocalized within the viral DNA replication centers in both OriR-transfected cells and virus-infected cells, highlighting a direct involvement of NP1 in viral DNA replication at OriR. Overall, our study revealed the characteristics of HBoV1 DNA replication at OriR, suggesting novel characteristics of autonomous parvovirus DNA replication. Human bocavirus 1 (HBoV1) causes acute respiratory tract infections in young children. The duplex HBoV1 genome replicates in HEK293 cells and produces progeny virions that are infectious in well-differentiated airway epithelial cells. A recombinant AAV2 vector pseudotyped with an HBoV1

Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts

International Nuclear Information System (INIS)

Kaufmann, W.K.; Cleaver, J.E.

1981-01-01

The inhibition of DNA replication in ultraviolet-irradiated human fibroblasts was characterized by quantitative analysis of radiation-induced alterations in the steady-state distribution of sizes of pulse-labeled, nascent DNA. Low, noncytotoxic fluences rapidly produced an inhibition of DNA synthesis in half-replicon-size replication intermediates. With time, the inhibition produced by low fluences spread progressively to include multi-replicon-size intermediates. The results indicate that ultraviolet radiation inhibits the initiation of DNA synthesis in replicons. Higher cytotoxic fluences inhibited DNA synthesis in operating replicons. Xeroderma pigmentosum fibroblasts with deficiencies in DNA excision repair exhibited an inhibition of replicon initiation after low radiation fluences, indicating the effect was not solely dependent upon operation of the nucleotidyl excision repair pathway. Owing to their inability to remove pyrimidine dimers ahead of DNA growing points, the repair-deficient cells also were more sensitive than normal cells to the ultraviolet-induced inhibition of chain elongation. Xeroderma pigmentosum cells belonging to the variant class were even more sensitive to inhibition of chain elongation despite their ability to remove pyrimidine dimers. The analysis suggested that normal and repair-deficient human fibroblasts either are able to rapidly bypass certain dimers or these dimers are not recognized by the chain elongation machinery. (author)

Identification of Persistent RNA-DNA Hybrid Structures within the Origin of Replication of Human Cytomegalovirus

OpenAIRE

Prichard, Mark N.; Jairath, Sanju; Penfold, Mark E. T.; Jeor, Stephen St.; Bohlman, Marlene C.; Pari, Gregory S.

1998-01-01

Human cytomegalovirus (HCMV) lytic-phase DNA replication initiates at the cis-acting origin of replication, oriLyt. oriLyt is a structurally complex region containing repeat elements and transcription factor binding sites. We identified two site-specific alkali-labile regions within oriLyt which flank an alkali-resistant DNA segment. These alkali-sensitive regions were the result of the degradation of two RNA species embedded within oriLyt and covalently linked to viral DNA. The virus-associa...

Single Cell Analysis of Human RAD18-Dependent DNA Post-Replication Repair by Alkaline Bromodeoxyuridine Comet Assay

Science.gov (United States)

Mórocz, Mónika; Gali, Himabindu; Raskó, István; Downes, C. Stephen; Haracska, Lajos

2013-01-01

Damage to DNA can block replication progression resulting in gaps in the newly synthesized DNA. Cells utilize a number of post-replication repair (PRR) mechanisms such as the RAD18 controlled translesion synthesis or template switching to overcome the discontinuities formed opposite the DNA lesions and to complete DNA replication. Gaining more insights into the role of PRR genes promotes better understanding of DNA damage tolerance and of how their malfunction can lead to increased genome instability and cancer. However, a simple and efficient method to characterise gene specific PRR deficiencies at a single cell level has not been developed. Here we describe the so named BrdU comet PRR assay to test the contribution of human RAD18 to PRR at a single cell level, by which we kinetically characterized the consequences of the deletion of human RAD18 on the replication of UV-damaged DNA. Moreover, we demonstrate the capability of our method to evaluate PRR at a single cell level in unsynchronized cell population. PMID:23936422

DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells

Science.gov (United States)

Sokol, Anna M.; Cruet-Hennequart, Séverine; Pasero, Philippe; Carty, Michael P.

2013-11-01

Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.

Iterated function systems for DNA replication

Science.gov (United States)

Gaspard, Pierre

2017-10-01

The kinetic equations of DNA replication are shown to be exactly solved in terms of iterated function systems, running along the template sequence and giving the statistical properties of the copy sequences, as well as the kinetic and thermodynamic properties of the replication process. With this method, different effects due to sequence heterogeneity can be studied, in particular, a transition between linear and sublinear growths in time of the copies, and a transition between continuous and fractal distributions of the local velocities of the DNA polymerase along the template. The method is applied to the human mitochondrial DNA polymerase γ without and with exonuclease proofreading.

The ESS and replicator equation in matrix games under time constraints.

Science.gov (United States)

Garay, József; Cressman, Ross; Móri, Tamás F; Varga, Tamás

2018-06-01

Recently, we introduced the class of matrix games under time constraints and characterized the concept of (monomorphic) evolutionarily stable strategy (ESS) in them. We are now interested in how the ESS is related to the existence and stability of equilibria for polymorphic populations. We point out that, although the ESS may no longer be a polymorphic equilibrium, there is a connection between them. Specifically, the polymorphic state at which the average strategy of the active individuals in the population is equal to the ESS is an equilibrium of the polymorphic model. Moreover, in the case when there are only two pure strategies, a polymorphic equilibrium is locally asymptotically stable under the replicator equation for the pure-strategy polymorphic model if and only if it corresponds to an ESS. Finally, we prove that a strict Nash equilibrium is a pure-strategy ESS that is a locally asymptotically stable equilibrium of the replicator equation in n-strategy time-constrained matrix games.

Differential replication of avian influenza H9N2 viruses in human alveolar epithelial A549 cells

Directory of Open Access Journals (Sweden)

Peiris Malik

2010-03-01

Full Text Available Abstract Avian influenza virus H9N2 isolates cause a mild influenza-like illness in humans. However, the pathogenesis of the H9N2 subtypes in human remains to be investigated. Using a human alveolar epithelial cell line A549 as host, we found that A/Quail/Hong Kong/G1/97 (H9N2/G1, which shares 6 viral "internal genes" with the lethal A/Hong Kong/156/97 (H5N1/97 virus, replicates efficiently whereas other H9N2 viruses, A/Duck/Hong Kong/Y280/97 (H9N2/Y280 and A/Chicken/Hong Kong/G9/97 (H9N2/G9, replicate poorly. Interestingly, we found that there is a difference in the translation of viral protein but not in the infectivity or transcription of viral genes of these H9N2 viruses in the infected cells. This difference may possibly be explained by H9N2/G1 being more efficient on viral protein production in specific cell types. These findings suggest that the H9N2/G1 virus like its counterpart H5N1/97 may be better adapted to the human host and replicates efficiently in human alveolar epithelial cells.

Cytotoxic effects of replication-competent adenoviruses on human esophageal carcinoma are enhanced by forced p53 expression

International Nuclear Information System (INIS)

Yang, Shan; Kawamura, Kiyoko; Okamoto, Shinya; Yamauchi, Suguru; Shingyoji, Masato; Sekine, Ikuo; Kobayashi, Hiroshi; Tada, Yuji; Tatsumi, Koichiro; Hiroshima, Kenzo; Shimada, Hideaki; Tagawa, Masatoshi

2015-01-01

Improvement of transduction and augmentation of cytotoxicity are crucial for adenoviruses (Ad)-mediated gene therapy for cancer. Down-regulated expression of type 5 Ad (Ad5) receptors on human tumors hampered Ad-mediated transduction. Furthermore, a role of the p53 pathways in cytotoxicity mediated by replication-competent Ad remained uncharacterized. We constructed replication-competent Ad5 of which the E1 region genes were activated by a transcriptional regulatory region of the midkine or the survivin gene, which is expressed preferentially in human tumors. We also prepared replication-competent Ad5 which were regulated by the same region but had a fiber-knob region derived from serotype 35 (AdF35). We examined the cytotoxicity of these Ad and a possible combinatory use of the replication-competent AdF35 and Ad5 expressing the wild-type p53 gene (Ad5/p53) in esophageal carcinoma cells. Expression levels of molecules involved in cell death, anti-tumor effects in vivo and production of viral progenies were also investigated. Replication-competent AdF35 in general achieved greater cytotoxic effects to esophageal carcinoma cells than the corresponding replication-competent Ad5. Infection with the AdF35 induced cleavages of caspases and increased sub-G1 fractions, but did not activate the autophagy pathway. Transduction with Ad5/p53 in combination with the replication-competent AdF35 further enhanced the cytotoxicity in a synergistic manner. We also demonstrated the combinatory effects in an animal model. Transduction with Ad5/p53 however suppressed production of replication-competent AdF35 progenies, but the combination augmented Ad5/p53-mediated p53 expression levels and the downstream pathways. Combination of replication-competent AdF35 and Ad5/p53 achieved synergistic cytotoxicity due to enhanced p53-mediated apoptotic pathways. The online version of this article (doi:10.1186/s12885-015-1482-8) contains supplementary material, which is available to authorized

Inhibition of DNA replication and repair by anthralin or danthron in cultured human cells

International Nuclear Information System (INIS)

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

1982-01-01

The comparative effects of the tumor promoter anthralin and its analog, danthron, on semiconservative DNA replication and DNA repair synthesis were studied in cultured human cells. Bromodeoxyuridine was used as density label together with 3 H-thymidine to distinguish replication from repair synthesis in isopycnic CsCl gradients. Anthralin at 1.1 microgram inhibited replication in T98G cells by 50%. In cells treated with 0.4 or 1.3 microM anthralin and additive effect was observed on the inhibition of replication by ultraviolet light (254 nm). In cells irradiated with 20 J/m2, 2.3 microM anthralin was required to inhibit repair synthesis by 50%. Thus there was no selective inhibitory effect of anthralin on repair synthesis. Danthron exhibited no detectable effect on either semiconservative replication or repair synthesis at concentrations below about 5.0 microM. Neither compound stimulated repair synthesis in the absence of ultraviolet irradiation. Thus, anthralin and danthron do not appear to react with DNA to form adducts that are subject to excision repair. Although both compounds appear to intercalate into supercoiled DNA in vitro to a limited extent, the degree of unwinding introduced by the respective drugs does not correlate with their relative effects on DNA synthesis in vivo. Therefore the inhibitory effect of anthralin on DNA replication and repair synthesis in T98G cells does not appear to result from the direct interaction of the drug with DNA

MicroRNA Regulation of Human Genes Essential for Influenza A (H7N9 Replication.

Directory of Open Access Journals (Sweden)

Stefan Wolf

Full Text Available Influenza A viruses are important pathogens of humans and animals. While seasonal influenza viruses infect humans every year, occasionally animal-origin viruses emerge to cause pandemics with significantly higher morbidity and mortality rates. In March 2013, the public health authorities of China reported three cases of laboratory confirmed human infection with avian influenza A (H7N9 virus, and subsequently there have been many cases reported across South East Asia and recently in North America. Most patients experience severe respiratory illness, and morbidity with mortality rates near 40%. No vaccine is currently available and the use of antivirals is complicated due the frequent emergence of drug resistant strains. Thus, there is an imminent need to identify new drug targets for therapeutic intervention. In the current study, a high-throughput screening (HTS assay was performed using microRNA (miRNA inhibitors to identify new host miRNA targets that reduce influenza H7N9 replication in human respiratory (A549 cells. Validation studies lead to a top hit, hsa-miR-664a-3p, that had potent antiviral effects in reducing H7N9 replication (TCID50 titers by two logs. In silico pathway analysis revealed that this microRNA targeted the LIF and NEK7 genes with effects on pro-inflammatory factors. In follow up studies using siRNAs, anti-viral properties were shown for LIF. Furthermore, inhibition of hsa-miR-664a-3p also reduced virus replication of pandemic influenza A strains H1N1 and H3N2.

Replication-competent human adenovirus 11p vectors can propagate in Vero cells

International Nuclear Information System (INIS)

Gokumakulapalle, Madhuri; Mei, Ya-Fang

2016-01-01

The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.

Replication-competent human adenovirus 11p vectors can propagate in Vero cells

Energy Technology Data Exchange (ETDEWEB)

Gokumakulapalle, Madhuri; Mei, Ya-Fang, E-mail: ya-fang.mei@umu.se

2016-08-15

The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.

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.

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

Science.gov (United States)

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

2012-01-01

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

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...

Human CD46-transgenic mice in studies involving replication-incompetent adenoviral type 35 vectors

NARCIS (Netherlands)

Verhaagh, S.; Jong, E. de; Goudsmit, J.; Lecollinet, S.; Gillissen, G.; Vries, M. de; Leuven, K. van; Que, I.; Ouwehand, K.; Mintardjo, R.; Weverling, G.J.; Radošević, K.; Richardson, J.; Eloit, M.; Lowik, C.; Quax, P.; Havenga, M.

2006-01-01

Wild-type strains of mice do not express CD46, a high-affinity receptor for human group B adenoviruses including type 35. Therefore, studies performed to date in mice using replication-incompetent Ad35 (rAd35) vaccine carriers may underestimate potency or result in altered vector distribution. Here,

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.

Human T Lymphocytes Are Permissive for Dengue Virus Replication.

Science.gov (United States)

Silveira, Guilherme F; Wowk, Pryscilla F; Cataneo, Allan H D; Dos Santos, Paula F; Delgobo, Murilo; Stimamiglio, Marco A; Lo Sarzi, Maria; Thomazelli, Ana Paula F S; Conchon-Costa, Ivete; Pavanelli, Wander R; Antonelli, Lis R V; Báfica, André; Mansur, Daniel S; Dos Santos, Claudia N Duarte; Bordignon, Juliano

2018-05-15

Dengue virus (DV) infection can cause either a self-limiting flu-like disease or a threatening hemorrhage that may evolve to shock and death. A variety of cell types, such as dendritic cells, monocytes, and B cells, can be infected by DV. However, despite the role of T lymphocytes in the control of DV replication, there remains a paucity of information on possible DV-T cell interactions during the disease course. In the present study, we have demonstrated that primary human naive CD4 + and CD8 + T cells are permissive for DV infection. Importantly, both T cell subtypes support viral replication and secrete viable virus particles. DV infection triggers the activation of both CD4 + and CD8 + T lymphocytes, but preactivation of T cells reduces the susceptibility of T cells to DV infection. Interestingly, the cytotoxicity-inducing protein granzyme A is highly secreted by human CD4 + but not CD8 + T cells after exposure to DV in vitro Additionally, using annexin V and polycaspase assays, we have demonstrated that T lymphocytes, in contrast to monocytes, are resistant to DV-induced apoptosis. Strikingly, both CD4 + and CD8 + T cells were found to be infected with DV in acutely infected dengue patients. Together, these results show that T cells are permissive for DV infection in vitro and in vivo , suggesting that this cell population may be a viral reservoir during the acute phase of the disease. IMPORTANCE Infection by dengue virus (DV) causes a flu-like disease that can evolve to severe hemorrhaging and death. T lymphocytes are important cells that regulate antibody secretion by B cells and trigger the death of infected cells. However, little is known about the direct interaction between DV and T lymphocytes. Here, we show that T lymphocytes from healthy donors are susceptible to infection by DV, leading to cell activation. Additionally, T cells seem to be resistant to DV-induced apoptosis, suggesting a potential role as a viral reservoir in humans. Finally, we show

Extended local similarity analysis (eLSA) of microbial community and other time series data with replicates.

Science.gov (United States)

Xia, Li C; Steele, Joshua A; Cram, Jacob A; Cardon, Zoe G; Simmons, Sheri L; Vallino, Joseph J; Fuhrman, Jed A; Sun, Fengzhu

2011-01-01

The increasing availability of time series microbial community data from metagenomics and other molecular biological studies has enabled the analysis of large-scale microbial co-occurrence and association networks. Among the many analytical techniques available, the Local Similarity Analysis (LSA) method is unique in that it captures local and potentially time-delayed co-occurrence and association patterns in time series data that cannot otherwise be identified by ordinary correlation analysis. However LSA, as originally developed, does not consider time series data with replicates, which hinders the full exploitation of available information. With replicates, it is possible to understand the variability of local similarity (LS) score and to obtain its confidence interval. We extended our LSA technique to time series data with replicates and termed it extended LSA, or eLSA. Simulations showed the capability of eLSA to capture subinterval and time-delayed associations. We implemented the eLSA technique into an easy-to-use analytic software package. The software pipeline integrates data normalization, statistical correlation calculation, statistical significance evaluation, and association network construction steps. We applied the eLSA technique to microbial community and gene expression datasets, where unique time-dependent associations were identified. The extended LSA analysis technique was demonstrated to reveal statistically significant local and potentially time-delayed association patterns in replicated time series data beyond that of ordinary correlation analysis. These statistically significant associations can provide insights to the real dynamics of biological systems. The newly designed eLSA software efficiently streamlines the analysis and is freely available from the eLSA homepage, which can be accessed at http://meta.usc.edu/softs/lsa.

DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes, a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase*

Science.gov (United States)

Bharti, Sanjay Kumar; Sommers, Joshua A.; Zhou, Jun; Kaplan, Daniel L.; Spelbrink, Johannes N.; Mergny, Jean-Louis; Brosh, Robert M.

2014-01-01

Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the “Pattern Finder” G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase. PMID:25193669

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.

Persistent Amplification of DNA Damage Signal Involved in Replicative Senescence of Normal Human Diploid Fibroblasts

Directory of Open Access Journals (Sweden)

Masatoshi Suzuki

2012-01-01

Full Text Available Foci of phosphorylated histone H2AX and ATM are the surrogate markers of DNA double strand breaks. We previously reported that the residual foci increased their size after irradiation, which amplifies DNA damage signals. Here, we addressed whether amplification of DNA damage signal is involved in replicative senescence of normal human diploid fibroblasts. Large phosphorylated H2AX foci (>1.5 μm diameter were specifically detected in presenescent cells. The frequency of cells with large foci was well correlated with that of cells positive for senescence-associated β-galactosidase staining. Hypoxic cell culture condition extended replicative life span of normal human fibroblast, and we found that the formation of large foci delayed in those cells. Our immuno-FISH analysis revealed that large foci partially localized at telomeres in senescent cells. Importantly, large foci of phosphorylated H2AX were always colocalized with phosphorylated ATM foci. Furthermore, Ser15-phosphorylated p53 showed colocalization with the large foci. Since the treatment of senescent cells with phosphoinositide 3-kinase inhibitor, wortmannin, suppressed p53 phosphorylation, it is suggested that amplification of DNA damage signaling sustains persistent activation of ATM-p53 pathway, which is essential for replicative senescence.

Interferon-alpha mediates restriction of human immunodeficiency virus type-1 replication in primary human macrophages at an early stage of replication.

Directory of Open Access Journals (Sweden)

Kelly M Cheney

2010-10-01

Full Text Available Type I interferons (IFNα and β are induced directly in response to viral infection, resulting in an antiviral state for the cell. In vitro studies have shown that IFNα is a potent inhibitor of viral replication; however, its role in HIV-1 infection is incompletely understood. In this study we describe the ability of IFNα to restrict HIV-1 infection in primary human macrophages in contrast to peripheral blood mononuclear cells and monocyte-derived dendritic cells. Inhibition to HIV-1 replication in cells pretreated with IFNα occurred at an early stage in the virus life cycle. Late viral events such as budding and subsequent rounds of infection were not affected by IFNα treatment. Analysis of early and late HIV-1 reverse transcripts and integrated proviral DNA confirmed an early post entry role for IFNα. First strand cDNA synthesis was slightly reduced but late and integrated products were severely depleted, suggesting that initiation or the nucleic acid intermediates of reverse transcription are targeted. The depletion of integrated provirus is disproportionally greater than that of viral cDNA synthesis suggesting the possibility of a least an additional later target. A role for either cellular protein APOBEC3G or tetherin in this IFNα mediated restriction has been excluded. Vpu, previously shown by others to rescue a viral budding restriction by tetherin, could not overcome this IFNα induced effect. Determining both the viral determinants and cellular proteins involved may lead to novel therapeutic approaches. Our results add to the understanding of HIV-1 restriction by IFNα.

Human CD134 (OX40) expressed on T cells plays a key role for human herpesvirus 6B replication after allogeneic hematopoietic stem cell transplantation.

Science.gov (United States)

Nagamata, Satoshi; Nagasaka, Miwako; Kawabata, Akiko; Kishimoto, Kenji; Hasegawa, Daiichiro; Kosaka, Yoshiyuki; Mori, Takeshi; Morioka, Ichiro; Nishimura, Noriyuki; Iijima, Kazumoto; Yamada, Hideto; Kawamoto, Shinichiro; Yakushijin, Kimikazu; Matsuoka, Hiroshi; Mori, Yasuko

2018-05-01

CD134 (OX40), which is a cellular receptor for human herpesvirus-6B (HHV-6B) and expresses on activated T cells, may play a key role for HHV-6B replication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Therefore, we examined the CD134 expression on T cells and HHV-6B replication after allo-HSCT, and analyzed the correlation between them. Twenty-three patients after allo-HSCT were enrolled. The percentages of CD134-positive cells within the CD4 + and CD8 + cell populations were measured by flow cytometry, and the viral copy number of HHV-6B was simultaneously quantified by real-time PCR. The correlation between CD134 and HHV-6B viral load was then statistically analyzed. HHV-6B reactivation occurred in 11 of 23 patients (47.8%). CD134 expression was seen on T cells and was coincident with the time of peak viral load. The percentage of CD134-positive cells decreased significantly when HHV-6B DNA disappeared (p = .005 in CD4 + T cells, p = .02 in CD8 + T cells). In the 4 patients who underwent umbilical cord blood transplantation (UCBT), the viral load varied with the percentage of CD134-positive cells. In the comparison between the HHV-6B reactivation group and non-reactivation group, maximum percentages of CD134-positive cells among CD4 + T cells in reactivation group were significantly higher than those in non-reactivation group (p = .04). This is the first study to show that a correlation of CD134 expression on T cells with HHV-6B replication after allo-HSCT, especially in UCBT. The results possibly indicate that CD134 on T cells plays a key role for HHV-6B replication after allo-HSCT. Copyright © 2018 Elsevier B.V. All rights reserved.

A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition

Science.gov (United States)

Gill, Martin R.; Harun, Siti Norain; Halder, Swagata; Boghozian, Ramon A.; Ramadan, Kristijan; Ahmad, Haslina; Vallis, Katherine A.

2016-08-01

Ruthenium(II) polypyridyl complexes can intercalate DNA with high affinity and prevent cell proliferation; however, the direct impact of ruthenium-based intercalation on cellular DNA replication remains unknown. Here we show the multi-intercalator [Ru(dppz)2(PIP)]2+ (dppz = dipyridophenazine, PIP = 2-(phenyl)imidazo[4,5-f][1,10]phenanthroline) immediately stalls replication fork progression in HeLa human cervical cancer cells. In response to this replication blockade, the DNA damage response (DDR) cell signalling network is activated, with checkpoint kinase 1 (Chk1) activation indicating prolonged replication-associated DNA damage, and cell proliferation is inhibited by G1-S cell-cycle arrest. Co-incubation with a Chk1 inhibitor achieves synergistic apoptosis in cancer cells, with a significant increase in phospho(Ser139) histone H2AX (γ-H2AX) levels and foci indicating increased conversion of stalled replication forks to double-strand breaks (DSBs). Normal human epithelial cells remain unaffected by this concurrent treatment. Furthermore, pre-treatment of HeLa cells with [Ru(dppz)2(PIP)]2+ before external beam ionising radiation results in a supra-additive decrease in cell survival accompanied by increased γ-H2AX expression, indicating the compound functions as a radiosensitizer. Together, these results indicate ruthenium-based intercalation can block replication fork progression and demonstrate how these DNA-binding agents may be combined with DDR inhibitors or ionising radiation to achieve more efficient cancer cell killing.

A Defective Interfering Influenza RNA Inhibits Infectious Influenza Virus Replication in Human Respiratory Tract Cells: A Potential New Human Antiviral

Directory of Open Access Journals (Sweden)

Claire M. Smith

2016-08-01

Full Text Available Defective interfering (DI viruses arise during the replication of influenza A virus and contain a non-infective version of the genome that is able to interfere with the production of infectious virus. In this study we hypothesise that a cloned DI influenza A virus RNA may prevent infection of human respiratory epithelial cells with infection by influenza A. The DI RNA (244/PR8 was derived by a natural deletion process from segment 1 of influenza A/PR/8/34 (H1N1; it comprises 395 nucleotides and is packaged in the DI virion in place of a full-length genome segment 1. Given intranasally, 244/PR8 DI virus protects mice and ferrets from clinical influenza caused by a number of different influenza A subtypes and interferes with production of infectious influenza A virus in cells in culture. However, evidence that DI influenza viruses are active in cells of the human respiratory tract is lacking. Here we show that 244/PR8 DI RNA is replicated by an influenza A challenge virus in human lung diploid fibroblasts, bronchial epithelial cells, and primary nasal basal cells, and that the yield of challenge virus is significantly reduced in a dose-dependent manner indicating that DI influenza virus has potential as a human antiviral.

Replicating animal mitochondrial DNA

Directory of Open Access Journals (Sweden)

Emily A. McKinney

2013-01-01

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

Genetic variations in the DNA replication origins of human papillomavirus family correlate with their oncogenic potential.

Science.gov (United States)

Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

2018-04-01

Human papillomaviruses (HPVs) encompass a large family of viruses that range from benign to highly carcinogenic. The crucial differences between benign and carcinogenic types of HPV remain unknown, except that the two HPV types differ in the frequency of DNA replication. We have systematically analyzed the mechanism of HPV DNA replication initiation in low-risk and high-risk HPVs. Our results demonstrate that HPV-encoded E2 initiator protein and its four binding sites in the replication origin play pivotal roles in determining the destiny of the HPV-infected cell. We have identified strain-specific single nucleotide variations in E2 binding sites found only in the high-risk HPVs. We have demonstrated that these variations result in attenuated formation of the E2-DNA complex. E2 binding to these sites is linked to the activation of the DNA replication origin as well as initiation of DNA replication. Both electrophoretic mobility shift assay and atomic force microscopy studies demonstrated that binding of E2 from either low- or high-risk HPVs with variant binding sequences lacked multimeric E2-DNA complex formation in vitro. These results provided a molecular basis of differential DNA replication in the two types of HPVs and pointed to a correlation with the development of cancer. Copyright © 2017. Published by Elsevier B.V.

Multiple DNA binding proteins contribute to timing of chromosome replication in E. coli

DEFF Research Database (Denmark)

Riber, Leise; Frimodt-Møller, Jakob; Charbon, Godefroid

2016-01-01

Chromosome replication in Escherichia coli is initiated from a single origin, oriC. Initiation involves a number of DNA binding proteins, but only DnaA is essential and specific for the initiation process. DnaA is an AAA+ protein that binds both ATP and ADP with similar high affinities. Dna...... replication is initiated, or the time window in which all origins present in a single cell are initiated, i.e. initiation synchrony, or both. Overall, these DNA binding proteins modulate the initiation frequency from oriC by: (i) binding directly to oriC to affect DnaA binding, (ii) altering the DNA topology...... in or around oriC, (iii) altering the nucleotide bound status of DnaA by interacting with non-coding chromosomal sequences, distant from oriC, that are important for DnaA activity. Thus, although DnaA is the key protein for initiation of replication, other DNA-binding proteins act not only on ori...

Evaluation of the minimal replication time of Cauliflower mosaic virus in different hosts

International Nuclear Information System (INIS)

Khelifa, Mounia; Masse, Delphine; Blanc, Stephane; Drucker, Martin

2010-01-01

Though the duration of a single round of replication is an important biological parameter, it has been determined for only few viruses. Here, this parameter was determined for Cauliflower mosaic virus (CaMV) in transfected protoplasts from different hosts: the highly susceptible Arabidopsis and turnip, and Nicotiana benthamiana, where CaMV accumulates only slowly. Four methods of differing sensitivity were employed: labelling of (1) progeny DNA and (2) capsid protein, (3) immunocapture PCR,, and (4) progeny-specific PCR. The first progeny virus was detected about 21 h after transfection. This value was confirmed by all methods, indicating that our estimate was not biased by the sensitivity of the detection method, and approximated the actual time required for one round of CaMV replication. Unexpectedly, the replication kinetics were similar in the three hosts; suggesting that slow accumulation of CaMV in Nicotiana plants is determined by non-optimal interactions in other steps of the infection cycle.

Ultrastructural Characterization of Zika Virus Replication Factories

Directory of Open Access Journals (Sweden)

Mirko Cortese

2017-02-01

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

Vaginal Lactobacillus Inhibits HIV-1 Replication in Human Tissues Ex Vivo

Directory of Open Access Journals (Sweden)

Rogers A. Ñahui Palomino

2017-05-01

Full Text Available Lactobacillus species, which dominate vaginal microbiota of healthy reproductive-age women, lower the risks of sexually transmitted infections, including the risk of human immunodeficiency virus (HIV acquisition. The exact mechanisms of this protection remain to be understood. Here, we investigated these mechanisms in the context of human cervico-vaginal and lymphoid tissues ex vivo. We found that all six Lactobacillus strains tested in these systems significantly suppressed HIV type-1 (HIV-1 infection. We identified at least three factors that mediated this suppression: (i Acidification of the medium. The pH of the undiluted medium conditioned by lactobacilli was between 3.8 and 4.6. Acidification of the culture medium with hydrochloric acid (HCl to this pH in control experiments was sufficient to abrogate HIV-1 replication. However, the pH of the Lactobacillus-conditioned medium (CM diluted fivefold, which reached ∼6.9, was also suppressive for HIV-1 infection, while in control experiments HIV-1 infection was not abrogated when the pH of the medium was brought to 6.9 through the use of HCl. This suggested the existence of other factors responsible for HIV-1 inhibition by lactobacilli. (ii Lactic acid. There was a correlation between the concentration of lactic acid in the Lactobacillus-CM and its ability to suppress HIV-1 infection in human tissues ex vivo. Addition of lactic acid isomers D and L to tissue culture medium at the concentration that corresponded to their amount released by lactobacilli resulted in HIV-1 inhibition. Isomer L was produced in higher quantities than isomer D and was mostly responsible for HIV-1 inhibition. These results indicate that lactic acid, in particular its L-isomer, inhibits HIV-1 independently of lowering of the pH. (iii Virucidal effect. Incubation of HIV-1 in Lactobacillus-CM significantly suppressed viral infectivity for human tissues ex vivo. Finally, lactobacilli adsorb HIV-1, serving as a sink

Inhibition of Avian Influenza A Virus Replication in Human Cells by Host Restriction Factor TUFM Is Correlated with Autophagy.

Science.gov (United States)

Kuo, Shu-Ming; Chen, Chi-Jene; Chang, Shih-Cheng; Liu, Tzu-Jou; Chen, Yi-Hsiang; Huang, Sheng-Yu; Shih, Shin-Ru

2017-06-13

Avian influenza A viruses generally do not replicate efficiently in human cells, but substitution of glutamic acid (Glu, E) for lysine (Lys, K) at residue 627 of avian influenza virus polymerase basic protein 2 (PB2) can serve to overcome host restriction and facilitate human infectivity. Although PB2 residue 627 is regarded as a species-specific signature of influenza A viruses, host restriction factors associated with PB2 627 E have yet to be fully investigated. We conducted immunoprecipitation, followed by differential proteomic analysis, to identify proteins associating with PB2 627 K (human signature) and PB2 627 E (avian signature) of influenza A/WSN/1933(H1N1) virus, and the results indicated that Tu elongation factor, mitochondrial (TUFM), had a higher binding affinity for PB2 627 E than PB2 627 K in transfected human cells. Stronger binding of TUFM to avian-signature PB2 590 G/ 591 Q and PB2 627 E in the 2009 swine-origin pandemic H1N1 and 2013 avian-origin H7N9 influenza A viruses was similarly observed. Viruses carrying avian-signature PB2 627 E demonstrated increased replication in TUFM-deficient cells, but viral replication decreased in cells overexpressing TUFM. Interestingly, the presence of TUFM specifically inhibited the replication of PB2 627 E viruses, but not PB2 627 K viruses. In addition, enhanced levels of interaction between TUFM and PB2 627 E were noted in the mitochondrial fraction of infected cells. Furthermore, TUFM-dependent autophagy was reduced in TUFM-deficient cells infected with PB2 627 E virus; however, autophagy remained consistent in PB2 627 K virus-infected cells. The results suggest that TUFM acts as a host restriction factor that impedes avian-signature influenza A virus replication in human cells in a manner that correlates with autophagy. IMPORTANCE An understanding of the mechanisms that influenza A viruses utilize to shift host tropism and the identification of host restriction factors that can limit infection are both

The AGIS metric and time of test: A replication study

OpenAIRE

Counsell, S; Swift, S; Tucker, A

2016-01-01

Visual Field (VF) tests and corresponding data are commonly used in clinical practices to manage glaucoma. The standard metric used to measure glaucoma severity is the Advanced Glaucoma Intervention Studies (AGIS) metric. We know that time of day when VF tests are applied can influence a patient’s AGIS metric value; a previous study showed that this was the case for a data set of 160 patients. In this paper, we replicate that study using data from 2468 patients obtained from Moorfields Eye Ho...

Assessing Human Immunodeficiency Virus Type 1 Tropism: Comparison of Assays Using Replication-Competent Virus versus Plasma-Derived Pseudotyped Virions ▿

Science.gov (United States)

Hosoya, Noriaki; Su, Zhaohui; Wilkin, Timothy; Gulick, Roy M.; Flexner, Charles; Hughes, Michael D.; Skolnik, Paul R.; Giguel, Françoise; Greaves, Wayne L.; Coakley, Eoin; Kuritzkes, Daniel R.

2009-01-01

Detection of CXCR4-using human immunodeficiency virus by the Trofile assay was compared to that by assays using virus isolates or replication-competent recombinants. Concordance with the Trofile assay was good, but assays using replicating viruses did not increase substantially the ability to detect the presence of CXCR4-using virus. PMID:19494074

Analysis of in vitro replicated human hepatitis C virus (HCV for the determination of genotypes and quasispecies

Directory of Open Access Journals (Sweden)

Chelyapov Nickolas

2006-09-01

Full Text Available Abstract Isolation and self-replication of infectious HCV has been a difficult task. However, this is needed for the purposes of developing rational drugs and for the analysis of the natural virus. Our recent report of an in vitro system for the isolation of human HCV from infected patients and their replication in tissue culture addresses this challenge. At California Institute of Molecular Medicine several isolates of HCV, called CIMM-HCV, were grown for over three years in cell culture. This is a report of the analysis of CIMM-HCV isolates for subtypes and quasispecies using a 269 bp segment of the 5'UTR. HCV RNA from three patients and eleven CIMM-HCV were analyzed for this purpose. All isolates were essentially identical. Isolates of HCV from one patient were serially transmitted into fresh cells up to eight times and the progeny viruses from each transmission were compared to each other and also to the primary isolates from the patient's serum. Some isolates were also transmitted to different cell types, while others were cultured continuously without retransmission for over three years. We noted minor sequence changes when HCV was cultured for extended periods of time. HCV in T-cells and non-committed lymphoid cells showed a few differences when compared to isolates obtained from immortalized B-cells. These viruses maintained close similarity despite repeated transmissions and passage of time. There were no subtypes or quasispecies noted in CIMM-HCV.

Bicyclams, selective antagonists of the human chemokine receptor CXCR4, potently inhibit feline immunodeficiency virus replication

NARCIS (Netherlands)

Horzinek, M.C.; Egberink, H.F.; Clercq, E. de; Vliet, A.L.W. van; Balzarini, J.; Bridger, G.J.; Henson, G.; Schols, D.

1999-01-01

Bicyclams are low-molecular-weight anti-human immunodeficiency virus (HIV) agents that have been shown to act as potent and selective CXC chemokine receptor 4 (CXCR4) antagonists. Here, we demonstrate that bicyclams are potent inhibitors of feline immunodeficiency virus (FIV) replication when

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.

Stochastic variation in telomere shortening rate causes heterogeneity of human fibroblast replicative life span.

Science.gov (United States)

Martin-Ruiz, Carmen; Saretzki, Gabriele; Petrie, Joanne; Ladhoff, Juliane; Jeyapalan, Jessie; Wei, Wenyi; Sedivy, John; von Zglinicki, Thomas

2004-04-23

The replicative life span of human fibroblasts is heterogeneous, with a fraction of cells senescing at every population doubling. To find out whether this heterogeneity is due to premature senescence, i.e. driven by a nontelomeric mechanism, fibroblasts with a senescent phenotype were isolated from growing cultures and clones by flow cytometry. These senescent cells had shorter telomeres than their cycling counterparts at all population doubling levels and both in mass cultures and in individual subclones, indicating heterogeneity in the rate of telomere shortening. Ectopic expression of telomerase stabilized telomere length in the majority of cells and rescued them from early senescence, suggesting a causal role of telomere shortening. Under standard cell culture conditions, there was a minor fraction of cells that showed a senescent phenotype and short telomeres despite active telomerase. This fraction increased under chronic mild oxidative stress, which is known to accelerate telomere shortening. It is possible that even high telomerase activity cannot fully compensate for telomere shortening in all cells. The data show that heterogeneity of the human fibroblast replicative life span can be caused by significant stochastic cell-to-cell variation in telomere shortening.

The human adenovirus type 5 E1B 55 kDa protein obstructs inhibition of viral replication by type I interferon in normal human cells.

Directory of Open Access Journals (Sweden)

Jasdave S Chahal

Full Text Available Vectors derived from human adenovirus type 5, which typically lack the E1A and E1B genes, induce robust innate immune responses that limit their therapeutic efficacy. We reported previously that the E1B 55 kDa protein inhibits expression of a set of cellular genes that is highly enriched for those associated with anti-viral defense and immune responses, and includes many interferon-sensitive genes. The sensitivity of replication of E1B 55 kDa null-mutants to exogenous interferon (IFN was therefore examined in normal human fibroblasts and respiratory epithelial cells. Yields of the mutants were reduced at least 500-fold, compared to only 5-fold, for wild-type (WT virus replication. To investigate the mechanistic basis of such inhibition, the accumulation of viral early proteins and genomes was compared by immunoblotting and qPCR, respectively, in WT- and mutant-infected cells in the absence or presence of exogenous IFN. Both the concentration of viral genomes detected during the late phase and the numbers of viral replication centers formed were strongly reduced in IFN-treated cells in the absence of the E1B protein, despite production of similar quantities of viral replication proteins. These defects could not be attributed to degradation of entering viral genomes, induction of apoptosis, or failure to reorganize components of PML nuclear bodies. Nor was assembly of the E1B- and E4 Orf6 protein- E3 ubiquitin ligase required to prevent inhibition of viral replication by IFN. However, by using RT-PCR, the E1B 55 kDa protein was demonstrated to be a potent repressor of expression of IFN-inducible genes in IFN-treated cells. We propose that a primary function of the previously described transcriptional repression activity of the E1B 55 kDa protein is to block expression of IFN- inducible genes, and hence to facilitate formation of viral replication centers and genome replication.

RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells

DEFF Research Database (Denmark)

Sleeth, Kate M; Sørensen, Claus Storgaard; Issaeva, Natalia

2007-01-01

The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited...... the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation...... and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role...

Timing of host feeding drives rhythms in parasite replication

KAUST Repository

Prior, Kimberley F.

2018-02-26

Circadian rhythms enable organisms to synchronise the processes underpinning survival and reproduction to anticipate daily changes in the external environment. Recent work shows that daily (circadian) rhythms also enable parasites to maximise fitness in the context of ecological interactions with their hosts. Because parasite rhythms matter for their fitness, understanding how they are regulated could lead to innovative ways to reduce the severity and spread of diseases. Here, we examine how host circadian rhythms influence rhythms in the asexual replication of malaria parasites. Asexual replication is responsible for the severity of malaria and fuels transmission of the disease, yet, how parasite rhythms are driven remains a mystery. We perturbed feeding rhythms of hosts by 12 hours (i.e. diurnal feeding in nocturnal mice) to desynchronise the host’s peripheral oscillators from the central, light-entrained oscillator in the brain and their rhythmic outputs. We demonstrate that the rhythms of rodent malaria parasites in day-fed hosts become inverted relative to the rhythms of parasites in night-fed hosts. Our results reveal that the host’s peripheral rhythms (associated with the timing of feeding and metabolism), but not rhythms driven by the central, light-entrained circadian oscillator in the brain, determine the timing (phase) of parasite rhythms. Further investigation reveals that parasite rhythms correlate closely with blood glucose rhythms. In addition, we show that parasite rhythms resynchronise to the altered host feeding rhythms when food availability is shifted, which is not mediated through rhythms in the host immune system. Our observations suggest that parasites actively control their developmental rhythms. Finally, counter to expectation, the severity of disease symptoms expressed by hosts was not affected by desynchronisation of their central and peripheral rhythms. Our study at the intersection of disease ecology and chronobiology opens up a new

Timing of host feeding drives rhythms in parasite replication

KAUST Repository

Prior, Kimberley F

2017-12-07

Circadian rhythms enable organisms to synchronise the processes underpinning survival and reproduction to anticipate daily changes in the external environment. Recent work shows that daily (circadian) rhythms also enable parasites to maximise fitness in the context of ecological interactions with their hosts. Because parasite rhythms matter for their fitness, understanding how they are regulated could lead to innovative ways to reduce the severity and spread of diseases. Here, we examine how host circadian rhythms influence rhythms in the asexual replication of malaria parasites. Asexual replication is responsible for the severity of malaria and fuels transmission of the disease, yet, how parasite rhythms are driven remains a mystery. We perturbed feeding rhythms of hosts by 12 hours (i.e. diurnal feeding in nocturnal mice) to desynchronise the host\\'s peripheral oscillators from the central, light-entrained oscillator in the brain and their rhythmic outputs. We demonstrate that the rhythms of rodent malaria parasites in day-fed hosts become inverted relative to the rhythms of parasites in night-fed hosts. Our results reveal that the host\\'s peripheral rhythms (associated with the timing of feeding and metabolism), but not rhythms driven by the central, light-entrained circadian oscillator in the brain, determine the timing (phase) of parasite rhythms. Further investigation reveals that parasite rhythms correlate closely with blood glucose rhythms. In addition, we show that parasite rhythms resynchronise to the altered host feeding rhythms when food availability is shifted, which is not mediated through rhythms in the host immune system. Our observations suggest that parasites actively control their developmental rhythms. Finally, counter to expectation, the severity of disease symptoms expressed by hosts was not affected by desynchronisation of their central and peripheral rhythms. Our study at the intersection of disease ecology and chronobiology opens up a new

Hierarchical Bayesian modelling of gene expression time series across irregularly sampled replicates and clusters.

Science.gov (United States)

Hensman, James; Lawrence, Neil D; Rattray, Magnus

2013-08-20

Time course data from microarrays and high-throughput sequencing experiments require simple, computationally efficient and powerful statistical models to extract meaningful biological signal, and for tasks such as data fusion and clustering. Existing methodologies fail to capture either the temporal or replicated nature of the experiments, and often impose constraints on the data collection process, such as regularly spaced samples, or similar sampling schema across replications. We propose hierarchical Gaussian processes as a general model of gene expression time-series, with application to a variety of problems. In particular, we illustrate the method's capacity for missing data imputation, data fusion and clustering.The method can impute data which is missing both systematically and at random: in a hold-out test on real data, performance is significantly better than commonly used imputation methods. The method's ability to model inter- and intra-cluster variance leads to more biologically meaningful clusters. The approach removes the necessity for evenly spaced samples, an advantage illustrated on a developmental Drosophila dataset with irregular replications. The hierarchical Gaussian process model provides an excellent statistical basis for several gene-expression time-series tasks. It has only a few additional parameters over a regular GP, has negligible additional complexity, is easily implemented and can be integrated into several existing algorithms. Our experiments were implemented in python, and are available from the authors' website: http://staffwww.dcs.shef.ac.uk/people/J.Hensman/.

Deciphering DNA replication dynamics in eukaryotic cell populations in relation with their averaged chromatin conformations

Science.gov (United States)

Goldar, A.; Arneodo, A.; Audit, B.; Argoul, F.; Rappailles, A.; Guilbaud, G.; Petryk, N.; Kahli, M.; Hyrien, O.

2016-03-01

We propose a non-local model of DNA replication that takes into account the observed uncertainty on the position and time of replication initiation in eukaryote cell populations. By picturing replication initiation as a two-state system and considering all possible transition configurations, and by taking into account the chromatin’s fractal dimension, we derive an analytical expression for the rate of replication initiation. This model predicts with no free parameter the temporal profiles of initiation rate, replication fork density and fraction of replicated DNA, in quantitative agreement with corresponding experimental data from both S. cerevisiae and human cells and provides a quantitative estimate of initiation site redundancy. This study shows that, to a large extent, the program that regulates the dynamics of eukaryotic DNA replication is a collective phenomenon that emerges from the stochastic nature of replication origins initiation.

Laser-induced heating integrated with a microfluidic platform for real-time DNA replication and detection

Science.gov (United States)

Hung, Min-Sheng; Ho, Chia-Chin; Chen, Chih-Pin

2016-08-01

This study developed a microfluidic platform for replicating and detecting DNA in real time by integrating a laser and a microfluidic device composed of polydimethylsiloxane. The design of the microchannels consisted of a laser-heating area and a detection area. An infrared laser was used as the heating source for DNA replication, and the laser power was adjusted to heat the solutions directly. In addition, strong biotin-avidin binding was used to capture and detect the replicated products. The biotin on one end was bound to avidin and anchored to the surface of the microchannels, whereas the biotin on the other end was bound to the quantum dots (Qdots). The results showed that the fluorescent intensity of the Qdots bound to the replicated products in the detection area increased with the number of thermal cycles created by the laser. When the number of thermal cycles was ≥10, the fluorescent intensity of the Qdots was directly detectable on the surface of the microchannels. The proposed method is more sensitive than detection methods entailing gel electrophoresis.

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.

Lycorine reduces mortality of human enterovirus 71-infected mice by inhibiting virus replication

Directory of Open Access Journals (Sweden)

Qin Chuan

2011-10-01

Full Text Available Abstract Human enterovirus 71 (EV71 infection causes hand, foot and mouth disease in children under 6 years old and this infection occasionally induces severe neurological complications. No vaccines or drugs are clinical available to control EV71 epidemics. In present study, we show that treatment with lycorine reduced the viral cytopathic effect (CPE on rhabdomyosarcoma (RD cells by inhibiting virus replication. Analysis of this inhibitory effect of lycorine on viral proteins synthesis suggests that lycorine blocks the elongation of the viral polyprotein during translation. Lycorine treatment of mice challenged with a lethal dose of EV71 resulted in reduction of mortality, clinical scores and pathological changes in the muscles of mice, which were achieved through inhibition of viral replication. When mice were infected with a moderate dose of EV71, lycorine treatment was able to protect them from paralysis. Lycorine may be a potential drug candidate for the clinical treatment of EV71-infected patients.

Miltefosine inhibits Chikungunya virus replication in human primary dermal fibroblasts [version 1; referees: 2 approved, 1 approved with reservations

Directory of Open Access Journals (Sweden)

Anuj Sharma

2018-01-01

Full Text Available Background: Chikungunya virus (CHIKV is a re-emerging pathogen that has caused widespread outbreaks affecting millions of people around the globe. Currently, there is no specific therapeutic drug against CHIKV, with symptomatic treatment only to manage the disease. Pi3-akt signaling has been implicated in infection of several viruses including that of CHIKV. Effect of Pi3-akt signaling inhibitors on CHIKV replication was evaluated in this study. Methods: Human primary dermal fibroblast cells were treated with inhibitors of the Pi3-akt signaling pathway. Suppression of CHIKV replication was evaluated as reduction in virus titer in cell supernatants. Effect of miltefosine (MF on CHIKV replication was evaluated in pre and post treatment regimen. Inhibition of virus replication was determined by cell growth, virus titer and western blot. Results: Inhibition of Akt-phosphorylation significantly inhibited CHIKV replication. No effect on CHIKV replication was observed after treatment with Pi3-kinase and mTOR activation inhibitors. Further, MF, an FDA-approved Akt-inhibitor, inhibited CHIKV replication in pre- and post-infection treatment regimens. Conclusion: Data suggests that Akt-phosphorylation can be an amenable target of therapy against CHIKV infection. This is the first study to show inhibition of CHIKV replication by MF, and presents a case for further development of MF as an anti-CHIKV drug.

A Study on Generic Representation of Skeletal Remains Replication of Prehistoric Burial

Directory of Open Access Journals (Sweden)

C.-W. Shao

2015-08-01

Full Text Available Generic representation of skeletal remains from burials consists of three dimensions which include physical anthropologists, replication technicians, and promotional educators. For the reason that archaeological excavation is irreversible and disruptive, detail documentation and replication technologies are surely needed for many purposes. Unearthed bones during the process of 3D digital scanning need to go through reverse procedure, 3D scanning, digital model superimposition, rapid prototyping, mould making, and the integrated errors generated from the presentation of colours and textures are important issues for the presentation of replicate skeleton remains among professional decisions conducted by physical anthropologists, subjective determination of makers, and the expectations of viewers. This study presents several cases and examines current issues on display and replication technologies for human skeletal remains of prehistoric burials. This study documented detail colour changes of human skeleton over time for the reference of reproduction. The tolerance errors of quantification and required technical qualification is acquired according to the precision of 3D scanning, the specification requirement of rapid prototyping machine, and the mould making process should following the professional requirement for physical anthropological study. Additionally, the colorimeter is adopted to record and analyse the “colour change” of the human skeletal remains from wet to dry condition. Then, the “colure change” is used to evaluate the “real” surface texture and colour presentation of human skeletal remains, and to limit the artistic presentation among the human skeletal remains reproduction. The“Lingdao man No.1”, is a well preserved burial of early Neolithic period (8300 B.P. excavated from Liangdao-Daowei site, Matsu, Taiwan , as the replicating object for this study. In this study, we examined the reproduction procedures step by

Human Neutralizing Monoclonal Antibody Inhibition of Middle East Respiratory Syndrome Coronavirus Replication in the Common Marmoset.

Science.gov (United States)

Chen, Zhe; Bao, Linlin; Chen, Cong; Zou, Tingting; Xue, Ying; Li, Fengdi; Lv, Qi; Gu, Songzhi; Gao, Xiaopan; Cui, Sheng; Wang, Jianmin; Qin, Chuan; Jin, Qi

2017-06-15

Middle East respiratory syndrome coronavirus (MERS-CoV) infection in humans is highly lethal, with a fatality rate of 35%. New prophylactic and therapeutic strategies to combat human infections are urgently needed. We isolated a fully human neutralizing antibody, MCA1, from a human survivor. The antibody recognizes the receptor-binding domain of MERS-CoV S glycoprotein and interferes with the interaction between viral S and the human cellular receptor human dipeptidyl peptidase 4 (DPP4). To our knowledge, this study is the first to report a human neutralizing monoclonal antibody that completely inhibits MERS-CoV replication in common marmosets. Monotherapy with MCA1 represents a potential alternative treatment for human infections with MERS-CoV worthy of evaluation in clinical settings. © Crown copyright 2017.

Structural determinants of phenotypic diversity and replication rate of human prions.

Directory of Open Access Journals (Sweden)

Jiri G Safar

2015-04-01

Full Text Available The infectious pathogen responsible for prion diseases is the misfolded, aggregated form of the prion protein, PrPSc. In contrast to recent progress in studies of laboratory rodent-adapted prions, current understanding of the molecular basis of human prion diseases and, especially, their vast phenotypic diversity is very limited. Here, we have purified proteinase resistant PrPSc aggregates from two major phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD, determined their conformational stability and replication tempo in vitro, as well as characterized structural organization using recently emerged approaches based on hydrogen/deuterium (H/D exchange coupled with mass spectrometry. Our data clearly demonstrate that these phenotypically distant prions differ in a major way with regard to their structural organization, both at the level of the polypeptide backbone (as indicated by backbone amide H/D exchange data as well as the quaternary packing arrangements (as indicated by H/D exchange kinetics for histidine side chains. Furthermore, these data indicate that, in contrast to previous observations on yeast and some murine prion strains, the replication rate of sCJD prions is primarily determined not by conformational stability but by specific structural features that control the growth rate of prion protein aggregates.

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

Science.gov (United States)

Smith, Owen K.; Aladjem, Mirit I.

2014-01-01

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

DNA replication stress: from molecular mechanisms to human disease.

Science.gov (United States)

Muñoz, Sergio; Méndez, Juan

2017-02-01

The genome of proliferating cells must be precisely duplicated in each cell division cycle. Chromosomal replication entails risks such as the possibility of introducing breaks and/or mutations in the genome. Hence, DNA replication requires the coordinated action of multiple proteins and regulatory factors, whose deregulation causes severe developmental diseases and predisposes to cancer. In recent years, the concept of "replicative stress" (RS) has attracted much attention as it impinges directly on genomic stability and offers a promising new avenue to design anticancer therapies. In this review, we summarize recent progress in three areas: (1) endogenous and exogenous factors that contribute to RS, (2) molecular mechanisms that mediate the cellular responses to RS, and (3) the large list of diseases that are directly or indirectly linked to RS.

Double silencing of relevant genes suggests the existence of the direct link between DNA replication/repair and central carbon metabolism in human fibroblasts.

Science.gov (United States)

Wieczorek, Aneta; Fornalewicz, Karolina; Mocarski, Łukasz; Łyżeń, Robert; Węgrzyn, Grzegorz

2018-04-15

Genetic evidence for a link between DNA replication and glycolysis has been demonstrated a decade ago in Bacillus subtilis, where temperature-sensitive mutations in genes coding for replication proteins could be suppressed by mutations in genes of glycolytic enzymes. Then, a strong influence of dysfunctions of particular enzymes from the central carbon metabolism (CCM) on DNA replication and repair in Escherichia coli was reported. Therefore, we asked if such a link occurs only in bacteria or it is a more general phenomenon. Here, we demonstrate that effects of silencing (provoked by siRNA) of expression of genes coding for proteins involved in DNA replication and repair (primase, DNA polymerase ι, ligase IV, and topoisomerase IIIβ) on these processes (less efficient entry into the S phase of the cell cycle and decreased level of DNA synthesis) could be suppressed by silencing of specific genes of enzymes from CMM. Silencing of other pairs of replication/repair and CMM genes resulted in enhancement of the negative effects of lower expression levels of replication/repair genes. We suggest that these results may be proposed as a genetic evidence for the link between DNA replication/repair and CMM in human cells, indicating that it is a common biological phenomenon, occurring from bacteria to humans. Copyright © 2018 Elsevier B.V. All rights reserved.

DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication.

Science.gov (United States)

Bristol, Molly L; Wang, Xu; Smith, Nathan W; Son, Minkyeong P; Evans, Michael R; Morgan, Iain M

2016-06-22

Human papillomaviruses (HPVs) are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR) pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs) could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II β binding protein I (TopBP1), does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer.

DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication

Directory of Open Access Journals (Sweden)

Molly L. Bristol

2016-06-01

Full Text Available Human papillomaviruses (HPVs are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II β binding protein I (TopBP1, does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer.

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...

The spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase β

Science.gov (United States)

Koag, Myong-Chul; Nam, Kwangho; Lee, Seongmin

2014-01-01

To provide molecular-level insights into the spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase β (polβ), we report four crystal structures of polβ complexed with dG•dTTP and dA•dCTP mismatches in the presence of Mg2+ or Mn2+. The Mg2+-bound ground-state structures show that the dA•dCTP-Mg2+ complex adopts an ‘intermediate’ protein conformation while the dG•dTTP-Mg2+ complex adopts an open protein conformation. The Mn2+-bound ‘pre-chemistry-state’ structures show that the dA•dCTP-Mn2+ complex is structurally very similar to the dA•dCTP-Mg2+ complex, whereas the dG•dTTP-Mn2+ complex undergoes a large-scale conformational change to adopt a Watson–Crick-like dG•dTTP base pair and a closed protein conformation. These structural differences, together with our molecular dynamics simulation studies, suggest that polβ increases replication fidelity via a two-stage mismatch discrimination mechanism, where one is in the ground state and the other in the closed conformation state. In the closed conformation state, polβ appears to allow only a Watson–Crick-like conformation for purine•pyrimidine base pairs, thereby discriminating the mismatched base pairs based on their ability to form the Watson–Crick-like conformation. Overall, the present studies provide new insights into the spontaneous replication error and the replication fidelity mechanisms of polβ. PMID:25200079

Piper betle L. Modulates Senescence-Associated Genes Expression in Replicative Senescent Human Diploid Fibroblasts

Directory of Open Access Journals (Sweden)

Lina Wati Durani

2017-01-01

Full Text Available Piper betle (PB is a traditional medicine that is widely used to treat different diseases around Asian region. The leaf extracts contain various bioactive compounds, which were reported to have antidiabetic, antibacterial, anti-inflammatory, antioxidant, and anticancer effects. In this study, the effect of PB aqueous extracts on replicative senescent human diploid fibroblasts (HDFs was investigated by determining the expressions of senescence-associated genes using quantitative PCR. Our results showed that PB extracts at 0.4 mg/ml can improve cell proliferation of young (143%, presenescent (127.3%, and senescent (157.3% HDFs. Increased expressions of PRDX6, TP53, CDKN2A, PAK2, and MAPK14 were observed in senescent HDFs compared to young and/or presenescent HDFs. Treatment with PB extracts modulates the transcriptional profile changes in senescent HDFs. By contrast, expressions of SOD1 increased, whereas GPX1, PRDX6, TP53, CDKN2A, PAK2, and MAPK14 were decreased in PB-treated senescent HDFs compared to untreated senescent HDFs. In conclusion, this study indicates the modulation of PB extracts on senescence-associated genes expression of replicative senescent HDFs. Further studies warrant determining the mechanism of PB in modulating replicative senescence of HDFs through these signaling pathways.

Piper betle L. Modulates Senescence-Associated Genes Expression in Replicative Senescent Human Diploid Fibroblasts.

Science.gov (United States)

Durani, Lina Wati; Khor, Shy Cian; Tan, Jen Kit; Chua, Kien Hui; Mohd Yusof, Yasmin Anum; Makpol, Suzana

2017-01-01

Piper betle (PB) is a traditional medicine that is widely used to treat different diseases around Asian region. The leaf extracts contain various bioactive compounds, which were reported to have antidiabetic, antibacterial, anti-inflammatory, antioxidant, and anticancer effects. In this study, the effect of PB aqueous extracts on replicative senescent human diploid fibroblasts (HDFs) was investigated by determining the expressions of senescence-associated genes using quantitative PCR. Our results showed that PB extracts at 0.4 mg/ml can improve cell proliferation of young (143%), presenescent (127.3%), and senescent (157.3%) HDFs. Increased expressions of PRDX6 , TP53 , CDKN2A , PAK2 , and MAPK14 were observed in senescent HDFs compared to young and/or presenescent HDFs. Treatment with PB extracts modulates the transcriptional profile changes in senescent HDFs. By contrast, expressions of SOD1 increased, whereas GPX1 , PRDX6 , TP53 , CDKN2A , PAK2 , and MAPK14 were decreased in PB-treated senescent HDFs compared to untreated senescent HDFs. In conclusion, this study indicates the modulation of PB extracts on senescence-associated genes expression of replicative senescent HDFs. Further studies warrant determining the mechanism of PB in modulating replicative senescence of HDFs through these signaling pathways.

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.

Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication

Directory of Open Access Journals (Sweden)

Candolfi Ermanno

2011-07-01

Full Text Available Abstract The Human Phosphate-Binding protein (HPBP is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.

Replication-dependent 65R→K reversion in human immunodeficiency virus type 1 reverse transcriptase double mutant K65R + L74V

International Nuclear Information System (INIS)

Sharma, Prem L.; Nurpeisov, Viktoria; Lee, Kimberly; Skaggs, Sara; Di San Filippo, Christina Amat; Schinazi, Raymond F.

2004-01-01

Understanding of the mechanisms of interaction among nucleoside reverse transcriptase inhibitor (NRTI)-selected mutations in the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) coding sequence is essential for the design of newer drugs and for enhancing our vision of the structure function relationship among amino acids of the polymerase domain of HIV-1. Although several nucleoside reverse transcriptase inhibitors select RT mutations K65R and L74V, the combination of 65R + 74V is rare in clinics. A novel NRTI (-)-β-D-dioxolane-guanosine (DXG) is known to select in vitro either the 65R or 74V mutant virus (Antimicrob. Agents Chemother. 44 (2000) 1783). These mutations were not selected together during repeated passaging of the HIV-1 in the presence of this drug. To analyze the impact of these RT mutations on viral replication, a double mutant containing K65R + L74V was created by site-directed mutagenesis in a pNL4-3 background. Replication kinetic assays revealed that the mutant K65R + L74V is unstable, and 65R→K reversion occurs during replication of virus in phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication kinetic assays in MT-2 cells demonstrated that double mutant 65R + 74V is highly attenuated for replication and the initiation of reversion is related to the increase in RT activity. Additionally, the suppression of viral replication in the presence of DXG or under suboptimal human recombinant interleukin-2 leads to minimal or no 65R→K reversion. These observations provide evidence that 65R→K reversion in the double mutant 65R + 74V is dependent on a specific rate of viral replication in a pNL4-3 background. A similar phenomenon may occur in vivo, which may have implications for treatment management strategies

Canine distemper virus isolated from a monkey efficiently replicates on Vero cells expressing non-human primate SLAM receptors but not human SLAM receptor.

Science.gov (United States)

Feng, Na; Liu, Yuxiu; Wang, Jianzhong; Xu, Weiwei; Li, Tiansong; Wang, Tiecheng; Wang, Lei; Yu, Yicong; Wang, Hualei; Zhao, Yongkun; Yang, Songtao; Gao, Yuwei; Hu, Guixue; Xia, Xianzhu

2016-08-02

In 2008, an outbreak of canine distemper virus (CDV) infection in monkeys was reported in China. We isolated CDV strain (subsequently named Monkey-BJ01-DV) from lung tissue obtained from a rhesus monkey that died in this outbreak. We evaluated the ability of this virus on Vero cells expressing SLAM receptors from dog, monkey and human origin, and analyzed the H gene of Monkey-BJ01-DV with other strains. The Monkey-BJ01-DV isolate replicated to the highest titer on Vero cells expressing dog-origin SLAM (10(5.2±0.2) TCID50/ml) and monkey-origin SLAM (10(5.4±0.1) TCID50/ml), but achieved markedly lower titers on human-origin SLAM cells (10(3.3±0.3) TCID50/ml). Phylogenetic analysis of the full-length H gene showed that Monkey-BJ01-DV was highly related to other CDV strains obtained during recent CDV epidemics among species of the Canidae family in China, and these Monkey strains CDV (Monkey-BJ01-DV, CYN07-dV, Monkey-KM-01) possessed a number of amino acid specific substitutions (E276V, Q392R, D435Y and I542F) compared to the H protein of CDV epidemic in other animals at the same period. Our results suggested that the monkey origin-CDV-H protein could possess specific substitutions to adapt to the new host. Monkey-BJ01-DV can efficiently use monkey- and dog-origin SLAM to infect and replicate in host cells, but further adaptation may be required for efficient replication in host cells expressing the human SLAM receptor.

Interaction of the retinoblastoma protein with Orc1 and its recruitment to human origins of DNA replication.

Directory of Open Access Journals (Sweden)

Ramiro Mendoza-Maldonado

Full Text Available BACKGROUND: The retinoblastoma protein (Rb is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1 specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. CONCLUSIONS/SIGNIFICANCE: The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of genes required for cell cycle progression to the activation of DNA replication.

Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

DEFF Research Database (Denmark)

Bartkova, J; Hamerlik, P; Stockhausen, Marie

2010-01-01

damage signalling in low- and high-grade human gliomas, and analyze the sources of such endogenous genotoxic stress. Based on analyses of human glioblastoma multiforme (GBM) cell lines, normal astrocytes and clinical specimens from grade II astrocytomas (n=41) and grade IV GBM (n=60), we conclude...... that the DDR machinery is constitutively activated in gliomas, as documented by phosphorylated histone H2AX (gammaH2AX), activation of the ATM-Chk2-p53 pathway, 53BP1 foci and other markers. Oxidative DNA damage (8-oxoguanine) was high in some GBM cell lines and many GBM tumors, while it was low in normal...... brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low...

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.

The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

Energy Technology Data Exchange (ETDEWEB)

Ziehr, Ben; Lenarcic, Erik; Cecil, Chad; Moorman, Nathaniel J., E-mail: nmoorman@med.unc.edu

2016-02-15

Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compound that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.

The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

International Nuclear Information System (INIS)

Ziehr, Ben; Lenarcic, Erik; Cecil, Chad; Moorman, Nathaniel J.

2016-01-01

Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compound that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.

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.

Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter.

Science.gov (United States)

Wang, Xiaohong; Liu, Haibin; Ge, Hui; Ajiro, Masahiko; Sharma, Nishi R; Meyers, Craig; Morozov, Pavel; Tuschl, Thomas; Klar, Amar; Court, Donald; Zheng, Zhi-Ming

2017-05-30

The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P 811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis -acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors. IMPORTANCE It has been known for decades that the activity of viral late promoters is associated with viral DNA replication among almost all DNA viruses. However, the mechanism of how DNA replication activates the viral late promoter and what components of the replication machinery are involved remain largely unknown. In this study, we characterized the P 811 promoter region of HPV18 and demonstrated that its activation depends on the orientation of DNA replication. Using single

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

Directory of Open Access Journals (Sweden)

Ghania Hammad

2018-01-01

Full Text Available 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 < 0.05 to the 2D-DIGE analyses revealed 81 differentially expressed protein spots, from which 123 proteins of interest were identified by mass spectrometry. We compared the changes in protein abundance for three different conditions: (i spots varying between young and presenescent cells, (ii spots varying in response to selenium concentration in young cells, and (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

Design Optimization of Time- and Cost-Constrained Fault-Tolerant Embedded Systems with Checkpointing and Replication

DEFF Research Database (Denmark)

Pop, Paul; Izosimov, Viacheslav; Eles, Petru

2009-01-01

We present an approach to the synthesis of fault-tolerant hard real-time systems for safety-critical applications. We use checkpointing with rollback recovery and active replication for tolerating transient faults. Processes and communications are statically scheduled. Our synthesis approach deci...

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.

‘The octet’: eight protein kinases that control mammalian DNA replication

Directory of Open Access Journals (Sweden)

Melvin L. Depamphilis

2012-09-01

Full Text Available Development of a fertilized human egg into an average sized adult requires about 29 trillion cell divisions, thereby producing enough DNA to stretch to the Sun and back 200 times (DePamphilis and Bell, 2011! Even more amazing is the fact that throughout these mitotic cell cycles, the human genome is duplicated once and only once each time a cell divides. If a cell accidentally begins to re-replicate its nuclear DNA prior to cell division, checkpoint pathways trigger apoptosis. And yet, some cells are developmentally programmed to respond to environmental cues by switching from mitotic cell cycles to endocycles, a process in which multiple S phases occur in the absence of either mitosis or cytokinesis. Endocycles allow production of viable, differentiated, polyploid cells that no longer proliferate. What is surprising is that among the 516 (Manning et al., 2002 to 557 (BioMart web site protein kinases encoded by the human genome, only eight regulate nuclear DNA replication directly. These are Cdk1, Cdk2, Cdk4, Cdk6, Cdk7, Cdc7, Chk1 and Chk2. Even more remarkable is the fact that only four of these enzymes (Cdk1, Cdk7, Cdc7 and Chk1 are essential for mammalian development. Here we describe how these protein kinases determine when DNA replication occurs during mitotic cell cycles, how mammalian cells switch from mitotic cell cycles to endocycles, and how cancer cells can be selectively targeted for destruction by inducing them to begin a second S phase before mitosis is complete.

Avian influenza viruses that cause highly virulent infections in humans exhibit distinct replicative properties in contrast to human H1N1 viruses

Science.gov (United States)

Simon, Philippe F.; de La Vega, Marc-Antoine; Paradis, Éric; Mendoza, Emelissa; Coombs, Kevin M.; Kobasa, Darwyn; Beauchemin, Catherine A. A.

2016-04-01

Avian influenza viruses present an emerging epidemiological concern as some strains of H5N1 avian influenza can cause severe infections in humans with lethality rates of up to 60%. These have been in circulation since 1997 and recently a novel H7N9-subtyped virus has been causing epizootics in China with lethality rates around 20%. To better understand the replication kinetics of these viruses, we combined several extensive viral kinetics experiments with mathematical modelling of in vitro infections in human A549 cells. We extracted fundamental replication parameters revealing that, while both the H5N1 and H7N9 viruses replicate faster and to higher titers than two low-pathogenicity H1N1 strains, they accomplish this via different mechanisms. While the H7N9 virions exhibit a faster rate of infection, the H5N1 virions are produced at a higher rate. Of the two H1N1 strains studied, the 2009 pandemic H1N1 strain exhibits the longest eclipse phase, possibly indicative of a less effective neuraminidase activity, but causes infection more rapidly than the seasonal strain. This explains, in part, the pandemic strain’s generally slower growth kinetics and permissiveness to accept mutations causing neuraminidase inhibitor resistance without significant loss in fitness. Our results highlight differential growth properties of H1N1, H5N1 and H7N9 influenza viruses.

Replication of avian, human and swine influenza viruses in porcine respiratory explants and association with sialic acid distribution

Directory of Open Access Journals (Sweden)

Nauwynck Hans J

2010-02-01

Full Text Available Abstract Background Throughout the history of human influenza pandemics, pigs have been considered the most likely "mixing vessel" for reassortment between human and avian influenza viruses (AIVs. However, the replication efficiencies of influenza viruses from various hosts, as well as the expression of sialic acid (Sia receptor variants in the entire porcine respiratory tract have never been studied in detail. Therefore, we established porcine nasal, tracheal, bronchial and lung explants, which cover the entire porcine respiratory tract with maximal similarity to the in vivo situation. Subsequently, we assessed virus yields of three porcine, two human and six AIVs in these explants. Since our results on virus replication were in disagreement with the previously reported presence of putative avian virus receptors in the trachea, we additionally studied the distribution of sialic acid receptors by means of lectin histochemistry. Human (Siaα2-6Gal and avian virus receptors (Siaα2-3Gal were identified with Sambucus Nigra and Maackia amurensis lectins respectively. Results Compared to swine and human influenza viruses, replication of the AIVs was limited in all cultures but most strikingly in nasal and tracheal explants. Results of virus titrations were confirmed by quantification of infected cells using immunohistochemistry. By lectin histochemistry we found moderate to abundant expression of the human-like virus receptors in all explant systems but minimal binding of the lectins that identify avian-like receptors, especially in the nasal, tracheal and bronchial epithelium. Conclusions The species barrier that restricts the transmission of influenza viruses from one host to another remains preserved in our porcine respiratory explants. Therefore this system offers a valuable alternative to study virus and/or host properties required for adaptation or reassortment of influenza viruses. Our results indicate that, based on the expression of Sia

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.

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

Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

International Nuclear Information System (INIS)

Dai Heqiao; Liu Jianying; Malkas, Linda H.; Catalano, Jennifer; Alagharu, Srilakshmi; Hickey, Robert J.

2009-01-01

Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC 50 of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases α, δ and ε is 15, 45 and 125 μM, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC 50 = 88 μM), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2-13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr(III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication

Distinct functions of human RecQ helicases during DNA replication

Czech Academy of Sciences Publication Activity Database

Urban, Václav; Dobrovolná, Jana; Janščák, Pavel

2017-01-01

Roč. 225, červen (2017), s. 20-26 ISSN 0301-4622 R&D Projects: GA ČR(CZ) GA14-05743S; GA MŠk LH14037 Institutional support: RVO:68378050 Keywords : DNA replication * Replication stress * RecQ helicases * Genomic instability * Cancer Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 2.402, year: 2016

Zinc binding activity of human metapneumovirus M2-1 protein is indispensable for viral replication and pathogenesis in vivo.

Science.gov (United States)

Cai, Hui; Zhang, Yu; Ma, Yuanmei; Sun, Jing; Liang, Xueya; Li, Jianrong

2015-06-01

Human metapneumovirus (hMPV) is a member of the Pneumovirinae subfamily in the Paramyxoviridae family that causes respiratory tract infections in humans. Unlike members of the Paramyxovirinae subfamily, the polymerase complex of pneumoviruses requires an additional cofactor, the M2-1 protein, which functions as a transcriptional antitermination factor. The M2-1 protein was found to incorporate zinc ions, although the specific role(s) of the zinc binding activity in viral replication and pathogenesis remains unknown. In this study, we found that the third cysteine (C21) and the last histidine (H25) in the zinc binding motif (CCCH) of hMPV M2-1 were essential for zinc binding activity, whereas the first two cysteines (C7 and C15) play only minor or redundant roles in zinc binding. In addition, the zinc binding motif is essential for the oligomerization of M2-1. Subsequently, recombinant hMPVs (rhMPVs) carrying mutations in the zinc binding motif were recovered. Interestingly, rhMPV-C21S and -H25L mutants, which lacked zinc binding activity, had delayed replication in cell culture and were highly attenuated in cotton rats. In contrast, rhMPV-C7S and -C15S strains, which retained 60% of the zinc binding activity, replicated as efficiently as rhMPV in cotton rats. Importantly, rhMPVs that lacked zinc binding activity triggered high levels of neutralizing antibody and provided complete protection against challenge with rhMPV. Taken together, these results demonstrate that zinc binding activity is indispensable for viral replication and pathogenesis in vivo. These results also suggest that inhibition of zinc binding activity may serve as a novel approach to rationally attenuate hMPV and perhaps other pneumoviruses for vaccine purposes. 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

A stochastic step model of replicative senescence explains ROS production rate in ageing cell populations.

Directory of Open Access Journals (Sweden)

Conor Lawless

Full Text Available Increases in cellular Reactive Oxygen Species (ROS concentration with age have been observed repeatedly in mammalian tissues. Concomitant increases in the proportion of replicatively senescent cells in ageing mammalian tissues have also been observed. Populations of mitotic human fibroblasts cultured in vitro, undergoing transition from proliferation competence to replicative senescence are useful models of ageing human tissues. Similar exponential increases in ROS with age have been observed in this model system. Tracking individual cells in dividing populations is difficult, and so the vast majority of observations have been cross-sectional, at the population level, rather than longitudinal observations of individual cells.One possible explanation for these observations is an exponential increase in ROS in individual fibroblasts with time (e.g. resulting from a vicious cycle between cellular ROS and damage. However, we demonstrate an alternative, simple hypothesis, equally consistent with these observations which does not depend on any gradual increase in ROS concentration: the Stochastic Step Model of Replicative Senescence (SSMRS. We also demonstrate that, consistent with the SSMRS, neither proliferation-competent human fibroblasts of any age, nor populations of hTERT overexpressing human fibroblasts passaged beyond the Hayflick limit, display high ROS concentrations. We conclude that longitudinal studies of single cells and their lineages are now required for testing hypotheses about roles and mechanisms of ROS increase during replicative senescence.

A stochastic step model of replicative senescence explains ROS production rate in ageing cell populations.

Science.gov (United States)

Lawless, Conor; Jurk, Diana; Gillespie, Colin S; Shanley, Daryl; Saretzki, Gabriele; von Zglinicki, Thomas; Passos, João F

2012-01-01

Increases in cellular Reactive Oxygen Species (ROS) concentration with age have been observed repeatedly in mammalian tissues. Concomitant increases in the proportion of replicatively senescent cells in ageing mammalian tissues have also been observed. Populations of mitotic human fibroblasts cultured in vitro, undergoing transition from proliferation competence to replicative senescence are useful models of ageing human tissues. Similar exponential increases in ROS with age have been observed in this model system. Tracking individual cells in dividing populations is difficult, and so the vast majority of observations have been cross-sectional, at the population level, rather than longitudinal observations of individual cells.One possible explanation for these observations is an exponential increase in ROS in individual fibroblasts with time (e.g. resulting from a vicious cycle between cellular ROS and damage). However, we demonstrate an alternative, simple hypothesis, equally consistent with these observations which does not depend on any gradual increase in ROS concentration: the Stochastic Step Model of Replicative Senescence (SSMRS). We also demonstrate that, consistent with the SSMRS, neither proliferation-competent human fibroblasts of any age, nor populations of hTERT overexpressing human fibroblasts passaged beyond the Hayflick limit, display high ROS concentrations. We conclude that longitudinal studies of single cells and their lineages are now required for testing hypotheses about roles and mechanisms of ROS increase during replicative senescence.

Evidence supporting a role for TopBP1 and Brd4 in the initiation but not continuation of human papillomavirus 16 E1/E2-mediated DNA replication.

Science.gov (United States)

Gauson, Elaine J; Donaldson, Mary M; Dornan, Edward S; Wang, Xu; Bristol, Molly; Bodily, Jason M; Morgan, Iain M

2015-05-01

To replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination. Human papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease

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...

Dengue virus replicates and accumulates in Aedes aegypti salivary glands

Energy Technology Data Exchange (ETDEWEB)

Raquin, Vincent, E-mail: vincent.raquin@univ-lyon1.fr [Insect-Virus Interactions Group, Department of Genomes and Genetics, Institut Pasteur, 75015 Paris (France); Centre National de la Recherche Scientifique, Unité de Recherche Associée 3012, 75015 Paris (France); Lambrechts, Louis, E-mail: louis.lambrechts@pasteur.fr [Insect-Virus Interactions Group, Department of Genomes and Genetics, Institut Pasteur, 75015 Paris (France); Centre National de la Recherche Scientifique, Unité de Recherche Associée 3012, 75015 Paris (France)

2017-07-15

Dengue virus (DENV) is an RNA virus transmitted among humans by mosquito vectors, mainly Aedes aegypti. DENV transmission requires viral dissemination from the mosquito midgut to the salivary glands. During this process the virus undergoes several population bottlenecks, which are stochastic reductions in population size that restrict intra-host viral genetic diversity and limit the efficiency of natural selection. Despite the implications for virus transmission and evolution, DENV replication in salivary glands has not been directly demonstrated. Here, we used a strand-specific quantitative RT-PCR assay to demonstrate that negative-strand DENV RNA is produced in Ae. aegypti salivary glands, providing conclusive evidence that viral replication occurs in this tissue. Furthermore, we showed that the concentration of DENV genomic RNA in salivary glands increases significantly over time, indicating that active replication likely replenishes DENV genetic diversity prior to transmission. These findings improve our understanding of the biological determinants of DENV fitness and evolution. - Highlights: •Strand-specific RT-qPCR allows accurate quantification of DENV (-) RNA in mosquito tissues. •Detection of DENV (-) RNA in salivary glands provides evidence of viral replication in this tissue. •Viral replication in salivary glands likely replenishes DENV genetic diversity prior to transmission.

Dengue virus replicates and accumulates in Aedes aegypti salivary glands

International Nuclear Information System (INIS)

Raquin, Vincent; Lambrechts, Louis

2017-01-01

Dengue virus (DENV) is an RNA virus transmitted among humans by mosquito vectors, mainly Aedes aegypti. DENV transmission requires viral dissemination from the mosquito midgut to the salivary glands. During this process the virus undergoes several population bottlenecks, which are stochastic reductions in population size that restrict intra-host viral genetic diversity and limit the efficiency of natural selection. Despite the implications for virus transmission and evolution, DENV replication in salivary glands has not been directly demonstrated. Here, we used a strand-specific quantitative RT-PCR assay to demonstrate that negative-strand DENV RNA is produced in Ae. aegypti salivary glands, providing conclusive evidence that viral replication occurs in this tissue. Furthermore, we showed that the concentration of DENV genomic RNA in salivary glands increases significantly over time, indicating that active replication likely replenishes DENV genetic diversity prior to transmission. These findings improve our understanding of the biological determinants of DENV fitness and evolution. - Highlights: •Strand-specific RT-qPCR allows accurate quantification of DENV (-) RNA in mosquito tissues. •Detection of DENV (-) RNA in salivary glands provides evidence of viral replication in this tissue. •Viral replication in salivary glands likely replenishes DENV genetic diversity prior to transmission.

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.

A human fecal contamination score for ranking recreational sites using the HF183/BacR287 quantitative real-time PCR method.

Science.gov (United States)

Cao, Yiping; Sivaganesan, Mano; Kelty, Catherine A; Wang, Dan; Boehm, Alexandria B; Griffith, John F; Weisberg, Stephen B; Shanks, Orin C

2018-01-01

Human fecal pollution of recreational waters remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality research and management. However, there are currently no standardized approaches for field implementation and interpretation of qPCR data. In this study, a standardized HF183/BacR287 qPCR method was combined with a water sampling strategy and a novel Bayesian weighted average approach to establish a human fecal contamination score (HFS) that can be used to prioritize sampling sites for remediation based on measured human waste levels. The HFS was then used to investigate 975 study design scenarios utilizing different combinations of sites with varying sampling intensities (daily to once per week) and number of qPCR replicates per sample (2-14 replicates). Findings demonstrate that site prioritization with HFS is feasible and that both sampling intensity and number of qPCR replicates influence reliability of HFS estimates. The novel data analysis strategy presented here provides a prescribed approach for the implementation and interpretation of human-associated HF183/BacR287 qPCR data with the goal of site prioritization based on human fecal pollution levels. In addition, information is provided for future users to customize study designs for optimal HFS performance. Published by Elsevier Ltd.

A DNA sequence element that advances replication origin activation time in Saccharomyces cerevisiae.

Science.gov (United States)

Pohl, Thomas J; Kolor, Katherine; Fangman, Walton L; Brewer, Bonita J; Raghuraman, M K

2013-11-06

Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.

A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells.

Science.gov (United States)

Li, Bing; Su, Trent; Ferrari, Roberto; Li, Jing-Yu; Kurdistani, Siavash K

2014-02-01

The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells.

Absence of specificity in inhibition of DNA repair replication by DNA-binding agents, cocarcinogens, and steroids in human cells

International Nuclear Information System (INIS)

Cleaver, J.E.; Painter, R.B.

1975-01-01

Although many chemicals, including cocarcinogens, DNA-binding agents, and steroids, inhibit repair replication of ultraviolet-induced damage to DNA in human lymphocytes and proliferating cells in culture, none of these chemicals is specific. Our results show that all the chemicals we tested inhibit normal DNA synthesis as much as or more than they inhibit repair replication. There is thus no evidence in our results to support the hypothesis that cocarcinogens are specific inhibitors of DNA repair or that any of the chemicals studied might be useful adjuncts to tumor therapy merely because of specific inhibition of radiation repair mechanisms

Adeno-associated virus type 2 enhances goose parvovirus replication in embryonated goose eggs

International Nuclear Information System (INIS)

Malkinson, Mertyn; Winocour, Ernest

2005-01-01

The autonomous goose parvovirus (GPV) and the human helper-dependent adeno-associated virus type 2 (AAV2) share a high degree of homology. To determine if this evolutionary relationship has a biological impact, we studied viral replication in human 293 cells and in embryonated goose eggs coinfected with both viruses. Similar experiments were performed with the minute virus of mice (MVM), an autonomous murine parvovirus with less homology to AAV2. In human 293 cells, both GPV and MVM augmented AAV2 replication. In contrast, AAV2 markedly enhanced GPV replication in embryonated goose eggs under conditions where a similar effect was not observed with MVM. AAV2 did not replicate in embryonated goose eggs and AAV2 inactivated by UV-irradiation also enhanced GPV replication. To our knowledge, this is the first report that a human helper-dependent member of the Parvoviridae can provide helper activity for an autonomous parvovirus in a natural host

Timeless links replication termination to mitotic kinase activation.

Directory of Open Access Journals (Sweden)

Jayaraju Dheekollu

2011-05-01

Full Text Available The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1. Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.

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)

Genetic characterization of an adapted pandemic 2009 H1N1 influenza virus that reveals improved replication rates in human lung epithelial cells

International Nuclear Information System (INIS)

Wörmann, Xenia; Lesch, Markus; Welke, Robert-William; Okonechnikov, Konstantin; Abdurishid, Mirshat; Sieben, Christian; Geissner, Andreas; Brinkmann, Volker; Kastner, Markus; Karner, Andreas; Zhu, Rong; Hinterdorfer, Peter; Anish, Chakkumkal; Seeberger, Peter H.; Herrmann, Andreas

2016-01-01

The 2009 influenza pandemic originated from a swine-origin H1N1 virus, which, although less pathogenic than anticipated, may acquire additional virulence-associated mutations in the future. To estimate the potential risk, we sequentially passaged the isolate A/Hamburg/04/2009 in A549 human lung epithelial cells. After passage 6, we observed a 100-fold increased replication rate. High-throughput sequencing of viral gene segments identified five dominant mutations, whose contribution to the enhanced growth was analyzed by reverse genetics. The increased replication rate was pinpointed to two mutations within the hemagglutinin (HA) gene segment (HA_1 D130E, HA_2 I91L), near the receptor binding site and the stem domain. The adapted virus also replicated more efficiently in mice in vivo. Enhanced replication rate correlated with increased fusion pH of the HA protein and a decrease in receptor affinity. Our data might be relevant for surveillance of pre-pandemic strains and development of high titer cell culture strains for vaccine production. - Highlights: • We observed a spontaneous mutation of a 2009-pandemic H1N1 influenza virus in vitro. • The adaptation led to a 100-fold rise in replication rate in human A549 cells. • Adaptation was caused by two mutations in the HA gene segment. • Adaptation correlates with increased fusion pH and decreased receptor affinity.

Genetic characterization of an adapted pandemic 2009 H1N1 influenza virus that reveals improved replication rates in human lung epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Wörmann, Xenia [Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin (Germany); Lesch, Markus [Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin (Germany); Steinbeis Innovation gGmbH, Center for Systems Biomedicine, Falkensee (Germany); Welke, Robert-William [Department of Biology, Molecular Biophysics, IRI Life Sciences, Humboldt-Universität zu Berlin (Germany); Okonechnikov, Konstantin; Abdurishid, Mirshat [Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin (Germany); Sieben, Christian [Department of Biology, Molecular Biophysics, IRI Life Sciences, Humboldt-Universität zu Berlin (Germany); Geissner, Andreas [Department for Biomolecular Systems, Max Planck Institute for Colloids and Interfaces, Potsdam (Germany); Institute of Chemistry and Biochemistry, Free University, Berlin (Germany); Brinkmann, Volker [Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin (Germany); Kastner, Markus [Institute for Biophysics, Johannes Kepler University, Linz (Austria); Karner, Andreas [Center for Advanced Bioanalysis GmbH (CBL), Linz (Austria); Zhu, Rong; Hinterdorfer, Peter [Institute for Biophysics, Johannes Kepler University, Linz (Austria); Anish, Chakkumkal [Department for Biomolecular Systems, Max Planck Institute for Colloids and Interfaces, Potsdam (Germany); Seeberger, Peter H. [Department for Biomolecular Systems, Max Planck Institute for Colloids and Interfaces, Potsdam (Germany); Institute of Chemistry and Biochemistry, Free University, Berlin (Germany); Herrmann, Andreas [Department of Biology, Molecular Biophysics, IRI Life Sciences, Humboldt-Universität zu Berlin (Germany); and others

2016-05-15

The 2009 influenza pandemic originated from a swine-origin H1N1 virus, which, although less pathogenic than anticipated, may acquire additional virulence-associated mutations in the future. To estimate the potential risk, we sequentially passaged the isolate A/Hamburg/04/2009 in A549 human lung epithelial cells. After passage 6, we observed a 100-fold increased replication rate. High-throughput sequencing of viral gene segments identified five dominant mutations, whose contribution to the enhanced growth was analyzed by reverse genetics. The increased replication rate was pinpointed to two mutations within the hemagglutinin (HA) gene segment (HA{sub 1} D130E, HA{sub 2} I91L), near the receptor binding site and the stem domain. The adapted virus also replicated more efficiently in mice in vivo. Enhanced replication rate correlated with increased fusion pH of the HA protein and a decrease in receptor affinity. Our data might be relevant for surveillance of pre-pandemic strains and development of high titer cell culture strains for vaccine production. - Highlights: • We observed a spontaneous mutation of a 2009-pandemic H1N1 influenza virus in vitro. • The adaptation led to a 100-fold rise in replication rate in human A549 cells. • Adaptation was caused by two mutations in the HA gene segment. • Adaptation correlates with increased fusion pH and decreased receptor affinity.

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.

Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes

International Nuclear Information System (INIS)

Hirose, Yukihiro; Nagahori, Hirohisa; Yamada, Tomoya; Deguchi, Yoshihito; Tomigahara, Yoshitaka; Nishioka, Kazuhiko; Uwagawa, Satoshi; Kawamura, Satoshi; Isobe, Naohiko; Lake, Brian G.; Okuno, Yasuyoshi

2009-01-01

High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 μM MTF and 50 μM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2'-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10-1000 μM MTF and 100-500 μM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5-50 ng/ml epidermal growth factor or 5-100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver

Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes.

Science.gov (United States)

Hirose, Yukihiro; Nagahori, Hirohisa; Yamada, Tomoya; Deguchi, Yoshihito; Tomigahara, Yoshitaka; Nishioka, Kazuhiko; Uwagawa, Satoshi; Kawamura, Satoshi; Isobe, Naohiko; Lake, Brian G; Okuno, Yasuyoshi

2009-04-05

High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 microM MTF and 50 microM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2'-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10-1000 microM MTF and 100-500 microM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5-50 ng/ml epidermal growth factor or 5-100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver.

Replicated Risk Nicotinic Cholinergic Receptor Genes for Nicotine Dependence

Directory of Open Access Journals (Sweden)

Lingjun Zuo

2016-11-01

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

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...

Regulation of SUMO2 Target Proteins by the Proteasome in Human Cells Exposed to Replication Stress

DEFF Research Database (Denmark)

Bursomanno, Sara; McGouran, Joanna F; Kessler, Benedikt M

2015-01-01

In human cells, SUMO2 is predominantly conjugated to target proteins in response to cellular stress. Previous studies suggested that proteins conjugated to SUMO2, but not to SUMO1, could be regulated by the ubiquitin-mediated proteasome system. Hence, we set out to understand the role...... of the proteasome in determining the fate of proteins conjugated to SUMO2 when cells are treated with DNA replication stress conditions. We conducted a quantitative proteomic analysis in a U2OS cell line stably expressing SUMO2(Q87R) tagged with StrepHA in the presence or absence of epoxomicin (EPOX), a proteasome...... inhibitor. We identified subgroups of putative SUMO2 targets that were either degraded or stabilized by EPOX upon SUMO2 conjugation in response to replication stress. Interestingly, the subgroup of proteins degraded upon SUMO2 conjugation was enriched in proteins playing roles in DNA damage repair...

How do walkers behave when crossing the way of a mobile robot that replicates human interaction rules?

Science.gov (United States)

Vassallo, Christian; Olivier, Anne-Hélène; Souères, Philippe; Crétual, Armel; Stasse, Olivier; Pettré, Julien

2018-02-01

Previous studies showed the existence of implicit interaction rules shared by human walkers when crossing each other. Especially, each walker contributes to the collision avoidance task and the crossing order, as set at the beginning, is preserved along the interaction. This order determines the adaptation strategy: the first arrived increases his/her advance by slightly accelerating and changing his/her heading, whereas the second one slows down and moves in the opposite direction. In this study, we analyzed the behavior of human walkers crossing the trajectory of a mobile robot that was programmed to reproduce this human avoidance strategy. In contrast with a previous study, which showed that humans mostly prefer to give the way to a non-reactive robot, we observed similar behaviors between human-human avoidance and human-robot avoidance when the robot replicates the human interaction rules. We discuss this result in relation with the importance of controlling robots in a human-like way in order to ease their cohabitation with humans. Copyright © 2017 Elsevier B.V. All rights reserved.

A SELEX-screened aptamer of human hepatitis B virus RNA encapsidation signal suppresses viral replication.

Directory of Open Access Journals (Sweden)

Hui Feng

Full Text Available BACKGROUND: The specific interaction between hepatitis B virus (HBV polymerase (P protein and the ε RNA stem-loop on pregenomic (pg RNA is crucial for viral replication. It triggers both pgRNA packaging and reverse transcription and thus represents an attractive antiviral target. RNA decoys mimicking ε in P protein binding but not supporting replication might represent novel HBV inhibitors. However, because generation of recombinant enzymatically active HBV polymerase is notoriously difficult, such decoys have as yet not been identified. METHODOLOGY/PRINCIPAL FINDINGS: Here we used a SELEX approach, based on a new in vitro reconstitution system exploiting a recombinant truncated HBV P protein (miniP, to identify potential ε decoys in two large ε RNA pools with randomized upper stem. Selection of strongly P protein binding RNAs correlated with an unexpected strong enrichment of A residues. Two aptamers, S6 and S9, displayed particularly high affinity and specificity for miniP in vitro, yet did not support viral replication when part of a complete HBV genome. Introducing S9 RNA into transiently HBV producing HepG2 cells strongly suppressed pgRNA packaging and DNA synthesis, indicating the S9 RNA can indeed act as an ε decoy that competitively inhibits P protein binding to the authentic ε signal on pgRNA. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the first successful identification of human HBV ε aptamers by an in vitro SELEX approach. Effective suppression of HBV replication by the S9 aptamer provides proof-of-principle for the ability of ε decoy RNAs to interfere with viral P-ε complex formation and suggests that S9-like RNAs may further be developed into useful therapeutics against chronic hepatitis B.

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.

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.

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

Science.gov (United States)

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

2017-12-01

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

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression.

Science.gov (United States)

Löb, D; Lengert, N; Chagin, V O; Reinhart, M; Casas-Delucchi, C S; Cardoso, M C; Drossel, B

2016-04-07

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase.

Targeting DNA Replication Stress for Cancer Therapy

Directory of Open Access Journals (Sweden)

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.

Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells.

Science.gov (United States)

Wehbe, Michel; Huguenin, Antoine; Leveque, Nicolas; Semler, Bert L; Hamze, Monzer; Andreoletti, Laurent; Bouin, Alexis

2016-04-01

Coxsackieviruses B (CV-B) (Picornaviridae) are a common infectious cause of acute myocarditis in children and young adults, a disease, which is a precursor to 10-20% of chronic myocarditis and dilated cardiomyopathy (DCM) cases. The mechanisms involved in the disease progression from acute to chronic myocarditis phase and toward the DCM clinical stage are not fully understood but are influenced by both viral and host factors. Subgenomic replicons of CV-B can be used to assess viral replication mechanisms in human cardiac cells and evaluate the effects of potential antiviral drugs on viral replication activities. Our objectives were to generate a reporter replicon from a cardiotropic prototype CV-B3/28 strain and to characterize its replication properties into human cardiac primary cells. To obtain this replicon, a cDNA plasmid containing the full CV-B3/28 genome flanked by a hammerhead ribozyme sequence and an MluI restriction site was generated and used as a platform for the insertion of sequences encoding emerald green fluorescent protein (EmGFP) in place of those encoding VP3. In vitro transcribed RNA from this plasmid was transfected into HeLa cells and human primary cardiac cells and was able to produce EmGFP and VP1-containing polypeptides. Moreover, non-structural protein biological activity was assessed by the specific cleavage of eIF4G1 by viral 2A(pro). Viral RNA replication was indirectly demonstrated by inhibition assays, fluoxetine was added to cell culture and prevented the EmGFP synthesis. Our results indicated that the EmGFP CV-B3 replicon was able to replicate and translate as well as the CV-B3/28 prototype strain. Our EmGFP CV-B3 replicon will be a valuable tool to readily investigate CV-B3 replication activities in human target cell models. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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

Rif1 acts through Protein Phosphatase 1 but independent of replication timing to suppress telomere extension in budding yeast.

Science.gov (United States)

Kedziora, Sylwia; Gali, Vamsi K; Wilson, Rosemary H C; Clark, Kate R M; Nieduszynski, Conrad A; Hiraga, Shin-Ichiro; Donaldson, Anne D

2018-05-04

The Rif1 protein negatively regulates telomeric TG repeat length in the budding yeast Saccharomyces cerevisiae, but how it prevents telomere over-extension is unknown. Rif1 was recently shown to control DNA replication by acting as a Protein Phosphatase 1 (PP1)-targeting subunit. Therefore, we investigated whether Rif1 controls telomere length by targeting PP1 activity. We find that a Rif1 mutant defective for PP1 interaction causes a long-telomere phenotype, similar to that of rif1Δ cells. Tethering PP1 at a specific telomere partially substitutes for Rif1 in limiting TG repeat length, confirming the importance of PP1 in telomere length control. Ablating Rif1-PP1 interaction is known to cause precocious activation of telomere-proximal replication origins and aberrantly early telomere replication. However, we find that Rif1 still limits telomere length even if late replication is forced through deletion of nearby replication origins, indicating that Rif1 can control telomere length independent of replication timing. Moreover we find that, even at a de novo telomere created after DNA synthesis during a mitotic block, Rif1-PP1 interaction is required to suppress telomere lengthening and prevent inappropriate recruitment of Tel1 kinase. Overall, our results show that Rif1 controls telomere length by recruiting PP1 to directly suppress telomerase-mediated TG repeat lengthening.

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

Differential effects of the extracellular microenvironment on human embryonic stem cell differentiation into keratinocytes and their subsequent replicative life span.

Science.gov (United States)

Movahednia, Mohammad Mehdi; Kidwai, Fahad Karim; Zou, Yu; Tong, Huei Jinn; Liu, Xiaochen; Islam, Intekhab; Toh, Wei Seong; Raghunath, Michael; Cao, Tong

2015-04-01

Culture microenvironment plays a critical role in the propagation and differentiation of human embryonic stem cells (hESCs) and their differentiated progenies. Although high efficiency of hESC differentiation to keratinocytes (hESC-Kert) has been achieved, little is known regarding the effects of early culture microenvironment and pertinent extracellular matrix (ECM) interactions during epidermal commitment on subsequent proliferative capacity of hESC-Kert. The aim of this study is to evaluate the effects of the different ECM microenvironments during hESC differentiation on subsequent replicative life span of hESC-Kert. In doing so, H1-hESCs were differentiated to keratinocytes (H1-Kert) in two differentiation systems. The first system employed autologous fibroblast feeder support, in which keratinocytes (H1-Kert(ACC)) were derived by coculture of hESCs with hESC-derived fibroblasts (H1-ebFs). The second system employed a novel decellularized matrix from H1-ebFs to create a dermoepidermal junction-like (DEJ) matrix. H1-Kert(AFF) were derived by differentiation of hESCs on the feeder-free system employing the DEJ matrix. Our study indicated that the feeder-free system with the use of DEJ matrix was more efficient in differentiation of hESCs toward epidermal progenitors. However, the feeder-free system was not sufficient to support the subsequent replicative capacity of differentiated keratinocytes. Of note, H1-Kert(AFF) showed limited replicative capacity with reduced telomere length and early cellular senescence. We further showed that the lack of cell-cell interactions during epidermal commitment led to heightened production of TGF-β1 by hESC-Kert during extended culture, which in turn was responsible for resulting in the limited replicative life span with cellular senescence of hESC-Kert derived under the feeder-free culture system. This study highlights for the first time the importance of the culture microenvironment and cell-ECM interactions during

Turning the Hands of Time Again: A Purely Confirmatory Replication Study and a Bayesian Analysis

Directory of Open Access Journals (Sweden)

Eric-Jan eWagenmakers

2015-04-01

Full Text Available In a series of four experiments, Topolinski and Sparenberg (2012; TS found support for the conjecture that clockwise movements induce psychological states of temporal progression and an orientation toward the future and novelty. Here we report the results of a preregistered replication attempt of Experiment 2 from TS. Participants turned kitchen rolls either clockwise or counterclockwise while answering items from a questionnaire assessing openness to experience. Data from 102 participants showed that the effect went slightly in the direction opposite to that predicted by TS, and a preregistered Bayes factor hypothesis test revealed that the data were 10.76 times more likely under the null hypothesis than under the alternative hypothesis. Our findings illustrate the theoretical importance and practical advantages of preregistered Bayes factor replication studies, both for psychological science and for empirical work in general.

Human geminin promotes pre-RC formation and DNA replication by stabilizing CDT1 in mitosis

DEFF Research Database (Denmark)

Ballabeni, Andrea; Melixetian, Marina; Zamponi, Raffaella

2004-01-01

-mediated degradation by inhibiting its ubiquitination. In particular, Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis. Consistently, inhibition of Geminin synthesis during M phase leads to impairment of pre-RC formation and DNA replication during the following cell cycle....... Moreover, we show that inhibition of CDK1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre-RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells. Taken together with recent data from our laboratory, our results demonstrate...

LuIII parvovirus selectively and efficiently targets, replicates in, and kills human glioma cells.

Science.gov (United States)

Paglino, Justin C; Ozduman, Koray; van den Pol, Anthony N

2012-07-01

Because productive infection by parvoviruses requires cell division and is enhanced by oncogenic transformation, some parvoviruses may have potential utility in killing cancer cells. To identify the parvovirus(es) with the optimal oncolytic effect against human glioblastomas, we screened 12 parvoviruses at a high multiplicity of infection (MOI). MVMi, MVMc, MVM-G17, tumor virus X (TVX), canine parvovirus (CPV), porcine parvovirus (PPV), rat parvovirus 1A (RPV1A), and H-3 were relatively ineffective. The four viruses with the greatest oncolytic activity, LuIII, H-1, MVMp, and MVM-G52, were tested for the ability, at a low MOI, to progressively infect the culture over time, causing cell death at a rate higher than that of cell proliferation. LuIII alone was effective in all five human glioblastomas tested. H-1 progressively infected only two of five; MVMp and MVM-G52 were ineffective in all five. To investigate the underlying mechanism of LuIII's phenotype, we used recombinant parvoviruses with the LuIII capsid replacing the MVMp capsid or with molecular alteration of the P4 promoter. The LuIII capsid enhanced efficient replication and oncolysis in MO59J gliomas cells; other gliomas tested required the entire LuIII genome to exhibit enhanced infection. LuIII selectively infected glioma cells over normal glial cells in vitro. In mouse models, human glioblastoma xenografts were selectively infected by LuIII when administered intratumorally; LuIII reduced tumor growth by 75%. LuIII also had the capacity to selectively infect subcutaneous or intracranial gliomas after intravenous inoculation. Intravenous or intracranial LuIII caused no adverse effects. Intracranial LuIII caused no infection of mature mouse neurons or glia in vivo but showed a modest infection of developing neurons.

MMSET is dynamically regulated during cell-cycle progression and promotes normal DNA replication.

Science.gov (United States)

Evans, Debra L; Zhang, Haoxing; Ham, Hyoungjun; Pei, Huadong; Lee, SeungBaek; Kim, JungJin; Billadeau, Daniel D; Lou, Zhenkun

2016-01-01

The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin Recognition Complex (ORC) binds to future replication origins, coordinating with multiple factors to load the minichromosome maintenance (MCM) complex onto future replication origins as part of the pre-replication complex (pre-RC). The pre-RC machinery, in turn, remains inactive until the subsequent S phase when it is required for replication fork formation, thereby initiating DNA replication. Multiple myeloma SET domain-containing protein (MMSET, a.k.a. WHSC1, NSD2) is a histone methyltransferase that is frequently overexpressed in aggressive cancers and is essential for normal human development. Several studies have suggested a role for MMSET in cell-cycle regulation; however, whether MMSET is itself regulated during cell-cycle progression has not been examined. In this study, we report that MMSET is degraded during S phase in a cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) and proteasome-dependent manner. Notably, we also report defects in DNA replication and a decreased association of pre-RC factors with chromatin in MMSET-depleted cells. Taken together, our results suggest a dynamic regulation of MMSET levels throughout the cell cycle, and further characterize the role of MMSET in DNA replication and cell-cycle progression.

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)

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

Science.gov (United States)

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

2017-01-01

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

Hili Inhibits HIV Replication in Activated T Cells.

Science.gov (United States)

Peterlin, B Matija; Liu, Pingyang; Wang, Xiaoyun; Cary, Daniele; Shao, Wei; Leoz, Marie; Hong, Tian; Pan, Tao; Fujinaga, Koh

2017-06-01

P-element-induced wimpy-like (Piwil) proteins restrict the replication of mobile genetic elements in the germ line. They are also expressed in many transformed cell lines. In this study, we discovered that the human Piwil 2 (Hili) protein can also inhibit HIV replication, especially in activated CD4 + T cells that are the preferred target cells for this virus in the infected host. Although resting cells did not express Hili, its expression was rapidly induced following T cell activation. In these cells and transformed cell lines, depletion of Hili increased levels of viral proteins and new viral particles. Further studies revealed that Hili binds to tRNA. Some of the tRNAs represent rare tRNA species, whose codons are overrepresented in the viral genome. Targeting tRNA Arg (UCU) with an antisense oligonucleotide replicated effects of Hili and also inhibited HIV replication. Finally, Hili also inhibited the retrotransposition of the endogenous intracysternal A particle (IAP) by a similar mechanism. Thus, Hili joins a list of host proteins that inhibit the replication of HIV and other mobile genetic elements. IMPORTANCE Piwil proteins inhibit the movement of mobile genetic elements in the germ line. In their absence, sperm does not form and male mice are sterile. This inhibition is thought to occur via small Piwi-interacting RNAs (piRNAs). However, in some species and in human somatic cells, Piwil proteins bind primarily to tRNA. In this report, we demonstrate that human Piwil proteins, especially Hili, not only bind to select tRNA species, including rare tRNAs, but also inhibit HIV replication. Importantly, T cell activation induces the expression of Hili in CD4 + T cells. Since Hili also inhibited the movement of an endogenous retrovirus (IAP), our finding shed new light on this intracellular resistance to exogenous and endogenous retroviruses as well as other mobile genetic elements. Copyright © 2017 American Society for Microbiology.

The cytoprotective enzyme heme oxygenase-1 suppresses Ebola virus replication.

Science.gov (United States)

Hill-Batorski, Lindsay; Halfmann, Peter; Neumann, Gabriele; Kawaoka, Yoshihiro

2013-12-01

Ebola virus (EBOV) is the causative agent of a severe hemorrhagic fever in humans with reported case fatality rates as high as 90%. There are currently no licensed vaccines or antiviral therapeutics to combat EBOV infections. Heme oxygenase-1 (HO-1), an enzyme that catalyzes the rate-limiting step in heme degradation, has antioxidative properties and protects cells from various stresses. Activated HO-1 was recently shown to have antiviral activity, potently inhibiting the replication of viruses such as hepatitis C virus and human immunodeficiency virus. However, the effect of HO-1 activation on EBOV replication remains unknown. To determine whether the upregulation of HO-1 attenuates EBOV replication, we treated cells with cobalt protoporphyrin (CoPP), a selective HO-1 inducer, and assessed its effects on EBOV replication. We found that CoPP treatment, pre- and postinfection, significantly suppressed EBOV replication in a manner dependent upon HO-1 upregulation and activity. In addition, stable overexpression of HO-1 significantly attenuated EBOV growth. Although the exact mechanism behind the antiviral properties of HO-1 remains to be elucidated, our data show that HO-1 upregulation does not attenuate EBOV entry or budding but specifically targets EBOV transcription/replication. Therefore, modulation of the cellular enzyme HO-1 may represent a novel therapeutic strategy against EBOV infection.

Intragenic origins due to short G1 phases underlie oncogene-induced DNA replication stress.

Science.gov (United States)

Macheret, Morgane; Halazonetis, Thanos D

2018-03-01

Oncogene-induced DNA replication stress contributes critically to the genomic instability that is present in cancer. However, elucidating how oncogenes deregulate DNA replication has been impeded by difficulty in mapping replication initiation sites on the human genome. Here, using a sensitive assay to monitor nascent DNA synthesis in early S phase, we identified thousands of replication initiation sites in cells before and after induction of the oncogenes CCNE1 and MYC. Remarkably, both oncogenes induced firing of a novel set of DNA replication origins that mapped within highly transcribed genes. These ectopic origins were normally suppressed by transcription during G1, but precocious entry into S phase, before all genic regions had been transcribed, allowed firing of origins within genes in cells with activated oncogenes. Forks from oncogene-induced origins were prone to collapse, as a result of conflicts between replication and transcription, and were associated with DNA double-stranded break formation and chromosomal rearrangement breakpoints both in our experimental system and in a large cohort of human cancers. Thus, firing of intragenic origins caused by premature S phase entry represents a mechanism of oncogene-induced DNA replication stress that is relevant for genomic instability in human cancer.

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.

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.

Conserved retinoblastoma protein-binding motif in human cytomegalovirus UL97 kinase minimally impacts viral replication but affects susceptibility to maribavir

Directory of Open Access Journals (Sweden)

Chou Sunwen

2009-01-01

Full Text Available Abstract The UL97 kinase has been shown to phosphorylate and inactivate the retinoblastoma protein (Rb and has three consensus Rb-binding motifs that might contribute to this activity. Recombinant viruses containing mutations in the Rb-binding motifs generally replicated well in human foreskin fibroblasts with only a slight delay in replication kinetics. Their susceptibility to the specific UL97 kinase inhibitor, maribavir, was also examined. Mutation of the amino terminal motif, which is involved in the inactivation of Rb, also renders the virus hypersensitive to the drug and suggests that the motif may play a role in its mechanism of action.

Fidelity of DNA Replication in Normal and Malignant Human Breast Cells.

Science.gov (United States)

1997-08-01

enzyme) into the multiple cloning site (MCS). This template will not only replicate inside a mammalian cell (utilizing the E-B virus origin), and...Maniatis, T. Commonly used techniques in molecular cloning . In: Molecular cloning : REFERENCES a laboratory manual, 2nd edition. Cold Spring Harbor...A vatit"Y Of DNA synthesis and the typt of DNA replica~tion Products " celular prca including DNA rsplicatlon. DNA repsair. R~NA formed in experiments

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.

DNA replication stress as a hallmark of cancer.

Science.gov (United States)

Macheret, Morgane; Halazonetis, Thanos D

2015-01-01

Human cancers share properties referred to as hallmarks, among which sustained proliferation, escape from apoptosis, and genomic instability are the most pervasive. The sustained proliferation hallmark can be explained by mutations in oncogenes and tumor suppressors that regulate cell growth, whereas the escape from apoptosis hallmark can be explained by mutations in the TP53, ATM, or MDM2 genes. A model to explain the presence of the three hallmarks listed above, as well as the patterns of genomic instability observed in human cancers, proposes that the genes driving cell proliferation induce DNA replication stress, which, in turn, generates genomic instability and selects for escape from apoptosis. Here, we review the data that support this model, as well as the mechanisms by which oncogenes induce replication stress. Further, we argue that DNA replication stress should be considered as a hallmark of cancer because it likely drives cancer development and is very prevalent.

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.

An RNA replication-center assay for high content image-based quantifications of human rhinovirus and coxsackievirus infections

Directory of Open Access Journals (Sweden)

Lötzerich Mark

2010-10-01

Full Text Available Abstract Background Picornaviruses are common human and animal pathogens, including polio and rhinoviruses of the enterovirus family, and hepatits A or food-and-mouth disease viruses. There are no effective countermeasures against the vast majority of picornaviruses, with the exception of polio and hepatitis A vaccines. Human rhinoviruses (HRV are the most prevalent picornaviruses comprising more than one hundred serotypes. The existing and also emerging HRVs pose severe health risks for patients with asthma or chronic obstructive pulmonary disease. Here, we developed a serotype-independent infection assay using a commercially available mouse monoclonal antibody (mabJ2 detecting double-strand RNA. Results Immunocytochemical staining for RNA replication centers using mabJ2 identified cells that were infected with either HRV1A, 2, 14, 16, 37 or coxsackievirus (CV B3, B4 or A21. MabJ2 labeled-cells were immunocytochemically positive for newly synthesized viral capsid proteins from HRV1A, 14, 16, 37 or CVB3, 4. We optimized the procedure for detection of virus replication in settings for high content screening with automated fluorescence microscopy and single cell analysis. Our data show that the infection signal was dependent on multiplicity, time and temperature of infection, and the mabJ2-positive cell numbers correlated with viral titres determined in single step growth curves. The mabJ2 infection assay was adapted to determine the efficacy of anti-viral compounds and small interfering RNAs (siRNAs blocking enterovirus infections. Conclusions We report a broadly applicable, rapid protocol to measure infection of cultured cells with enteroviruses at single cell resolution. This assay can be applied to a wide range of plus-sense RNA viruses, and hence allows comparative studies of viral infection biology without dedicated reagents or procedures. This protocol also allows to directly compare results from small compound or siRNA infection screens

Therapeutic Assessment for Preadolescent Boys with Oppositional Defiant Disorder: A Replicated Single-Case Time-Series Design

Science.gov (United States)

Smith, Justin D.; Handler, Leonard; Nash, Michael R.

2010-01-01

The Therapeutic Assessment (TA) model is a relatively new treatment approach that fuses assessment and psychotherapy. The study examines the efficacy of this model with preadolescent boys with oppositional defiant disorder and their families. A replicated single-case time-series design with daily measures is used to assess the effects of TA and to…

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 ...

Viral gene products and replication of the human immunodeficiency type 1 virus.

Science.gov (United States)

Morrow, C D; Park, J; Wakefield, J K

1994-05-01

The acquired immunodeficiency syndrome (AIDS) epidemic represents a modern-day plague that has not only resulted in a tragic loss of people from a wide spectrum of society but has reshaped our viewpoints regarding health care, the treatment of infectious diseases, and social issues regarding sexual behavior. There is little doubt now that the cause of the disease AIDS is a virus known as the human immunodeficiency virus (HIV). The HIV virus is a member of a large family of viruses termed retroviruses, which have as a hallmark the capacity to convert their RNA genome into a DNA form that then undergoes a process of integration into the host cell chromosome, followed by the expression of the viral genome and translation of viral proteins in the infected cell. This review describes the organization of the HIV-1 viral genome, the expression of viral proteins, as well as the functions of the accessory viral proteins in HIV replication. The replication of the viral genome is divided into two phases, the early phase and the late phase. The early phase consists of the interaction of the virus with the cell surface receptor (CD4 molecule in most cases), the uncoating and conversion of the viral RNA genome into a DNA form, and the integration into the host cell chromosome. The late phase consists of the expression of the viral proteins from the integrated viral genome, the translation of viral proteins, and the assembly and release of the virus. Points in the HIV-1 life cycle that are targets for therapeutic intervention are also discussed.

Timely binding of IHF and Fis to DARS2 regulates ATP–DnaA production and replication initiation

Science.gov (United States)

Kasho, Kazutoshi; Fujimitsu, Kazuyuki; Matoba, Toshihiro; Oshima, Taku; Katayama, Tsutomu

2014-01-01

In Escherichia coli, the ATP-bound form of DnaA (ATP–DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP–DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP–DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP–DnaA was fully active in replication initiation and underwent DnaA–ATP hydrolysis. ADP–DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP–DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP–DnaA production, thereby promoting timely initiation. Moreover, we show that IHF–DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP–DnaA and replication initiation in coordination with the cell cycle and growth phase. PMID:25378325

Timely binding of IHF and Fis to DARS2 regulates ATP-DnaA production and replication initiation.

Science.gov (United States)

Kasho, Kazutoshi; Fujimitsu, Kazuyuki; Matoba, Toshihiro; Oshima, Taku; Katayama, Tsutomu

2014-12-01

In Escherichia coli, the ATP-bound form of DnaA (ATP-DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP-DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP-DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP-DnaA was fully active in replication initiation and underwent DnaA-ATP hydrolysis. ADP-DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP-DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP-DnaA production, thereby promoting timely initiation. Moreover, we show that IHF-DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP-DnaA and replication initiation in coordination with the cell cycle and growth phase. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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...

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.

How MCM loading and spreading specify eukaryotic DNA replication initiation sites.

Science.gov (United States)

Hyrien, Olivier

2016-01-01

DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

Cyclophilin B facilitates the replication of Orf virus

OpenAIRE

Zhao, Kui; Li, Jida; He, Wenqi; Song, Deguang; Zhang, Ximu; Zhang, Di; Zhou, Yanlong; Gao, Feng

2017-01-01

Background 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 f...

Long Terminal Repeat Circular DNA as Markers of Active Viral Replication of Human T Lymphotropic Virus-1 in Vivo

Directory of Open Access Journals (Sweden)

James M Fox

2016-03-01

Full Text Available Clonal expansion of human T-lymphotropic virus type-1 (HTLV-1 infected cells in vivo is well documented. Unlike human immunodeficiency virus type 1 (HIV-1, HTLV-1 plasma RNA is sparse. The contribution of the “mitotic” spread of HTLV-1 compared with infectious spread of the virus to HTLV-1 viral burden in established infection is uncertain. Since extrachromosomal long terminal repeat (LTR DNA circles are indicators of viral replication in HIV-1 carriers with undetectable plasma HIV RNA, we hypothesised that HTLV-1 LTR circles could indicate reverse transcriptase (RT usage and infectious activity. 1LTR and 2LTR DNA circles were measured in HTLV-1 cell lines and peripheral blood mononuclear cells (PBMC of asymptomatic carriers (ACs and patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP or adult T cell leukaemia/lymphoma (ATLL. 1LTR DNA circles were detected in 14/20 patients at a mean of 1.38/100 PBMC but did not differentiate disease status nor correlate with HTLV-1 DNA copies. 2LTR DNA circles were detected in 30/31 patients and at higher concentrations in patients with HTLV-1-associated diseases, independent of HTLV-1 DNA load. In an incident case the 2LTR DNA circle concentration increased 2.1 fold at the onset of HAM/TSP compared to baseline. Detectable and fluctuating levels of HTLV-1 DNA circles in patients indicate viral RT usage and virus replication. Our results indicate HTLV-1 viral replication capacity is maintained in chronic infection and may be associated with disease onset.

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....

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.

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.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the central zinc-binding domain of the human Mcm10 DNA-replication factor

International Nuclear Information System (INIS)

Jung, Nam Young; Bae, Won Jin; Chang, Jeong Ho; Kim, Young Chang; Cho, Yunje

2008-01-01

Mcm10 is a highly conserved nuclear protein that plays a key role in the initiation and elongation processes of DNA replication by providing a physical link between the Mcm2–7 complex and DNA polymerases. In this study, the central domain of human Mcm10 was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 3350. The initiation of eukaryotic DNA replication requires the tightly controlled assembly of a set of replication factors. Mcm10 is a highly conserved nuclear protein that plays a key role in the initiation and elongation processes of DNA replication by providing a physical link between the Mcm2–7 complex and DNA polymerases. The central domain, which contains the CCCH zinc-binding motif, is most conserved within Mcm10 and binds to DNA and several proteins, including proliferative cell nuclear antigen. In this study, the central domain of human Mcm10 was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 3350. An X-ray diffraction data set was collected to a resolution of 2.6 Å on a synchrotron beamline. The crystals formed belonged to space group R3, with unit-cell parameters a = b = 99.5, c = 133.0 Å. According to Matthews coefficient calculations, the crystals were predicted to contain six MCM10 central domain molecules in the asymmetric unit

DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells

Science.gov (United States)

Spada, Fabio; Haemmer, Andrea; Kuch, David; Rothbauer, Ulrich; Schermelleh, Lothar; Kremmer, Elisabeth; Carell, Thomas; Längst, Gernot; Leonhardt, Heinrich

2007-01-01

DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigen–binding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interference–mediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required. PMID:17312023

Photosensitized UVA-Induced Cross-Linking between Human DNA Repair and Replication Proteins and DNA Revealed by Proteomic Analysis

Science.gov (United States)

2016-01-01

Long wavelength ultraviolet radiation (UVA, 320–400 nm) interacts with chromophores present in human cells to induce reactive oxygen species (ROS) that damage both DNA and proteins. ROS levels are amplified, and the damaging effects of UVA are exacerbated if the cells are irradiated in the presence of UVA photosensitizers such as 6-thioguanine (6-TG), a strong UVA chromophore that is extensively incorporated into the DNA of dividing cells, or the fluoroquinolone antibiotic ciprofloxacin. Both DNA-embedded 6-TG and ciprofloxacin combine synergistically with UVA to generate high levels of ROS. Importantly, the extensive protein damage induced by these photosensitizer+UVA combinations inhibits DNA repair. DNA is maintained in intimate contact with the proteins that effect its replication, transcription, and repair, and DNA–protein cross-links (DPCs) are a recognized reaction product of ROS. Cross-linking of DNA metabolizing proteins would compromise these processes by introducing physical blocks and by depleting active proteins. We describe a sensitive and statistically rigorous method to analyze DPCs in cultured human cells. Application of this proteomics-based analysis to cells treated with 6-TG+UVA and ciprofloxacin+UVA identified proteins involved in DNA repair, replication, and gene expression among those most vulnerable to cross-linking under oxidative conditions. PMID:27654267

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.

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.

Rotating Arc Jet Test Model: Time-Accurate Trajectory Heat Flux Replication in a Ground Test Environment

Science.gov (United States)

Laub, Bernard; Grinstead, Jay; Dyakonov, Artem; Venkatapathy, Ethiraj

2011-01-01

Though arc jet testing has been the proven method employed for development testing and certification of TPS and TPS instrumentation, the operational aspects of arc jets limit testing to selected, but constant, conditions. Flight, on the other hand, produces timevarying entry conditions in which the heat flux increases, peaks, and recedes as a vehicle descends through an atmosphere. As a result, we are unable to "test as we fly." Attempts to replicate the time-dependent aerothermal environment of atmospheric entry by varying the arc jet facility operating conditions during a test have proven to be difficult, expensive, and only partially successful. A promising alternative is to rotate the test model exposed to a constant-condition arc jet flow to yield a time-varying test condition at a point on a test article (Fig. 1). The model shape and rotation rate can be engineered so that the heat flux at a point on the model replicates the predicted profile for a particular point on a flight vehicle. This simple concept will enable, for example, calibration of the TPS sensors on the Mars Science Laboratory (MSL) aeroshell for anticipated flight environments.

Effects of Replication and Transcription on DNA Structure-Related Genetic Instability.

Science.gov (United States)

Wang, Guliang; Vasquez, Karen M

2017-01-05

Many repetitive sequences in the human genome can adopt conformations that differ from the canonical B-DNA double helix (i.e., non-B DNA), and can impact important biological processes such as DNA replication, transcription, recombination, telomere maintenance, viral integration, transposome activation, DNA damage and repair. Thus, non-B DNA-forming sequences have been implicated in genetic instability and disease development. In this article, we discuss the interactions of non-B DNA with the replication and/or transcription machinery, particularly in disease states (e.g., tumors) that can lead to an abnormal cellular environment, and how such interactions may alter DNA replication and transcription, leading to potential conflicts at non-B DNA regions, and eventually result in genetic stability and human disease.

Temporal organization of cellular self-replication

Science.gov (United States)

Alexandrov, Victor; Pugatch, Rami

Recent experiments demonstrate that single cells grow exponentially in time. A coarse grained model of cellular self-replication is presented based on a novel concept - the cell is viewed as a self-replicating queue. This allows to have a more fundamental look into various temporal organizations and, importantly, the inherent non-Markovianity of noise distributions. As an example, the distribution of doubling times can be inferred and compared to single cell experiments in bacteria. We observe data collapse upon scaling by the average doubling time for different environments and present an inherent task allocation trade-off. Support from the Simons Center for Systems Biology, IAS, Princeon.

Timing, Coordination, and Rhythm : Acrobatics at the DNA Replication Fork

NARCIS (Netherlands)

Hamdan, Samir M.; Oijen, Antoine M. van

2010-01-01

In DNA replication, the antiparallel nature of the parental duplex imposes certain constraints on the activity of the DNA polymerases that synthesize new DNA. The leading-strand polymerase advances in a continuous fashion, but the lagging-strand polymerase is forced to restart at short intervals. In

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

Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA

NARCIS (Netherlands)

Fontijn, R.; Hop, C.; Brinkman, H. J.; Slater, R.; Westerveld, A.; van Mourik, J. A.; Pannekoek, H.

1995-01-01

Primary human vascular endothelial cells were immortalized by the integration of a single DNA copy of an amphotrophic, replication-deficient retrovirus containing the E6/E7 genes of human papilloma virus. To date, the resulting cell lines, designated EC-RF7 and EC-RF24, have been cultured for more

Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS

Directory of Open Access Journals (Sweden)

Ciervo Alessandra

2007-10-01

Full Text Available Abstract Background Treatment of feline immunodeficiency virus (FIV infection has been hampered by the absence of a specific combination antiretroviral treatment (ART. Integrase strand transfer inhibitors (INSTIs are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. Methods Phylogenetic analysis of lentiviral integrase (IN sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD. Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. Results The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. Conclusion We report a drug class (other than nucleosidic reverse transcriptase inhibitors that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound

Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS

Science.gov (United States)

Savarino, Andrea; Pistello, Mauro; D'Ostilio, Daniela; Zabogli, Elisa; Taglia, Fabiana; Mancini, Fabiola; Ferro, Stefania; Matteucci, Donatella; De Luca, Laura; Barreca, Maria Letizia; Ciervo, Alessandra; Chimirri, Alba; Ciccozzi, Massimo; Bendinelli, Mauro

2007-01-01

Background Treatment of feline immunodeficiency virus (FIV) infection has been hampered by the absence of a specific combination antiretroviral treatment (ART). Integrase strand transfer inhibitors (INSTIs) are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. Methods Phylogenetic analysis of lentiviral integrase (IN) sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD) was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD). Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. Results The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. Conclusion We report a drug class (other than nucleosidic reverse transcriptase inhibitors) that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound successfully tested in

Investigation of radiation enhanced reactivation of cytoplasmic replicating human virus

International Nuclear Information System (INIS)

Bockstahler, L.E.; Haynes, K.F.; Stafford, J.E.

1976-01-01

When monolayers of CV-1 monkey kidney cells were exposed to ultraviolet (uv) radiation (0 to 200 erg/nm 2 ) or x rays (0 to 10 krads) before infection with uv-irradiated herpes simplex virus, an increase in the infectivity of this nuclear replicating virus occurred as measured by plaque formation. These phenomena are known as uv (Weigle) reactivation (WR) and x-ray reactivation (x-ray R). In this study the presence of WR and x-ray R was examined in CV-1 cells infected with uv-irradiated vaccinia virus or poliovirus, both cytoplasmic replicating viruses. Little or no WR or x-ray R was observed for either of these viruses. These results suggest that WR and x-ray R in mammalian cells may be restricted to viruses which are synthesized in the cell nucleus

Roles of polypyrimidine tract binding proteins in major immediate-early gene expression and viral replication of human cytomegalovirus.

Science.gov (United States)

Cosme, Ruth S Cruz; Yamamura, Yasuhiro; Tang, Qiyi

2009-04-01

Human cytomegalovirus (HCMV), a member of the beta subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns.

Relationship between DNA replication and DNA repair in human lymphocytes proliferating in vitro in the presence and in absence of mutagen

International Nuclear Information System (INIS)

Szyfter, K.; Wielgosz, M.Sz.; Kujawski, M.; Jaloszynski, P.; Zajaczek, S.

1995-01-01

The effects of mutagens on DNA replication and DNA repair were studied in peripheral blood lymphocytes (PBL) obtained from 21 healthy subjects, 2 samples from healthy heterozygote of ''Xeroderma pigmentosum'' (XP) and 2 samples from patient with clinically recognised XP. Inter-individual variations were found in DNA replication and in the level of spontaneous DNA repair measured under standard culture condition. Exposure of human PBL proliferating in vitro to B(a)P was followed by a partial inhibition of replicative DNA synthesis in all subjects and by an induction of DNA repair in healthy subjects. In XP patients DNA repair synthesis remained at the level attributed to spontaneous DNA repair. The response to mutagen varied individually. Results were analysed statistically. It was established that the studied indices of DNA synthesis correlate well with each other. The highest correlation was found between the levels of spontaneous and B(a)P-induced DNA repair. It is concluded that the level of spontaneous DNA repair is predictive for an estimation of cells ability to repair DNA damage. Inter-individual variations in the inhibition of DNA replication and in DNA repair synthesis are also dependent on the type of mutagen as shown by effects of other mutagens. Different effects of mutagen exposure on the inhibition of DNA replicative synthesis and induction of DNA repair can be explained by genetically controlled differences in the activity of enzymes responsible for mutagen processing and lesion removal. (author). 37 refs, 2 figs, 2 tabs

Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts

International Nuclear Information System (INIS)

Smith, C.A.; Hanawalt, P.C.

1978-01-01

The repair mode of DNA replication has been demonstrated in isolated nuclei from uv-irradiated human cells. Nuclei are incubated in a mixture containing [ 3 H]thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The 3 H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is uv-dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg 2+ -dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote uv-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms

An Alternative Form of Replication Protein A Prevents Viral Replication in Vitro*

OpenAIRE

Mason, Aaron C.; Haring, Stuart J.; Pryor, John M.; Staloch, Cathy A.; Gan, Tze Fei; Wold, Marc S.

2009-01-01

Replication protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism. The “canonical” RPA is composed of three subunits (RPA1, RPA2, and RPA3); however, there is a human homolog to the RPA2 subunit, called RPA4, that can substitute for RPA2 in complex formation. We demonstrate that the resulting “alternative” RPA (aRPA) complex has solution and DNA binding properties indistinguishable from the c...

How Human Papillomavirus Replication and Immune Evasion Strategies Take Advantage of the Host DNA Damage Repair Machinery.

Science.gov (United States)

Bordignon, Valentina; Di Domenico, Enea Gino; Trento, Elisabetta; D'Agosto, Giovanna; Cavallo, Ilaria; Pontone, Martina; Pimpinelli, Fulvia; Mariani, Luciano; Ensoli, Fabrizio

2017-12-19

The DNA damage response (DDR) is a complex signalling network activated when DNA is altered by intrinsic or extrinsic agents. DDR plays important roles in genome stability and cell cycle regulation, as well as in tumour transformation. Viruses have evolved successful life cycle strategies in order to ensure a chronic persistence in the host, virtually avoiding systemic sequelae and death. This process promotes the periodic shedding of large amounts of infectious particles to maintain a virus reservoir in individual hosts, while allowing virus spreading within the community. To achieve such a successful lifestyle, the human papilloma virus (HPV) needs to escape the host defence systems. The key to understanding how this is achieved is in the virus replication process that provides by itself an evasion mechanism by inhibiting and delaying the host immune response against the viral infection. Numerous studies have demonstrated that HPV exploits both the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and rad3-related (ATR) DDR pathways to replicate its genome and maintain a persistent infection by downregulating the innate and cell-mediated immunity. This review outlines how HPV interacts with the ATM- and ATR-dependent DDR machinery during the viral life cycle to create an environment favourable to viral replication, and how the interaction with the signal transducers and activators of transcription (STAT) protein family and the deregulation of the Janus kinase (JAK)-STAT pathways may impact the expression of interferon-inducible genes and the innate immune responses.

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

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.

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...

Non‐Canonical Replication Initiation: You’re Fired!

Directory of Open Access Journals (Sweden)

Bazilė Ravoitytė

2017-01-01

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

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.

Defects of mtDNA Replication Impaired Mitochondrial Biogenesis During Trypanosoma cruzi Infection in Human Cardiomyocytes and Chagasic Patients: The Role of Nrf1/2 and Antioxidant Response

Science.gov (United States)

Wan, Xianxiu; Gupta, Shivali; Zago, Maria P.; Davidson, Mercy M.; Dousset, Pierre; Amoroso, Alejandro; Garg, Nisha Jain

2012-01-01

Background Mitochondrial dysfunction is a key determinant in chagasic cardiomyopathy development in mice; however, its relevance in human Chagas disease is not known. We determined if defects in mitochondrial biogenesis and dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1)–regulated transcriptional pathways constitute a mechanism or mechanisms underlying mitochondrial oxidative-phosphorylation (OXPHOS) deficiency in human Chagas disease. Methods and Results We utilized human cardiomyocytes and left-ventricular tissue from chagasic and other cardiomyopathy patients and healthy donors (n>6/group). We noted no change in citrate synthase activity, yet mRNA and/or protein levels of subunits of the respiratory complexes were significantly decreased in Trypanosoma cruzi–infected cardiomyocytes (0 to 24 hours) and chagasic hearts. We observed increased mRNA and decreased nuclear localization of PGC-1-coactivated transcription factors, yet the expression of genes for PPARγ-regulated fatty acid oxidation and nuclear respiratory factor (NRF1/2)–regulated mtDNA replication and transcription machinery was enhanced in infected cardiomyocytes and chagasic hearts. The D-loop formation was normal or higher, but mtDNA replication and mtDNA content were decreased by 83% and 40% to 65%, respectively. Subsequently, we noted that reactive oxygen species (ROS), oxidative stress, and mtDNA oxidation were significantly increased, yet NRF1/2-regulated antioxidant gene expression remained compromised in infected cardiomyocytes and chagasic hearts. Conclusions The replication of mtDNA was severely compromised, resulting in a significant loss of mtDNA and expression of OXPHOS genes in T cruzi–infected cardiomyocytes and chagasic hearts. Our data suggest increased ROS generation and selective functional incapacity of NRF2-mediated antioxidant gene expression played a role in the defects in mtDNA replication and unfitness of mtDNA for

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

Directory of Open Access Journals (Sweden)

Yuichi Hirata

Full Text Available Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2 encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

Science.gov (United States)

Hirata, Yuichi; Ikeda, Kazutaka; Sudoh, Masayuki; Tokunaga, Yuko; Suzuki, Akemi; Weng, Leiyun; Ohta, Masatoshi; Tobita, Yoshimi; Okano, Ken; Ozeki, Kazuhisa; Kawasaki, Kenichi; Tsukuda, Takuo; Katsume, Asao; Aoki, Yuko; Umehara, Takuya; Sekiguchi, Satoshi; Toyoda, Tetsuya; Shimotohno, Kunitada; Soga, Tomoyoshi; Nishijima, Masahiro; Taguchi, Ryo; Kohara, Michinori

2012-01-01

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Proteome-wide analysis of SUMO2 targets in response to pathological DNA replication stress in human cells

DEFF Research Database (Denmark)

Bursomanno, Sara; Beli, Petra; Khan, Asif M

2015-01-01

SUMOylation is a form of post-translational modification involving covalent attachment of SUMO (Small Ubiquitin-like Modifier) polypeptides to specific lysine residues in the target protein. In human cells, there are four SUMO proteins, SUMO1-4, with SUMO2 and SUMO3 forming a closely related subf......, and that excessive replication stress is a hallmark of pre-neoplastic and tumor cells, our characterization of SUMO2 targets during a perturbed S-phase should provide a valuable resource for future functional studies in the fields of DNA metabolism and cancer biology....

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.)

Replication protein A and γ-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks

International Nuclear Information System (INIS)

Balajee, Adayabalam S.; Geard, Charles R.

2004-01-01

Human replication protein A (RPA p34), a crucial component of diverse DNA excision repair pathways, is implicated in DNA double-strand break (DSB) repair. To evaluate its role in DSB repair, the intranuclear dynamics of RPA was investigated after DNA damage and replication blockage in human cells. Using two different agents [ionizing radiation (IR) and hydroxyurea (HU)] to generate DSBs, we found that RPA relocated into distinct nuclear foci and colocalized with a well-known DSB binding factor, γ-H2AX, at the sites of DNA damage in a time-dependent manner. Colocalization of RPA and γ-H2AX foci peaked at 2 h after IR treatment and subsequently declined with increasing postrecovery times. The time course of RPA and γ-H2AX foci association correlated well with the DSB repair activity detected by a neutral comet assay. A phosphatidylinositol-3 (PI-3) kinase inhibitor, wortmannin, completely abolished both RPA and γ-H2AX foci formation triggered by IR. Additionally, radiosensitive ataxia telangiectasia (AT) cells harboring mutations in ATM gene product were found to be deficient in RPA and γ-H2AX colocalization after IR. Transfection of AT cells with ATM cDNA fully restored the association of RPA foci with γ-H2AX illustrating the requirement of ATM gene product for this process. The exact coincidence of RPA and γ-H2AX in response to HU specifically in S-phase cells supports their role in DNA replication checkpoint control. Depletion of RPA by small interfering RNA (SiRNA) substantially elevated the frequencies of IR-induced micronuclei (MN) and apoptosis in human cells suggestive of a role for RPA in DSB repair. We propose that RPA in association with γ-H2AX contributes to both DNA damage checkpoint control and repair in response to strand breaks and stalled replication forks in human cells

Time-varying Entry Heating Profile Replication with a Rotating Arc Jet Test Article

Science.gov (United States)

Grinstead, Jay Henderson; Venkatapathy, Ethiraj; Noyes, Eric A.; Mach, Jeffrey J.; Empey, Daniel M.; White, Todd R.

2014-01-01

A new approach for arc jet testing of thermal protection materials at conditions approximating the time-varying conditions of atmospheric entry was developed and demonstrated. The approach relies upon the spatial variation of heat flux and pressure over a cylindrical test model. By slowly rotating a cylindrical arc jet test model during exposure to an arc jet stream, each point on the test model will experience constantly changing applied heat flux. The predicted temporal profile of heat flux at a point on a vehicle can be replicated by rotating the cylinder at a prescribed speed and direction. An electromechanical test model mechanism was designed, built, and operated during an arc jet test to demonstrate the technique.

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.

Replication stress, DNA damage signalling, and cytomegalovirus infection in human medulloblastomas

DEFF Research Database (Denmark)

Bartek, Jiri; Fornara, Olesja; Merchut-Maya, Joanna Maria

2017-01-01

suppressor activation, across our medulloblastoma cohort. Most tumours showed high proliferation (Ki67 marker), variable oxidative DNA damage (8-oxoguanine lesions) and formation of 53BP1 nuclear 'bodies', the latter indicating (along with ATR-Chk1 signalling) endogenous replication stress. The bulk...... cell replication stress and DNA repair. Collectively, the scenario we report here likely fuels genomic instability and evolution of medulloblastoma resistance to standard-of-care genotoxic treatments....... eight established immunohistochemical markers to assess the status of the DDR machinery, we found pronounced endogenous DNA damage signalling (γH2AX marker) and robust constitutive activation of both the ATM-Chk2 and ATR-Chk1 DNA damage checkpoint kinase cascades, yet unexpectedly modest p53 tumour...

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.

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

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts.

Science.gov (United States)

Bernstein, Diana L; Le Lay, John E; Ruano, Elena G; Kaestner, Klaus H

2015-05-01

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator-like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics.

Replicating rather than nonreplicating adenovirus-human immunodeficiency virus recombinant vaccines are better at eliciting potent cellular immunity and priming high-titer antibodies.

Science.gov (United States)

Peng, Bo; Wang, Liqun Rejean; Gómez-Román, Victor Raúl; Davis-Warren, Alberta; Montefiori, David C; Kalyanaraman, V S; Venzon, David; Zhao, Jun; Kan, Elaine; Rowell, Thomas J; Murthy, Krishna K; Srivastava, Indresh; Barnett, Susan W; Robert-Guroff, Marjorie

2005-08-01

A major challenge in combating the human immunodeficiency virus (HIV) epidemic is the development of vaccines capable of inducing potent, persistent cellular immunity and broadly reactive neutralizing antibody responses to HIV type 1 (HIV-1). We report here the results of a preclinical trial using the chimpanzee model to investigate a combination vaccine strategy involving sequential priming immunizations with different serotypes of adenovirus (Ad)/HIV-1(MN)env/rev recombinants and boosting with an HIV envelope subunit protein, oligomeric HIV(SF162) gp140deltaV2. The immunogenicities of replicating and nonreplicating Ad/HIV-1(MN)env/rev recombinants were compared. Replicating Ad/HIV recombinants were better at eliciting HIV-specific cellular immune responses and better at priming humoral immunity against HIV than nonreplicating Ad-HIV recombinants carrying the same gene insert. Enhanced cellular immunity was manifested by a greater frequency of HIV envelope-specific gamma interferon-secreting peripheral blood lymphocytes and better priming of T-cell proliferative responses. Enhanced humoral immunity was seen in higher anti-envelope binding and neutralizing antibody titers and better induction of antibody-dependent cellular cytotoxicity. More animals primed with replicating Ad recombinants mounted neutralizing antibodies against heterologous R5 viruses after one or two booster immunizations with the mismatched oligomeric HIV-1(SF162) gp140deltaV2 protein. These results support continued development of the replicating Ad-HIV recombinant vaccine approach and suggest that the use of replicating vectors for other vaccines may prove fruitful.

Epigenetically-inherited centromere and neocentromere DNA replicates earliest in S-phase.

Directory of Open Access Journals (Sweden)

Amnon Koren

2010-08-01

Full Text Available Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position.

Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis

DEFF Research Database (Denmark)

Natsume, Toyoaki; Nishimura, Kohei; Minocherhomji, Sheroy

2017-01-01

stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks...

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.

Roles of Polypyrimidine Tract Binding Proteins in Major Immediate-Early Gene Expression and Viral Replication of Human Cytomegalovirus▿

Science.gov (United States)

Cosme, Ruth S. Cruz; Yamamura, Yasuhiro; Tang, Qiyi

2009-01-01

Human cytomegalovirus (HCMV), a member of the β subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns. PMID:19144709

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

Science.gov (United States)

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

2015-06-19

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

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.

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

Science.gov (United States)

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

1995-01-01

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

Initiation of DNA replication requires actin dynamics and formin activity.

Science.gov (United States)

Parisis, Nikolaos; Krasinska, Liliana; Harker, Bethany; Urbach, Serge; Rossignol, Michel; Camasses, Alain; Dewar, James; Morin, Nathalie; Fisher, Daniel

2017-11-02

Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin- and redox-dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)-cargo release from RanGTP-importin complexes. Nuclear formin activity is further required to promote loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms. © 2017 The Authors.

Replication of genetic associations as pseudoreplication due to shared genealogy.

Science.gov (United States)

Rosenberg, Noah A; Vanliere, Jenna M

2009-09-01

The genotypes of individuals in replicate genetic association studies have some level of correlation due to shared descent in the complete pedigree of all living humans. As a result of this genealogical sharing, replicate studies that search for genotype-phenotype associations using linkage disequilibrium between marker loci and disease-susceptibility loci can be considered as "pseudoreplicates" rather than true replicates. We examine the size of the pseudoreplication effect in association studies simulated from evolutionary models of the history of a population, evaluating the excess probability that both of a pair of studies detect a disease association compared to the probability expected under the assumption that the two studies are independent. Each of nine combinations of a demographic model and a penetrance model leads to a detectable pseudoreplication effect, suggesting that the degree of support that can be attributed to a replicated genetic association result is less than that which can be attributed to a replicated result in a context of true independence.

Effect of low-dose gamma radiation on HIV replication in human peripheral blood mononuclear cells

Energy Technology Data Exchange (ETDEWEB)

Xu, Y. [British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada); Conway, B. [British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada)]|[British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada). Dept. of Medicine; Montaner, J.S.G. [British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada)]|[British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada). Dept. of Medicine]|[Canadian HIV Trials Network, Vancouver (Canada); O`Shaughnessy, M.V. [British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada)]|[British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada). Faculty of Medicine]|[Canadian HIV Trials Network, Vancouver (Canada); Greenstock, C.L. [AECL Research, Chalk River, Ontario (Canada). Radiation Biology and Health Physics Branch

1996-08-01

Recent studies have demonstrated that UV light and x-irradiation enhance human immunodeficiency virus (HIV) gene expression. There are few published data on related effects of {gamma}-radiation. This may be of clinical relevance, as radiotherapy has been used extensively for the treatment of acquired immunodeficiency syndrome associated conditions. We have studied the effects of {gamma}-radiation on HIV replication in mono-nuclear cells (MC). These cells were obtained from five seronegative healthy donors, exposed to 0-200 cGy {gamma}-radiation, stimulated with phytohemagglutinin-P (PHA-P) for 24 h, infected with a laboratory strain of HIV (HTLV-IIIB, multiplicity of infection = 0.001), then carried in culture for 14 days. Overall, when considering p24 antigen levels on days 7 and 11 in cultures established from cells exposed to 50 cGy, the maximal levels were significantly higher than those measured in the parallel control cultures taken as a whole (P < 0.05), with viral replication enhanced as much as 1000-fold in one case. No significant cytotoxicity was observed following exposure to doses up to 50 cGy. The mechanism of the observed effect remains unknown but may relate to direct gene activation and/or free radical generation, leading to such activation. To date, there is no evidence that viral stimulation occurs following therapeutic radiation in a clinical setting. (author).

Effect of low-dose gamma radiation on HIV replication in human peripheral blood mononuclear cells

International Nuclear Information System (INIS)

Xu, Y.; Conway, B.; O'Shaughnessy, M.V.; Greenstock, C.L.

1996-01-01

Recent studies have demonstrated that UV light and x-irradiation enhance human immunodeficiency virus (HIV) gene expression. There are few published data on related effects of γ-radiation. This may be of clinical relevance, as radiotherapy has been used extensively for the treatment of acquired immunodeficiency syndrome associated conditions. We have studied the effects of γ-radiation on HIV replication in mono-nuclear cells (MC). These cells were obtained from five seronegative healthy donors, exposed to 0-200 cGy γ-radiation, stimulated with phytohemagglutinin-P (PHA-P) for 24 h, infected with a laboratory strain of HIV (HTLV-IIIB, multiplicity of infection = 0.001), then carried in culture for 14 days. Overall, when considering p24 antigen levels on days 7 and 11 in cultures established from cells exposed to 50 cGy, the maximal levels were significantly higher than those measured in the parallel control cultures taken as a whole (P < 0.05), with viral replication enhanced as much as 1000-fold in one case. No significant cytotoxicity was observed following exposure to doses up to 50 cGy. The mechanism of the observed effect remains unknown but may relate to direct gene activation and/or free radical generation, leading to such activation. To date, there is no evidence that viral stimulation occurs following therapeutic radiation in a clinical setting. (author)

Cathepsin B & L are not required for ebola virus replication.

Science.gov (United States)

Marzi, Andrea; Reinheckel, Thomas; Feldmann, Heinz

2012-01-01

Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d'Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB(-/-) and catL(-/-) mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.

Cathepsin B & L are not required for ebola virus replication.

Directory of Open Access Journals (Sweden)

Andrea Marzi

Full Text Available Ebola virus (EBOV, family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV. EBOV encodes one viral surface glycoprotein (GP, which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL, which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d'Ivoire, Reston and Sudan ebolavirus was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control, catB(-/- and catL(-/- mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.

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

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.

Identification of 30 protein species involved in replicative senescence and stress-induced premature senescence

DEFF Research Database (Denmark)

Dierick, Jean François; Kalume, Dário E; Wenders, Frédéric

2002-01-01

Exposure of human proliferative cells to subcytotoxic stress triggers stress-induced premature senescence (SIPS) which is characterized by many biomarkers of replicative senescence. Proteomic comparison of replicative senescence and stress-induced premature senescence indicates that, at the level...

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

A microhomology-mediated break-induced replication model for the origin of human copy number variation.

Directory of Open Access Journals (Sweden)

P J Hastings

2009-01-01

Full Text Available Chromosome structural changes with nonrecurrent endpoints associated with genomic disorders offer windows into the mechanism of origin of copy number variation (CNV. A recent report of nonrecurrent duplications associated with Pelizaeus-Merzbacher disease identified three distinctive characteristics. First, the majority of events can be seen to be complex, showing discontinuous duplications mixed with deletions, inverted duplications, and triplications. Second, junctions at endpoints show microhomology of 2-5 base pairs (bp. Third, endpoints occur near pre-existing low copy repeats (LCRs. Using these observations and evidence from DNA repair in other organisms, we derive a model of microhomology-mediated break-induced replication (MMBIR for the origin of CNV and, ultimately, of LCRs. We propose that breakage of replication forks in stressed cells that are deficient in homologous recombination induces an aberrant repair process with features of break-induced replication (BIR. Under these circumstances, single-strand 3' tails from broken replication forks will anneal with microhomology on any single-stranded DNA nearby, priming low-processivity polymerization with multiple template switches generating complex rearrangements, and eventual re-establishment of processive replication.

RIPK1 and PGAM5 Control Leishmania Replication through Distinct Mechanisms.

Science.gov (United States)

Farias Luz, Nivea; Balaji, Sakthi; Okuda, Kendi; Barreto, Aline Silva; Bertin, John; Gough, Peter J; Gazzinelli, Ricardo; Almeida, Roque P; Bozza, Marcelo T; Borges, Valeria M; Chan, Francis Ka-Ming

2016-06-15

Leishmaniasis is an important parasitic disease found in the tropics and subtropics. Cutaneous and visceral leishmaniasis affect an estimated 1.5 million people worldwide. Despite its human health relevance, relatively little is known about the cell death pathways that control Leishmania replication in the host. Necroptosis is a recently identified form of cell death with potent antiviral effects. Receptor interacting protein kinase 1 (RIPK1) is a critical kinase that mediates necroptosis downstream of death receptors and TLRs. Heme, a product of hemoglobin catabolism during certain intracellular pathogen infections, is also a potent inducer of macrophage necroptosis. We found that human visceral leishmaniasis patients exhibit elevated serum levels of heme. Therefore, we examined the impact of heme and necroptosis on Leishmania replication. Indeed, heme potently inhibited Leishmania replication in bone marrow-derived macrophages. Moreover, we found that inhibition of RIPK1 kinase activity also enhanced parasite replication in the absence of heme. We further found that the mitochondrial phosphatase phosphoglycerate mutase family member 5 (PGAM5), a putative downstream effector of RIPK1, was also required for inhibition of Leishmania replication. In mouse infection, both PGAM5 and RIPK1 kinase activity are required for IL-1β expression in response to Leishmania However, PGAM5, but not RIPK1 kinase activity, was directly responsible for Leishmania-induced IL-1β secretion and NO production in bone marrow-derived macrophages. Collectively, these results revealed that RIPK1 and PGAM5 function independently to exert optimal control of Leishmania replication in the host. Copyright © 2016 by The American Association of Immunologists, Inc.

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.

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.

Fidelity of DNA Replication in Normal and Malignant Human Breast Cells

National Research Council Canada - National Science Library

Sekowski, Jennifer

1998-01-01

In order to determine the degree to which the accumulation of mutations in breast cancer cells is due to a change in the fidelity of the cellular DNA replication machinery we have completed a series...

STAT2 Knockout Syrian Hamsters Support Enhanced Replication and Pathogenicity of Human Adenovirus, Revealing an Important Role of Type I Interferon Response in Viral Control.

Directory of Open Access Journals (Sweden)

Karoly Toth

2015-08-01

Full Text Available Human adenoviruses have been studied extensively in cell culture and have been a model for studies in molecular, cellular, and medical biology. However, much less is known about adenovirus replication and pathogenesis in vivo in a permissive host because of the lack of an adequate animal model. Presently, the most frequently used permissive immunocompetent animal model for human adenovirus infection is the Syrian hamster. Species C human adenoviruses replicate in these animals and cause pathology that is similar to that seen with humans. Here, we report findings with a new Syrian hamster strain in which the STAT2 gene was functionally knocked out by site-specific gene targeting. Adenovirus-infected STAT2 knockout hamsters demonstrated an accentuated pathology compared to the wild-type control animals, and the virus load in the organs of STAT2 knockout animals was 100- to 1000-fold higher than that in wild-type hamsters. Notably, the adaptive immune response to adenovirus is not adversely affected in STAT2 knockout hamsters, and surviving hamsters cleared the infection by 7 to 10 days post challenge. We show that the Type I interferon pathway is disrupted in these hamsters, revealing the critical role of interferon-stimulated genes in controlling adenovirus infection. This is the first study to report findings with a genetically modified Syrian hamster infected with a virus. Further, this is the first study to show that the Type I interferon pathway plays a role in inhibiting human adenovirus replication in a permissive animal model. Besides providing an insight into adenovirus infection in humans, our results are also interesting from the perspective of the animal model: STAT2 knockout Syrian hamster may also be an important animal model for studying other viral infections, including Ebola-, hanta-, and dengue viruses, where Type I interferon-mediated innate immunity prevents wild type hamsters from being effectively infected to be used as

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.)

Pyrimidine dimers block simian virus 40 replication forks

International Nuclear Information System (INIS)

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

1986-01-01

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

Initiation preference at a yeast origin of replication.

Science.gov (United States)

Brewer, B J; Fangman, W L

1994-04-12

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

Effects of age, replicative lifespan and growth rate of human nucleus pulposus cells on selecting age range for cell-based biological therapies for degenerative disc diseases.

Science.gov (United States)

Lee, J S; Lee, S M; Jeong, S W; Sung, Y G; Lee, J H; Kim, K W

2016-07-01

Autologous disc cell implantation, growth factors and gene therapy appear to be promising therapies for disc regeneration. Unfortunately, the replicative lifespan and growth kinetics of human nucleus pulposus (NP) cells related to host age are unclear. We investigated the potential relations among age, replicative lifespan and growth rate of NP cells, and determined the age range that is suitable for cell-based biological therapies for degenerative disc diseases. We used NP tissues classified by decade into five age groups: 30s, 40s, 50s, 60s and 70s. The mean cumulative population doubling level (PDL) and population doubling rate (PDR) of NP cells were assessed by decade. We also investigated correlations between cumulative PDL and age, and between PDR and age. The mean cumulative PDL and PDR decreased significantly in patients in their 60s. The mean cumulative PDL and PDR in the younger groups (30s, 40s and 50s) were significantly higher than those in the older groups (60s and 70s). There also were significant negative correlations between cumulative PDL and age, and between PDR and age. We found that the replicative lifespan and growth rate of human NP cells decreased with age. The replicative potential of NP cells decreased significantly in patients 60 years old and older. Young individuals less than 60 years old may be suitable candidates for NP cell-based biological therapies for treating degenerative disc diseases.

Soluble human CD4 elicits an antibody response in rhesus monkeys that inhibits simian immunodeficiency virus replication

International Nuclear Information System (INIS)

Watanabe, Mamoru; Chen, Zheng W.; Tsubota, Hiroshi; Lord, C.I.; Levine, C.G.; Letvin, N.L.

1991-01-01

Rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIV mac ) demonstrate significant virologic and clinical improvement as a result of treatment with human recombinant soluble CD4 (rsCD4). The authors show that human rsCD4 does not efficiently inhibit SIV mac replication in bone marrow macrophages of rhesus monkeys and does not significantly augment bone marrow hematopoietic colony formation in vitro. However, plasma of human rsCD4-treated rhesus monkeys does exhibit significant anti-SIV mac activity in vitro. Plasma of these animals efficiently blocks SIV mac replicaton in peripheral blood lymphocytes and bone marrow macrophages. It also increases granulocyte/macrophage colony formation in vitro by bone marrow cells of SIV mac -infected monkeys. This plasma and the IgG fraction of plasma from a rhesus monkey immunized with human rsCD4 in adjuvant demonstrate reactivity with a soluble form of the rhesus monkey CD4 molecule, exhibit binding to CD4 + but not CD8 + concanavalin A-activated rhesus monkey peripheral blood lymphocytes, and precipitate the CD4 molecule from surface-labeled activated rhesus monkey peripheral blood lymphocytes. Moreover, anti-viral activity is demonstrable in the IgG fraction of plasma from a human rsCD4-immunized monkey. These studies raise the possibility that a modified human CD4 molecule serving as an immunogen might elicit an antibody response that could potentially induce a beneficial therapeutic response in human immunodeficiency virus-infected individuals

Database Replication

CERN Document Server

Kemme, Bettina

2010-01-01

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

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

Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

International Nuclear Information System (INIS)

Lu, Li; Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin; Song, Wen-Hui; Yan, Ba-Yi; Yang, Gui-Jiao; Li, Ang; Yang, Wu-Lin

2014-01-01

Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5

Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

Energy Technology Data Exchange (ETDEWEB)

Lu, Li, E-mail: luli7300@126.com [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Wen-Hui [Department of Orthopaedics, The Second Affiliated Hospital of Shanxi Medical University, Taiyuan 030001 (China); Yan, Ba-Yi; Yang, Gui-Jiao [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Li, Ang [Department of Medicine, University of Hong Kong Faculty of Medicine, Hong Kong (Hong Kong); Department of Anatomy, University of Hong Kong Faculty of Medicine, Hong Kong (Hong Kong); Yang, Wu-Lin, E-mail: wulinyoung@163.com [School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium (SBIC), Agency for Science, Technology and Research - A*STAR (Singapore)

2014-01-24

Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5.

New insights into HCV replication in original cells from Aedes mosquitoes.

Science.gov (United States)

Fallecker, Catherine; Caporossi, Alban; Rechoum, Yassine; Garzoni, Frederic; Larrat, Sylvie; François, Olivier; Fender, Pascal; Morand, Patrice; Berger, Imre; Petit, Marie-Anne; Drouet, Emmanuel

2017-08-22

The existing literature about HCV association with, and replication in mosquitoes is extremely poor. To fill this gap, we performed cellular investigations aimed at exploring (i) the capacity of HCV E1E2 glycoproteins to bind on Aedes mosquito cells and (ii) the ability of HCV serum particles (HCVsp) to replicate in these cell lines. First, we used purified E1E2 expressing baculovirus-derived HCV pseudo particles (bacHCVpp) so we could investigate their association with mosquito cell lines from Aedes aegypti (Aag-2) and Aedes albopictus (C6/36). We initiated a series of infections of both mosquito cells (Ae aegypti and Ae albopictus) with the HCVsp (Lat strain - genotype 3) and we observed the evolution dynamics of viral populations within cells over the course of infection via next-generation sequencing (NGS) experiments. Our binding assays revealed bacHCVpp an association with the mosquito cells, at comparable levels obtained with human hepatocytes (HepaRG cells) used as a control. In our infection experiments, the HCV RNA (+) were detectable by RT-PCR in the cells between 21 and 28 days post-infection (p.i.). In human hepatocytes HepaRG and Ae aegypti insect cells, NGS experiments revealed an increase of global viral diversity with a selection for a quasi-species, suggesting a structuration of the population with elimination of deleterious mutations. The evolutionary pattern in Ae albopictus insect cells is different (stability of viral diversity and polymorphism). These results demonstrate for the first time that natural HCV could really replicate within Aedes mosquitoes, a discovery which may have major consequences for public health as well as in vaccine development.

The multi-replication protein A (RPA) system--a new perspective.

Science.gov (United States)

Sakaguchi, Kengo; Ishibashi, Toyotaka; Uchiyama, Yukinobu; Iwabata, Kazuki

2009-02-01

Replication protein A (RPA) complex has been shown, using both in vivo and in vitro approaches, to be required for most aspects of eukaryotic DNA metabolism: replication, repair, telomere maintenance and homologous recombination. Here, we review recent data concerning the function and biological importance of the multi-RPA complex. There are distinct complexes of RPA found in the biological kingdoms, although for a long time only one type of RPA complex was believed to be present in eukaryotes. Each complex probably serves a different role. In higher plants, three distinct large and medium subunits are present, but only one species of the smallest subunit. Each of these protein subunits forms stable complexes with their respective partners. They are paralogs as complex. Humans possess two paralogs and one analog of RPA. The multi-RPA system can be regarded as universal in eukaryotes. Among eukaryotic kingdoms, paralogs, orthologs, analogs and heterologs of many DNA synthesis-related factors, including RPA, are ubiquitous. Convergent evolution seems to be ubiquitous in these processes. Using recent findings, we review the composition and biological functions of RPA complexes.

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.

Effect of ethanol on innate antiviral pathways and HCV replication in human liver cells

Directory of Open Access Journals (Sweden)

Fausto Nelson

2005-12-01

Full Text Available Abstract Alcohol abuse reduces response rates to IFN therapy in patients with chronic hepatitis C. To model the molecular mechanisms behind this phenotype, we characterized the effects of ethanol on Jak-Stat and MAPK pathways in Huh7 human hepatoma cells, in HCV replicon cell lines, and in primary human hepatocytes. High physiological concentrations of acute ethanol activated the Jak-Stat and p38 MAPK pathways and inhibited HCV replication in several independent replicon cell lines. Moreover, acute ethanol induced Stat1 serine phosphorylation, which was partially mediated by the p38 MAPK pathway. In contrast, when combined with exogenously applied IFN-α, ethanol inhibited the antiviral actions of IFN against HCV replication, involving inhibition of IFN-induced Stat1 tyrosine phosphorylation. These effects of alcohol occurred independently of i alcohol metabolism via ADH and CYP2E1, and ii cytotoxic or cytostatic effects of ethanol. In this model system, ethanol directly perturbs the Jak-Stat pathway, and HCV replication. Infection with Hepatitis C virus is a significant cause of morbidity and mortality throughout the world. With a propensity to progress to chronic infection, approximately 70% of patients with chronic viremia develop histological evidence of chronic liver diseases including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The situation is even more dire for patients who abuse ethanol, where the risk of developing end stage liver disease is significantly higher as compared to HCV patients who do not drink 12. Recombinant interferon alpha (IFN-α therapy produces sustained responses (ie clearance of viremia in 8–12% of patients with chronic hepatitis C 3. Significant improvements in response rates can be achieved with IFN plus ribavirin combination 456 and pegylated IFN plus ribavirin 78 therapies. However, over 50% of chronically infected patients still do not clear viremia. Moreover, HCV-infected patients who abuse

Effects of integration and replication on transcription of the HIV-1 long terminal repeat

NARCIS (Netherlands)

Jeang, K. T.; Berkhout, B.; Dropulic, B.

1993-01-01

The activity of a promoter is influenced by chromosomal and cell cycle/replication context. We analyzed the influences of integration and replication on transcription of the human immunodeficiency virus (HIV)-1 long terminal repeat (LTR). We found that one requirement for Tat trans-activated

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...

Human Immunodeficiency Viruses Appear Compartmentalized to the Female Genital Tract in Cross-Sectional Analyses but Genital Lineages Do Not Persist Over Time

OpenAIRE

Bull, Marta E.; Heath, Laura M.; McKernan-Mullin, Jennifer L.; Kraft, Kelli M.; Acevedo, Luis; Hitti, Jane E.; Cohn, Susan E.; Tapia, Kenneth A.; Holte, Sarah E.; Dragavon, Joan A.; Coombs, Robert W.; Mullins, James I.; Frenkel, Lisa M.

2013-01-01

Background. Whether unique human immunodeficiency type 1 (HIV) genotypes occur in the genital tract is important for vaccine development and management of drug resistant viruses. Multiple cross-sectional studies suggest HIV is compartmentalized within the female genital tract. We hypothesize that bursts of HIV replication and/or proliferation of infected cells captured in cross-sectional analyses drive compartmentalization but over time genital-specific viral lineages do not form; rather viru...

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

Directory of Open Access Journals (Sweden)

Richard A. Klein

2014-04-01

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

Maintaining replication origins in the face of genomic change.

Science.gov (United States)

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.

Low cost management of replicated data in fault-tolerant distributed systems

Science.gov (United States)

Joseph, Thomas A.; Birman, Kenneth P.

1990-01-01

Many distributed systems replicate data for fault tolerance or availability. In such systems, a logical update on a data item results in a physical update on a number of copies. The synchronization and communication required to keep the copies of replicated data consistent introduce a delay when operations are performed. A technique is described that relaxes the usual degree of synchronization, permitting replicated data items to be updated concurrently with other operations, while at the same time ensuring that correctness is not violated. The additional concurrency thus obtained results in better response time when performing operations on replicated data. How this technique performs in conjunction with a roll-back and a roll-forward failure recovery mechanism is also discussed.

Replicative senescence of T cells: does the Hayflick Limit lead to immune exhaustion?

Science.gov (United States)

Effros, R B; Pawelec, G

1997-09-01

Extensive in vitro research on fibroblasts has defined numerous genetic and phenotypic changes associated with replicative senescence. Identification of T-cell replicative senescence as a feature of human immunodeficiency virus (HIV) disease and ageing suggests this phenomenon merits more careful consideration by immunologists, especially with regard to chronic infection, memory and adoptive immunotherapy.

Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA

DEFF Research Database (Denmark)

Reddy, Madhava C; Christensen, Jesper; Vasquez, Karen M

2005-01-01

-DNA interstrand cross-link (ICL) to a specific site to determine the effect of HMGB proteins on recognition of these lesions. Our results reveal that human HMGB1 (but not HMGB2) binds with high affinity and specificity to psoralen ICLs, and interacts with the essential NER protein, replication protein A (RPA......), at these lesions. RPA, shown previously to bind tightly to these lesions, also binds in the presence of HMGB1, without displacing HMGB1. A discrete ternary complex is formed, containing HMGB1, RPA, and psoralen-damaged DNA. Thus, HMGB1 has the ability to recognize ICLs, can cooperate with RPA in doing so...

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.

Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress

Science.gov (United States)

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

2016-01-01

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

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.

Inter-Fork Strand Annealing causes genomic deletions during the termination of DNA replication.

Science.gov (United States)

Morrow, Carl A; Nguyen, Michael O; Fower, Andrew; Wong, Io Nam; Osman, Fekret; Bryer, Claire; Whitby, Matthew C

2017-06-06

Problems that arise during DNA replication can drive genomic alterations that are instrumental in the development of cancers and many human genetic disorders. Replication fork barriers are a commonly encountered problem, which can cause fork collapse and act as hotspots for replication termination. Collapsed forks can be rescued by homologous recombination, which restarts replication. However, replication restart is relatively slow and, therefore, replication termination may frequently occur by an active fork converging on a collapsed fork. We find that this type of non-canonical fork convergence in fission yeast is prone to trigger deletions between repetitive DNA sequences via a mechanism we call Inter-Fork Strand Annealing (IFSA) that depends on the recombination proteins Rad52, Exo1 and Mus81, and is countered by the FANCM-related DNA helicase Fml1. Based on our findings, we propose that IFSA is a potential threat to genomic stability in eukaryotes.

Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus

Directory of Open Access Journals (Sweden)

Mittra Arjun

2011-03-01

Full Text Available Abstract Introduction Oncolytic viruses show promise for treating cancer. However, to assess therapeutic efficacy and potential toxicity, a noninvasive imaging modality is needed. This study aimed to determine if insertion of the human sodium iodide symporter (hNIS cDNA as a marker for non-invasive imaging of virotherapy alters the replication and oncolytic capability of a novel vaccinia virus, GLV-1h153. Methods GLV-1h153 was modified from parental vaccinia virus GLV-1h68 to carry hNIS via homologous recombination. GLV-1h153 was tested against human pancreatic cancer cell line PANC-1 for replication via viral plaque assays and flow cytometry. Expression and transportation of hNIS in infected cells was evaluated using Westernblot and immunofluorescence. Intracellular uptake of radioiodide was assessed using radiouptake assays. Viral cytotoxicity and tumor regression of treated PANC-1tumor xenografts in nude mice was also determined. Finally, tumor radiouptake in xenografts was assessed via positron emission tomography (PET utilizing carrier-free 124I radiotracer. Results GLV-1h153 infected, replicated within, and killed PANC-1 cells as efficiently as GLV-1h68. GLV-1h153 provided dose-dependent levels of hNIS expression in infected cells. Immunofluorescence detected transport of the protein to the cell membrane prior to cell lysis, enhancing hNIS-specific radiouptake (P In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts (P 124I-PET. Conclusion Insertion of the hNIS gene does not hinder replication or oncolytic capability of GLV-1h153, rendering this novel virus a promising new candidate for the noninvasive imaging and tracking of oncolytic viral therapy.

Molecular analysis of the replication program in unicellular model organisms

OpenAIRE

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 o...

Identifying Cancer Driver Genes Using Replication-Incompetent Retroviral Vectors

Directory of Open Access Journals (Sweden)

Victor M. Bii

2016-10-01

Full Text Available Identifying novel genes that drive tumor metastasis and drug resistance has significant potential to improve patient outcomes. High-throughput sequencing approaches have identified cancer genes, but distinguishing driver genes from passengers remains challenging. Insertional mutagenesis screens using replication-incompetent retroviral vectors have emerged as a powerful tool to identify cancer genes. Unlike replicating retroviruses and transposons, replication-incompetent retroviral vectors lack additional mutagenesis events that can complicate the identification of driver mutations from passenger mutations. They can also be used for almost any human cancer due to the broad tropism of the vectors. Replication-incompetent retroviral vectors have the ability to dysregulate nearby cancer genes via several mechanisms including enhancer-mediated activation of gene promoters. The integrated provirus acts as a unique molecular tag for nearby candidate driver genes which can be rapidly identified using well established methods that utilize next generation sequencing and bioinformatics programs. Recently, retroviral vector screens have been used to efficiently identify candidate driver genes in prostate, breast, liver and pancreatic cancers. Validated driver genes can be potential therapeutic targets and biomarkers. In this review, we describe the emergence of retroviral insertional mutagenesis screens using replication-incompetent retroviral vectors as a novel tool to identify cancer driver genes in different cancer types.

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…

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...

Quantitative live imaging of endogenous DNA replication in mammalian cells.

Directory of Open Access Journals (Sweden)

Andrew Burgess

Full Text Available Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.

Accessory genes confer a high replication rate to virulent feline immunodeficiency virus.

Science.gov (United States)

Troyer, Ryan M; Thompson, Jesse; Elder, John H; VandeWoude, Sue

2013-07-01

Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.

The nucleolar phosphoprotein B23 targets Newcastle disease virus matrix protein to the nucleoli and facilitates viral replication.

Science.gov (United States)

Duan, Zhiqiang; Chen, Jian; Xu, Haixu; Zhu, Jie; Li, Qunhui; He, Liang; Liu, Huimou; Hu, Shunlin; Liu, Xiufan

2014-03-01

The cellular nucleolar proteins are reported to facilitate the replication cycles of some human and animal viruses by interaction with viral proteins. In this study, a nucleolar phosphoprotein B23 was identified to interact with Newcastle disease virus (NDV) matrix (M) protein. We found that NDV M protein accumulated in the nucleolus by binding B23 early in infection, but resulted in the redistribution of B23 from the nucleoli to the nucleoplasm later in infection. In vitro binding studies utilizing deletion mutants indicated that amino acids 30-60 of M and amino acids 188-245 of B23 were required for binding. Furthermore, knockdown of B23 by siRNA or overexpression of B23 or M-binding B23-derived polypeptides remarkably reduced cytopathic effect and inhibited NDV replication. Collectively, we show that B23 facilitates NDV replication by targeting M to the nucleolus, demonstrating for the first time a direct role for nucleolar protein B23 in a paramyxovirus replication process. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Human cellular restriction factors that target HIV-1 replication

Directory of Open Access Journals (Sweden)

Jeang Kuan-Teh

2009-09-01

Full Text Available Abstract Recent findings have highlighted roles played by innate cellular factors in restricting intracellular viral replication. In this review, we discuss in brief the activities of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, bone marrow stromal cell antigen 2 (BST-2, cyclophilin A, tripartite motif protein 5 alpha (Trim5α, and cellular microRNAs as examples of host restriction factors that target HIV-1. We point to countermeasures encoded by HIV-1 for moderating the potency of these cellular restriction functions.

Intrinsic Stability of Episomal Circles Formed during Human Immunodeficiency Virus Type 1 Replication

Science.gov (United States)

Pierson, TheodoreC.; Kieffer, Tara L.; Ruff, Christian T.; Buck, Christopher; Gange, Stephen J.; Siliciano, Robert F.

2002-01-01

The development of surrogate markers capable of detecting residual ongoing human immunodeficiency virus type 1 (HIV-1) replication in patients receiving highly active antiretroviral therapy is an important step in understanding viral dynamics and in developing new treatment strategies. In this study, we evaluated the utility of circular forms of the viral genome for the detection of recent infection of cells by HIV-1. We measured the fate of both one-long terminal repeat (1-LTR) and 2-LTR circles following in vitro infection of logarithmically growing CD4+ T cells under conditions in which cell death was not a significant contributing factor. Circular forms of the viral genome were found to be highly stable and to decrease in concentration only as a function of dilution resulting from cell division. We conclude that these DNA circles are not intrinsically unstable in all cell types and suggest that the utility of 2-LTR circle assays in measuring recent HIV-1 infection of susceptible cells in vivo needs to be reevaluated. PMID:11907256

Efficiency of Human Epiphyseal Chondrocytes with Differential Replication Numbers for Cellular Therapy Products

Directory of Open Access Journals (Sweden)

Michiyo Nasu

2016-01-01

Full Text Available The cell-based therapy for cartilage or bone requires a large number of cells; serial passages of chondrocytes are, therefore, needed. However, fates of expanded chondrocytes from extra fingers remain unclarified. The chondrocytes from human epiphyses morphologically changed from small polygonal cells to bipolar elongated spindle cells and to large polygonal cells with degeneration at early passages. Gene of type II collagen was expressed in the cells only at a primary culture (Passage 0 and Passage 1 (P1 cells. The nodules by implantation of P0 to P8 cells were composed of cartilage and perichondrium. The cartilage consisted of chondrocytes with round nuclei and type II collagen-positive matrix, and the perichondrium consisted of spindle cells with type I collage-positive matrix. The cartilage and perichondrium developed to bone with marrow cavity through enchondral ossification. Chondrogenesis and osteogenesis by epiphyseal chondrocytes depended on replication number in culture. It is noteworthy to take population doubling level in correlation with pharmaceutical efficacy into consideration when we use chondrocytes for cell-based therapies.

Pandemic Swine-Origin H1N1 Influenza Virus Replicates to Higher Levels and Induces More Fever and Acute Inflammatory Cytokines in Cynomolgus versus Rhesus Monkeys and Can Replicate in Common Marmosets.

Directory of Open Access Journals (Sweden)

Petra Mooij

Full Text Available The close immunological and physiological resemblance with humans makes non-human primates a valuable model for studying influenza virus pathogenesis and immunity and vaccine efficacy against infection. Although both cynomolgus and rhesus macaques are frequently used in influenza virus research, a direct comparison of susceptibility to infection and disease has not yet been performed. In the current study a head-to-head comparison was made between these species, by using a recently described swine-origin pandemic H1N1 strain, A/Mexico/InDRE4487/2009. In comparison to rhesus macaques, cynomolgus macaques developed significantly higher levels of virus replication in the upper airways and in the lungs, involving both peak level and duration of virus production, as well as higher increases in body temperature. In contrast, clinical symptoms, including respiratory distress, were more easily observed in rhesus macaques. Expression of sialyl-α-2,6-Gal saccharides, the main receptor for human influenza A viruses, was 50 to 73 times more abundant in trachea and bronchus of cynomolgus macaques relative to rhesus macaques. The study also shows that common marmosets, a New World non-human primate species, are susceptible to infection with pandemic H1N1. The study results favor the cynomolgus macaque as model for pandemic H1N1 influenza virus research because of the more uniform and high levels of virus replication, as well as temperature increases, which may be due to a more abundant expression of the main human influenza virus receptor in the trachea and bronchi.

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

Hydroxyurea-Induced Replication Stress

Directory of Open Access Journals (Sweden)

Kenza Lahkim Bennani-Belhaj

2010-01-01

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

Transcription-replication conflicts at chromosomal fragile sites—consequences in M phase and beyond

DEFF Research Database (Denmark)

Østergaard, Vibe Hallundbæk; Lisby, Michael

2017-01-01

transcription and replication patterns. At the same time, these chromosomal fragile sites engage in aberrant DNA structures in mitosis. Here, we discuss the mechanistic details of transcription–replication conflicts including putative scenarios for R-loop-induced replication inhibition to understand how...... transcription–replication conflicts transition from S phase into various aberrant DNA structures in mitosis....

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.

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast.

Directory of Open Access Journals (Sweden)

Joseph C Sanchez

2017-10-01

Full Text Available A form of dwarfism known as Meier-Gorlin syndrome (MGS is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5. These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45. The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C. We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast.

Science.gov (United States)

Sanchez, Joseph C; Kwan, Elizabeth X; Pohl, Thomas J; Amemiya, Haley M; Raghuraman, M K; Brewer, Bonita J

2017-10-01

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.

Human CST Facilitates Genome-wide RAD51 Recruitment to GC-Rich Repetitive Sequences in Response to Replication Stress.

Science.gov (United States)

Chastain, Megan; Zhou, Qing; Shiva, Olga; Fadri-Moskwik, Maria; Whitmore, Leanne; Jia, Pingping; Dai, Xueyu; Huang, Chenhui; Ye, Ping; Chai, Weihang

2016-08-02

The telomeric CTC1/STN1/TEN1 (CST) complex has been implicated in promoting replication recovery under replication stress at genomic regions, yet its precise role is unclear. Here, we report that STN1 is enriched at GC-rich repetitive sequences genome-wide in response to hydroxyurea (HU)-induced replication stress. STN1 deficiency exacerbates the fragility of these sequences under replication stress, resulting in chromosome fragmentation. We find that upon fork stalling, CST proteins form distinct nuclear foci that colocalize with RAD51. Furthermore, replication stress induces physical association of CST with RAD51 in an ATR-dependent manner. Strikingly, CST deficiency diminishes HU-induced RAD51 foci formation and reduces RAD51 recruitment to telomeres and non-telomeric GC-rich fragile sequences. Collectively, our findings establish that CST promotes RAD51 recruitment to GC-rich repetitive sequences in response to replication stress to facilitate replication restart, thereby providing insights into the mechanism underlying genome stability maintenance. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Parental Leave Policies, Intra-household Time Allocations and Children's Human Capital

OpenAIRE

Bernal, Raquel; Fruttero, Anna

2008-01-01

This paper uses a general equilibrium model of marriage and divorce to assess how public policies on parental leave and leave benefits affect intra-household decision making, family structure, intergenerational mobility, and the distribution of income. The benchmark economy is calibrated to US data to replicate some characteristics relevant to the interaction between the marriage and labor markets. The effects of unpaid leave, paid leave benefits, and mandated leave on human capital investmen...

In vitro assessment of attachment pattern and replication efficiency of H5N1 influenza A viruses with altered receptor specificity.

Science.gov (United States)

Chutinimitkul, Salin; van Riel, Debby; Munster, Vincent J; van den Brand, Judith M A; Rimmelzwaan, Guus F; Kuiken, Thijs; Osterhaus, Albert D M E; Fouchier, Ron A M; de Wit, Emmie

2010-07-01

The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, alpha2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to alpha2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with alpha2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished.

In Vitro Assessment of Attachment Pattern and Replication Efficiency of H5N1 Influenza A Viruses with Altered Receptor Specificity▿

Science.gov (United States)

Chutinimitkul, Salin; van Riel, Debby; Munster, Vincent J.; van den Brand, Judith M. A.; Rimmelzwaan, Guus F.; Kuiken, Thijs; Osterhaus, Albert D. M. E.; Fouchier, Ron A. M.; de Wit, Emmie

2010-01-01

The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, α2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to α2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with α2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished. PMID:20392847

Augmentation of DHCR24 expression by hepatitis C virus infection facilitates viral replication in hepatocytes.

Science.gov (United States)

Takano, Takashi; Tsukiyama-Kohara, Kyoko; Hayashi, Masahiro; Hirata, Yuichi; Satoh, Masaaki; Tokunaga, Yuko; Tateno, Chise; Hayashi, Yukiko; Hishima, Tsunekazu; Funata, Nobuaki; Sudoh, Masayuki; Kohara, Michinori

2011-09-01

We characterized the role of 24-dehydrocholesterol reductase (DHCR24) in hepatitis C virus infection (HCV). DHCR24 is a cholesterol biosynthetic enzyme and cholesterol is a major component of lipid rafts, which is reported to play an important role in HCV replication. Therefore, we examined the potential of DHCR24 as a target for novel HCV therapeutic agents. We examined DHCR24 expression in human hepatocytes in both the livers of HCV-infected patients and those of chimeric mice with human hepatocytes. We targeted DHCR24 with siRNA and U18666A which is an inhibitor of both DHCR24 and cholesterol synthesis. We measured the level of HCV replication in these HCV replicon cell lines and HCV infected cells. U18666A was administrated into chimeric mice with humanized liver, and anti-viral effects were assessed. Expression of DHCR24 was induced by HCV infection in human hepatocytes in vitro, and in human hepatocytes of chimeric mouse liver. Silencing of DHCR24 by siRNA decreased HCV replication in replicon cell lines and HCV JFH-1 strain-infected cells. Treatment with U18666A suppressed HCV replication in the replicon cell lines. Moreover, to evaluate the anti-viral effect of U18666A in vivo, we administrated U18666A with or without pegylated interferon to chimeric mice and observed an inhibitory effect of U18666A on HCV infection and a synergistic effect with interferon. DHCR24 is an essential host factor which augmented its expression by HCV infection, and plays a significant role in HCV replication. DHCR24 may serve as a novel anti-HCV drug target. Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Identification of rep-associated factors in herpes simplex virus type 1-induced adeno-associated virus type 2 replication compartments.

Science.gov (United States)

Nicolas, Armel; Alazard-Dany, Nathalie; Biollay, Coline; Arata, Loredana; Jolinon, Nelly; Kuhn, Lauriane; Ferro, Myriam; Weller, Sandra K; Epstein, Alberto L; Salvetti, Anna; Greco, Anna

2010-09-01

Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase delta was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.

Hierarchical Data Replication and Service Monitoring Methods in a Scientific Data Grid

Directory of Open Access Journals (Sweden)

Weizhong Lu

2009-04-01

Full Text Available In a grid and distributed computing environment, data replication is an effective way to improve data accessibility and data accessing efficiency. It is also significant in developing a real-time service monitoring system for a Chinese Scientific Data Grid to guarantee the system stability and data availability. Hierarchical data replication and service monitoring methods are proposed in this paper. The hierarchical data replication method divides the network into different domains and replicates data in local domains. The nodes in a local domain are classified into hierarchies to improve data accessibility according to bandwidth and storage memory space. An extensible agent-based prototype of a hierarchical service monitoring system is presented. The status information of services in the Chinese Scientific Data Grid is collected from the grid nodes based on agent technology and then is transformed into real-time operational pictures for management needs. This paper presents frameworks of the hierarchical data replication and service monitoring methods and gives detailed resolutions. Simulation analyses have demonstrated improved data accessing efficiency and verified the effectiveness of the methods at the same time.

Replication and transmission of H9N2 influenza viruses in ferrets: evaluation of pandemic potential.

Directory of Open Access Journals (Sweden)

Hongquan Wan

2008-08-01

Full Text Available H9N2 avian influenza A viruses are endemic in poultry of many Eurasian countries and have caused repeated human infections in Asia since 1998. To evaluate the potential threat of H9N2 viruses to humans, we investigated the replication and transmission efficiency of H9N2 viruses in the ferret model. Five wild-type (WT H9N2 viruses, isolated from different avian species from 1988 through 2003, were tested in vivo and found to replicate in ferrets. However these viruses achieved mild peak viral titers in nasal washes when compared to those observed with a human H3N2 virus. Two of these H9N2 viruses transmitted to direct contact ferrets, however no aerosol transmission was detected in the virus displaying the most efficient direct contact transmission. A leucine (Leu residue at amino acid position 226 in the hemagglutinin (HA receptor-binding site (RBS, responsible for human virus-like receptor specificity, was found to be important for the transmission of the H9N2 viruses in ferrets. In addition, an H9N2 avian-human reassortant virus, which contains the surface glycoprotein genes from an H9N2 virus and the six internal genes of a human H3N2 virus, showed enhanced replication and efficient transmission to direct contacts. Although no aerosol transmission was observed, the virus replicated in multiple respiratory tissues and induced clinical signs similar to those observed with the parental human H3N2 virus. Our results suggest that the establishment and prevalence of H9N2 viruses in poultry pose a significant threat for humans.

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.

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

Science.gov (United States)

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

2017-01-05

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

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.

Virtual Machine Replication on Achieving Energy-Efficiency in a Cloud

Directory of Open Access Journals (Sweden)

Subrota K. Mondal

2016-07-01

Full Text Available The rapid growth in cloud service demand has led to the establishment of large-scale virtualized data centers in which virtual machines (VMs are used to handle user requests for service. A user’s request cannot be completed if the VM fails. Replication mechanisms can be used to mitigate the impact of failures. Further, data centers consume a large amount of energy resulting in high operating costs and contributing to significant greenhouse gas (GHG emissions. In this paper, we focus on Infrastructure as a Service (IaaS cloud where user job requests are processed by VMs and analyze the effectiveness of VM replications in terms of job completion time performance as well as energy consumption. Three different schemes: cold, warm, and hot replications are considered. The trade-offs between job completion time and energy consumption in different replication schemes are characterized through comprehensive analytical models which capture VM state transitions and associated power consumption patterns. The effectiveness of replication schemes are demonstrated through experimental results. To verify the validity of the proposed analytical models, we extend the widely used cloud simulator CloudSim and compare the simulation results with analytical solutions.

Novel host restriction factors implicated in HIV-1 replication.

Science.gov (United States)

Ghimire, Dibya; Rai, Madhu; Gaur, Ritu

2018-04-01

Human immunodeficiency virus-1 (HIV-1) is known to interact with multiple host cellular proteins during its replication in the target cell. While many of these host cellular proteins facilitate viral replication, a number of them are reported to inhibit HIV-1 replication at various stages of its life cycle. These host cellular proteins, which are known as restriction factors, constitute an integral part of the host's first line of defence against the viral pathogen. Since the discovery of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G) as an HIV-1 restriction factor, several human proteins have been identified that exhibit anti-HIV-1 restriction. While each restriction factor employs a distinct mechanism of inhibition, the HIV-1 virus has equally evolved complex counter strategies to neutralize their inhibitory effect. APOBEC3G, tetherin, sterile alpha motif and histidine-aspartate domain 1 (SAMHD1), and trim-5α are some of the best known HIV-1 restriction factors that have been studied in great detail. Recently, six novel restriction factors were discovered that exhibit significant antiviral activity: endoplasmic reticulum α1,2-mannosidase I (ERManI), translocator protein (TSPO), guanylate-binding protein 5 (GBP5), serine incorporator (SERINC3/5) and zinc-finger antiviral protein (ZAP). The focus of this review is to discuss the antiviral mechanism of action of these six restriction factors and provide insights into the probable counter-evasion strategies employed by the HIV-1 virus. The recent discovery of new restriction factors substantiates the complex host-pathogen interactions occurring during HIV-1 pathogenesis and makes it imperative that further investigations are conducted to elucidate the molecular basis of HIV-1 replication.

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.

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

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

How MCM loading and spreading specify eukaryotic DNA replication initiation sites [version 1; referees: 4 approved

Directory of Open Access Journals (Sweden)

Olivier Hyrien

2016-08-01

Full Text Available DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs, the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC, they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

Human prostate supports more efficient replication of HIV-1 R5 than X4 strains ex vivo

Directory of Open Access Journals (Sweden)

Denis Hélène

2008-12-01

Full Text Available Abstract Background In order to determine whether human prostate can be productively infected by HIV-1 strains with different tropism, and thus represent a potential source of HIV in semen, an organotypic culture of prostate from men undergoing prostatic adenomectomy for benign prostate hypertrophy (BPH was developed. The presence of potential HIV target cells in prostate tissues was investigated using immunohistochemistry. The infection of prostate explants following exposures with HIV-1 R5, R5X4 and X4 strains was analyzed through the measure of RT activity in culture supernatants, the quantification of HIV DNA in the explants and the detection of HIV RNA+ cells in situ. Results The overall prostate characteristics were retained for 21/2 weeks in culture. Numerous potential HIV-1 target cells were detected in the prostate stroma. Whilst HIV-1 R5SF162 strain consistently productively infected prostatic T lymphocytes and macrophages, the prototypic X4IIIB strain and a primary R5X4 strain showed less efficient replication in this organ. Conclusion The BPH prostate is a site of HIV-1 R5 replication that could contribute virus to semen. A limited spreading of HIV-1 X4 and R5X4 in this organ could participate to the preferential sexual transmission of HIV-1 R5 strains.

Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines.

Directory of Open Access Journals (Sweden)

Anne Frentzen

2011-04-01

Full Text Available Hepatitis C virus (HCV is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model.

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...

Replicative DNA polymerase mutations in cancer☆

Science.gov (United States)

Heitzer, Ellen; Tomlinson, Ian

2014-01-01

Three DNA polymerases — Pol α, Pol δ and Pol ɛ — are essential for DNA replication. After initiation of DNA synthesis by Pol α, Pol δ or Pol ɛ take over on the lagging and leading strand respectively. Pol δ and Pol ɛ perform the bulk of replication with very high fidelity, which is ensured by Watson–Crick base pairing and 3′exonuclease (proofreading) activity. Yeast models have shown that mutations in the exonuclease domain of Pol δ and Pol ɛ homologues can cause a mutator phenotype. Recently, we identified germline exonuclease domain mutations (EDMs) in human POLD1 and POLE that predispose to ‘polymerase proofreading associated polyposis’ (PPAP), a disease characterised by multiple colorectal adenomas and carcinoma, with high penetrance and dominant inheritance. Moreover, somatic EDMs in POLE have also been found in sporadic colorectal and endometrial cancers. Tumors with EDMs are microsatellite stable and show an ‘ultramutator’ phenotype, with a dramatic increase in base substitutions. PMID:24583393

Prolonged control of replication-competent dual- tropic human immunodeficiency virus-1 following cessation of highly active antiretroviral therapy

Directory of Open Access Journals (Sweden)

Salgado Maria

2011-12-01

Full Text Available Abstract Background While initiation of highly active antiretroviral therapy (HAART during primary HIV-1 infection occasionally results in transient control of viral replication after treatment interruption, the vast majority of patients eventually experience a rebound in plasma viremia. Results Here we report a case of a patient who was started on HAART during symptomatic primary infection and who has subsequently maintained viral loads of + T cells. In addition, he does not have any known protective HLA alleles. Thus it is unlikely that he was destined to become a natural elite controller or suppressor. The mechanism of control of viral replication is unclear; he is infected with a CCR5/CXCR4 dual-tropic virus that is fully replication-competent in vitro. In addition, his spouse, who transmitted the virus to him, developed AIDS. The patient's CD4+ T cells are fully susceptible to HIV-1 infection, and he has low titers of neutralizing antibodies to heterologous and autologous HIV-1 isolates. Furthermore, his CD8+ T cells do not have potent HIV suppressive activity. Conclusion This report suggests that some patients may be capable of controlling pathogenic HIV-1 isolates for extended periods of time after the cessation of HAART through a mechanism that is distinct from the potent cytotoxic T lymphocyte (CTL mediated suppression that has been reported in many elite suppressors.

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…

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.

Plasticity of DNA replication initiation in Epstein-Barr virus episomes.

Directory of Open Access Journals (Sweden)

Paolo Norio

2004-06-01

Full Text Available In mammalian cells, the activity of the sites of initiation of DNA replication appears to be influenced epigenetically, but this regulation is not fully understood. Most studies of DNA replication have focused on the activity of individual initiation sites, making it difficult to evaluate the impact of changes in initiation activity on the replication of entire genomic loci. Here, we used single molecule analysis of replicated DNA (SMARD to study the latent duplication of Epstein-Barr virus (EBV episomes in human cell lines. We found that initiation sites are present throughout the EBV genome and that their utilization is not conserved in different EBV strains. In addition, SMARD shows that modifications in the utilization of multiple initiation sites occur across large genomic regions (tens of kilobases in size. These observations indicate that individual initiation sites play a limited role in determining the replication dynamics of the EBV genome. Long-range mechanisms and the genomic context appear to play much more important roles, affecting the frequency of utilization and the order of activation of multiple initiation sites. Finally, these results confirm that initiation sites are extremely redundant elements of the EBV genome. We propose that these conclusions also apply to mammalian chromosomes.

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....

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…

Endogenous MOV10 inhibits the retrotransposition of endogenous retroelements but not the replication of exogenous retroviruses.

Science.gov (United States)

Arjan-Odedra, Shetal; Swanson, Chad M; Sherer, Nathan M; Wolinsky, Steven M; Malim, Michael H

2012-06-22

The identification of cellular factors that regulate the replication of exogenous viruses and endogenous mobile elements provides fundamental understanding of host-pathogen relationships. MOV10 is a superfamily 1 putative RNA helicase that controls the replication of several RNA viruses and whose homologs are necessary for the repression of endogenous mobile elements. Here, we employ both ectopic expression and gene knockdown approaches to analyse the role of human MOV10 in the replication of a panel of exogenous retroviruses and endogenous retroelements. MOV10 overexpression substantially decreased the production of infectious retrovirus particles, as well the propagation of LTR and non-LTR endogenous retroelements. Most significantly, RNAi-mediated silencing of endogenous MOV10 enhanced the replication of both LTR and non-LTR endogenous retroelements, but not the production of infectious retrovirus particles demonstrating that natural levels of MOV10 suppress retrotransposition, but have no impact on infection by exogenous retroviruses. Furthermore, functional studies showed that MOV10 is not necessary for miRNA or siRNA-mediated mRNA silencing. We have identified novel specificity for human MOV10 in the control of retroelement replication and hypothesise that MOV10 may be a component of a cellular pathway or process that selectively regulates the replication of endogenous retroelements in somatic cells.

ATM and KAT5 safeguard replicating chromatin against formaldehyde damage

Science.gov (United States)

Ortega-Atienza, Sara; Wong, Victor C.; DeLoughery, Zachary; Luczak, Michal W.; Zhitkovich, Anatoly

2016-01-01

Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA. PMID:26420831

A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells

Directory of Open Access Journals (Sweden)

Ina Ersing

2017-05-01

Full Text Available Epstein-Barr virus (EBV replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication.

Impact of Mutations in the Hemagglutinin of H10N7 Viruses Isolated from Seals on Virus Replication in Avian and Human Cells

Directory of Open Access Journals (Sweden)

Anne Dittrich

2018-02-01

Full Text Available Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L to enhance replication in mammals and retained replication efficiency in the original avian host.

Impact of Mutations in the Hemagglutinin of H10N7 Viruses Isolated from Seals on Virus Replication in Avian and Human Cells.

Science.gov (United States)

Dittrich, Anne; Scheibner, David; Salaheldin, Ahmed H; Veits, Jutta; Gischke, Marcel; Mettenleiter, Thomas C; Abdelwhab, Elsayed M

2018-02-14

Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA) of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering) in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L) to enhance replication in mammals and retained replication efficiency in the original avian host.

Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses.

Science.gov (United States)

Hamperl, Stephan; Bocek, Michael J; Saldivar, Joshua C; Swigut, Tomek; Cimprich, Karlene A

2017-08-10

Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states. Copyright © 2017 Elsevier Inc. All rights reserved.

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...

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.

Effects of Cigarette Smoke Condensate on Oxidative Stress, Apoptotic Cell Death, and HIV Replication in Human Monocytic Cells.

Directory of Open Access Journals (Sweden)

Pss Rao

Full Text Available While cigarette smoking is prevalent amongst HIV-infected patients, the effects of cigarette smoke constituents in cells of myeloid lineage are poorly known. Recently, we have shown that nicotine induces oxidative stress through cytochrome P450 (CYP 2A6-mediated pathway in U937 monocytic cells. The present study was designed to examine the effect of cigarette smoke condensate (CSC, which contains majority of tobacco constituents, on oxidative stress, cytotoxicity, expression of CYP1A1, and/or HIV-1 replication in HIV-infected (U1 and uninfected U937 cells. The effects of CSC on induction of CYP1 enzymes in HIV-infected primary macrophages were also analyzed. The results showed that the CSC-mediated increase in production of reactive oxygen species (ROS in U937 cells is dose- and time-dependent. Moreover, CSC treatment was found to induce cytotoxicity in U937 cells through the apoptotic pathway via activation of caspase-3. Importantly, pretreatment with vitamin C blocked the CSC-mediated production of ROS and induction of caspase-3 activity. In U1 cells, acute treatment of CSC increased ROS production at 6H (>2-fold and both ROS (>2 fold and HIV-1 replication (>3-fold after chronic treatment. The CSC mediated effects were associated with robust induction in the expression of CYP1A1 mRNA upon acute CSC treatment of U937 and U1 cells (>20-fold, and upon chronic CSC treatment to U1 cells (>30-fold. In addition, the CYP1A1 induction in U937 cells was mediated through the aromatic hydrocarbon receptor pathway. Lastly, CSC, which is known to increase viral replication in primary macrophages, was also found to induce CYP1 enzymes in HIV-infected primary macrophages. While mRNA levels of both CYP1A1 and CYP1B1 were elevated following CSC treatment, only CYP1B1 protein levels were increased in HIV-infected primary macrophages. In conclusion, these results suggest a possible association between oxidative stress, CYP1 expression, and viral replication in

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

Science.gov (United States)

Xu, Kai; Nagy, Peter D

2017-04-01

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

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...

Reliability analysis of a replication with limited number of journaling files

International Nuclear Information System (INIS)

Kimura, Mitsutaka; Imaizumi, Mitsuhiro; Nakagawa, Toshio

2013-01-01

Recently, replication mechanisms using journaling files have been widely used for the server systems. We have already discussed the model of asynchronous replication system using journaling files [8]. This paper formulates a stochastic model of a server system with replication considering the number of transmitting journaling files. The server updates the storage database and transmits the journaling file when a client requests the data update. The server transmits the database content to a backup site either at a constant time or after a constant number of transmitting journaling files. We derive the expected number of the replication and of transmitting journaling files. Further, we calculate the expected cost and discuss optimal replication interval to minimize it. Finally, numerical examples are given

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

Replication stress interferes with histone recycling and predeposition marking of new histones

DEFF Research Database (Denmark)

Jasencakova, Zuzana; Scharf, Annette N D; Ask, Katrine

2010-01-01

To restore chromatin on new DNA during replication, recycling of histones evicted ahead of the fork is combined with new histone deposition. The Asf1 histone chaperone, which buffers excess histones under stress, is a key player in this process. Yet how histones handled by human Asf1 are modified...... remains unclear. Here we identify marks on histones H3-H4 bound to Asf1 and changes induced upon replication stress. In S phase, distinct cytosolic and nuclear Asf1b complexes show ubiquitous H4K5K12diAc and heterogeneous H3 marks, including K9me1, K14ac, K18ac, and K56ac. Upon acute replication arrest......, the predeposition mark H3K9me1 and modifications typical of chromatin accumulate in Asf1 complexes. In parallel, ssDNA is generated at replication sites, consistent with evicted histones being trapped with Asf1. During recovery, histones stored with Asf1 are rapidly used as replication resumes. This shows...

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.

Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes.

Directory of Open Access Journals (Sweden)

Nunya Chotiwan

2018-02-01

Full Text Available We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several

Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes.

Science.gov (United States)

Chotiwan, Nunya; Andre, Barbara G; Sanchez-Vargas, Irma; Islam, M Nurul; Grabowski, Jeffrey M; Hopf-Jannasch, Amber; Gough, Erik; Nakayasu, Ernesto; Blair, Carol D; Belisle, John T; Hill, Catherine A; Kuhn, Richard J; Perera, Rushika

2018-02-01

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted

Isolation and characterization of highly replicable hepatitis C virus genotype 1a strain HCV-RMT.

Science.gov (United States)

Arai, Masaaki; Tokunaga, Yuko; Takagi, Asako; Tobita, Yoshimi; Hirata, Yuichi; Ishida, Yuji; Tateno, Chise; Kohara, Michinori

2013-01-01

Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV) clone called H77. The replication ability of some of these clones has been confirmed in vitro and in vivo, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient's serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between in vitro and in vivo replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication in vitro and in vivo.

Isolation and characterization of highly replicable hepatitis C virus genotype 1a strain HCV-RMT.

Directory of Open Access Journals (Sweden)

Masaaki Arai

Full Text Available Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV clone called H77. The replication ability of some of these clones has been confirmed in vitro and in vivo, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient's serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between in vitro and in vivo replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication in vitro and in vivo.

Autoradiographic studies of chromosome replication during the cell cycle of Streptococcus faecium

International Nuclear Information System (INIS)

Higgins, M.L.; Koch, A.L.; Dicker, D.T.; Daneo-Moore, L.

1986-01-01

Analysis of the distribution of autoradiographic grains around cells of Streptococcus faecium which had been either continuously or pulse-labeled with tritiated thymidine (mass doubling time, 90 min) showed a non-Poisson distribution even when the distribution of cell sizes in the populations studied was taken into account. These non-Poisson distributions of grains were assumed to reflect the discontinuous nature of chromosome replication. To study this discontinuous process further, an equation was fitted to the grain distribution observed for the pulse-labeled cells that assumed that in any population of cells there were subpopulations in which there were zero, one, or two replicating chromosomes. This analysis predicted an average time for chromosome replication and for the period between completion of rounds of chromosome replication and division of 55 and 43 min, respectively, which were in excellent agreement with estimates made by other techniques. The present investigation extended past studies in indicating that the initiation and completion of rounds of chromosome replication are poorly phased with increases in cell volume and that the amount of chromosome replication may be different in different cell halves

Enhanced replication of damaged SV40 DNA in carcinogen-treated monkey cells

International Nuclear Information System (INIS)

Maga, J.A.; Dixon, K.

1984-01-01

Treatment of mammalian cells with certain chemical or physical carcinogens prior to infection with ultraviolet-irradiated virus results in enhanced survival or reactivation of the damaged virus. To investigate the molecular basis of this enhanced reactivation (ER), Simian virus 40 DNA replication in carcinogen-treated cells was examined. Treatment of monkey kidney cells with N-acetoxy-2-acetylamino-fluorene or UV radiation 24 h prior to infection with ultraviolet-irradiated Simian virus 40 leads to enhancement of viral DNA replication measured at 36 h after infection by [ 3 H]thymidine incorporation or hybridization. The enhancement of DNA replication is observed when cells are treated from 1 to 60 h before infection or 1 to 16 h after infection. The fact that enhancement is observed also when cells are treated after infection rules out the possiblity that enhancement occurs at the level of adsorption or penetration of the virus. Measurements of the time course of viral DNA replication indicate that pretreatment of cells does not alter the time of onset of viral DNA replication. It is concluded that ER of Simain virus 40 occurs at the level of viral DNA replication. (author)

DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair.

Science.gov (United States)

de Laat, W L; Appeldoorn, E; Sugasawa, K; Weterings, E; Jaspers, N G; Hoeijmakers, J H

1998-08-15

The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5' side of its binding region, a weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning of the excision repair nucleases XPG and ERCC1-XPF on the DNA. With the 3'-oriented side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1-XPF, whereas the 5'-oriented side of hRPA at a DNA junction allows stable binding of XPG to hRPA. Our data pinpoint hRPA to the undamaged strand during nucleotide excision repair. Polarity of hRPA on ssDNA is likely to contribute to the directionality of other hRPA-dependent processes as well.

Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells.

Science.gov (United States)

Kumar, S; Peng, X; Daley, J; Yang, L; Shen, J; Nguyen, N; Bae, G; Niu, H; Peng, Y; Hsieh, H-J; Wang, L; Rao, C; Stephan, C C; Sung, P; Ira, G; Peng, G

2017-04-17

Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. DNA2 nuclease is an evolutionarily conserved essential enzyme in replication and HR repair. Here we demonstrate that DNA2 is overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90-95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA double strand break end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anticancer drugs by targeting druggable nuclease DNA2. We propose DNA2 inhibition as new strategy in cancer therapy by targeting

Social learning and the replication process: an experimental investigation.

Science.gov (United States)

Derex, Maxime; Feron, Romain; Godelle, Bernard; Raymond, Michel

2015-06-07

Human cultural traits typically result from a gradual process that has been described as analogous to biological evolution. This observation has led pioneering scholars to draw inspiration from population genetics to develop a rigorous and successful theoretical framework of cultural evolution. Social learning, the mechanism allowing information to be transmitted between individuals, has thus been described as a simple replication mechanism. Although useful, the extent to which this idealization appropriately describes the actual social learning events has not been carefully assessed. Here, we used a specifically developed computer task to evaluate (i) the extent to which social learning leads to the replication of an observed behaviour and (ii) the consequences it has for fitness landscape exploration. Our results show that social learning does not lead to a dichotomous choice between disregarding and replicating social information. Rather, it appeared that individuals combine and transform information coming from multiple sources to produce new solutions. As a consequence, landscape exploration was promoted by the use of social information. These results invite us to rethink the way social learning is commonly modelled and could question the validity of predictions coming from models considering this process as replicative. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

Codon and amino acid usage in two major human pathogens of genus Bartonella--optimization between replicational-transcriptional selection, translational control and cost minimization.

Science.gov (United States)

Das, Sabyasachi; Paul, Sandip; Chatterjee, Sanjib; Dutta, Chitra

2005-01-01

Intra-genomic variation in synonymous codon and amino acid usage in two human pathogens Bartonella henselae and B. quintana has been carried out through multivariate analysis. Asymmetric mutational bias, coupled with replicational-transcriptional selection, has been identified as the prime selection force behind synonymous codon selection--a characteristic of the genus Bartonella, not exhibited by any other alpha-proteobacterial genome. Distinct codon usage patterns and low synonymous divergence values between orthologous sequences of highly expressed genes from the two Bartonella species indicate that there exists a residual intra-strand synonymous codon bias in the highly expressed genes, possibly operating at the level of translation. In the case of amino acid usage, the mean hydropathy level and aromaticity are the major sources of variation, both having nearly equal impact, while strand-specific mutational pressure and gene expressivity strongly influence the inter-strand variations. In both species under study, the highly expressed gene products tend not to contain heavy and/or aromatic residues, following the cost-minimization hypothesis in spite of their intracellular lifestyle. The codon and amino acid usage in these two human pathogens are, therefore, consequences of a complex balance between replicational-transcriptional selection, translational control, protein hydropathy and cost minimization.

Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag.

Science.gov (United States)

Coleman, John W; Wright, Kevin J; Wallace, Olivia L; Sharma, Palka; Arendt, Heather; Martinez, Jennifer; DeStefano, Joanne; Zamb, Timothy P; Zhang, Xinsheng; Parks, Christopher L

2015-03-01

Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert. Copyright © 2014 Elsevier B.V. All rights reserved.

Active RNA replication of hepatitis C virus downregulates CD81 expression.

Science.gov (United States)

Ke, Po-Yuan; Chen, Steve S-L

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

Active RNA replication of hepatitis C virus downregulates CD81 expression.

Directory of Open Access Journals (Sweden)

Po-Yuan Ke

Full Text Available So far how hepatitis C virus (HCV replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp infection and downregulated cell surface level of CD81, a critical HCV entry (coreceptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner.

Science.gov (United States)

Loutre, Romuald; Heckel, Anne-Marie; Jeandard, Damien; Tarassov, Ivan; Entelis, Nina

2018-01-01

Mutations in mitochondrial DNA are an important source of severe and incurable human diseases. The vast majority of these mutations are heteroplasmic, meaning that mutant and wild-type genomes are present simultaneously in the same cell. Only a very high proportion of mutant mitochondrial DNA (heteroplasmy level) leads to pathological consequences. We previously demonstrated that mitochondrial targeting of small RNAs designed to anneal with mutant mtDNA can decrease the heteroplasmy level by specific inhibition of mutant mtDNA replication, thus representing a potential therapy. We have also shown that 5S ribosomal RNA, partially imported into human mitochondria, can be used as a vector to deliver anti-replicative oligoribonucleotides into human mitochondria. So far, the efficiency of cellular expression of recombinant 5S rRNA molecules bearing therapeutic insertions remained very low. In the present study, we designed new versions of anti-replicative recombinant 5S rRNA targeting a large deletion in mitochondrial DNA which causes the KSS syndrome, analyzed their specific annealing to KSS mitochondrial DNA and demonstrated their import into mitochondria of cultured human cells. To obtain an increased level of the recombinant 5S rRNA stable expression, we created transmitochondrial cybrid cell line bearing a site for Flp-recombinase and used this system for the recombinase-mediated integration of genes coding for the anti-replicative recombinant 5S rRNAs into nuclear genome. We demonstrated that stable expression of anti-replicative 5S rRNA versions in human transmitochondrial cybrid cells can induce a shift in heteroplasmy level of KSS mutation in mtDNA. This shift was directly dependent on the level of the recombinant 5S rRNA expression and the sequence of the anti-replicative insertion. Quantification of mtDNA copy number in transfected cells revealed the absence of a non-specific effect on wild type mtDNA replication, indicating that the decreased proportion

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…

Using Model Replication to Improve the Reliability of Agent-Based Models

Science.gov (United States)

Zhong, Wei; Kim, Yushim

The basic presupposition of model replication activities for a computational model such as an agent-based model (ABM) is that, as a robust and reliable tool, it must be replicable in other computing settings. This assumption has recently gained attention in the community of artificial society and simulation due to the challenges of model verification and validation. Illustrating the replication of an ABM representing fraudulent behavior in a public service delivery system originally developed in the Java-based MASON toolkit for NetLogo by a different author, this paper exemplifies how model replication exercises provide unique opportunities for model verification and validation process. At the same time, it helps accumulate best practices and patterns of model replication and contributes to the agenda of developing a standard methodological protocol for agent-based social simulation.

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

Endogenous MOV10 inhibits the retrotransposition of endogenous retroelements but not the replication of exogenous retroviruses

Science.gov (United States)

2012-01-01

Background The identification of cellular factors that regulate the replication of exogenous viruses and endogenous mobile elements provides fundamental understanding of host-pathogen relationships. MOV10 is a superfamily 1 putative RNA helicase that controls the replication of several RNA viruses and whose homologs are necessary for the repression of endogenous mobile elements. Here, we employ both ectopic expression and gene knockdown approaches to analyse the role of human MOV10 in the replication of a panel of exogenous retroviruses and endogenous retroelements. Results MOV10 overexpression substantially decreased the production of infectious retrovirus particles, as well the propagation of LTR and non-LTR endogenous retroelements. Most significantly, RNAi-mediated silencing of endogenous MOV10 enhanced the replication of both LTR and non-LTR endogenous retroelements, but not the production of infectious retrovirus particles demonstrating that natural levels of MOV10 suppress retrotransposition, but have no impact on infection by exogenous retroviruses. Furthermore, functional studies showed that MOV10 is not necessary for miRNA or siRNA-mediated mRNA silencing. Conclusions We have identified novel specificity for human MOV10 in the control of retroelement replication and hypothesise that MOV10 may be a component of a cellular pathway or process that selectively regulates the replication of endogenous retroelements in somatic cells. PMID:22727223

Determination of selenium in food matrices by replicate sample neutron activation analysis

International Nuclear Information System (INIS)

Ventura, M.G.; Freitas, M.C.; Ventura, M.G.; Pacheco, A.M.G.

2009-01-01

The replicate sample instrumental neutron activation method was optimized and used for the determination of selenium in foodstuffs. The method was reliable, yielding accurate results. Lower detections limits were obtained after each successive irradiation. Different irradiation conditions were used depending on the type of sample. For samples with higher selenium contents (meat, fish, eggs), the measured selenium in the first replicate is in all cases larger than the detection limit, but a better accuracy was obtained with a larger number of replicates (2-3 replicates). For samples with extremely low selenium contents (vegetable samples), at least seven replicates were necessary to obtain a concentration value two times larger than the detection limit. (author)

Enhanced transduction and replication of RGD-fiber modified adenovirus in primary T cells.

Directory of Open Access Journals (Sweden)

Sadhak Sengupta

2011-03-01

Full Text Available Adenoviruses are often used as vehicles to mediate gene delivery for therapeutic purposes, but their research scope in hematological cells remains limited due to a narrow choice of host cells that express the adenoviral receptor (CAR. T cells, which are attractive targets for gene therapy of numerous diseases, remain resistant to adenoviral infection because of the absence of CAR expression. Here, we demonstrate that this resistance can be overcome when murine or human T cells are transduced with an adenovirus incorporating the RGD-fiber modification (Ad-RGD.A luciferase-expressing replication-deficient Ad-RGD infected 3-fold higher number of activated primary T cells than an adenovirus lacking the RGD-fiber modification in vitro. Infection with replication-competent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells. Transduction with oncolytic Ad-RGD also resulted in higher titers of progeny virus and enhanced the killing of T cells. In vivo, 35-45% of splenic T cells were transduced by Ad-RGD.Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary T cells of both murine and human origin.

Human hepatocytes apoptosis induced by replication of hepatitis B virus subgenotypes F1b and F4: Role of basal core promoter and preCore mutations.

Science.gov (United States)

Elizalde, María Mercedes; Sevic, Ina; González López Ledesma, María Mora; Campos, Rodolfo Héctor; Barbini, Luciana; Flichman, Diego Martin

2018-01-01

In the context of pathogenesis of HBV infection, HBV genotypes and mutants have been shown to affect the natural course of chronic infection and treatment outcomes. In this work, we studied the induction of apoptosis by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. Both subgenotypes F1b and F4 HBV genome transfections induced cell death by apoptosis in human hepatocytes. The BCPdm (A1762T/G1764A) and preCore (G1896A) mutants induced higher levels of apoptosis than the wt virus. This increase in apoptosis was not associated with the enhanced viral replication of the variants. HBV-mediated apoptosis was independent of viral subgenotypes, and associated with the modulation of members of the regulatory Bcl-2 family proteins expression in the mitochondrial apoptotic pathway. Finally, the apoptosis induction increase observed for the preCore mutants suggests that HBeAg might have an anti-apoptotic effect in human hepatocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibition of influenza virus replication by targeting broad host cell pathways.

Directory of Open Access Journals (Sweden)

Isabelle Marois

Full Text Available Antivirals that are currently used to treat influenza virus infections target components of the virus which can mutate rapidly. Consequently, there has been an increase in the number of resistant strains to one or many antivirals in recent years. Here we compared the antiviral effects of lysosomotropic alkalinizing agents (LAAs and calcium modulators (CMs, which interfere with crucial events in the influenza virus replication cycle, against avian, swine, and human viruses of different subtypes in MDCK cells. We observed that treatment with LAAs, CMs, or a combination of both, significantly inhibited viral replication. Moreover, the drugs were effective even when they were administered 8 h after infection. Finally, analysis of the expression of viral acidic polymerase (PA revealed that both drugs classes interfered with early events in the viral replication cycle. This study demonstrates that targeting broad host cellular pathways can be an efficient strategy to inhibit influenza replication. Furthermore, it provides an interesting avenue for drug development where resistance by the virus might be reduced since the virus is not targeted directly.

Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq.

Science.gov (United States)

Marchal, Claire; Sasaki, Takayo; Vera, Daniel; Wilson, Korey; Sima, Jiao; Rivera-Mulia, Juan Carlos; Trevilla-García, Claudia; Nogues, Coralin; Nafie, Ebtesam; Gilbert, David M

2018-05-01

This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and subnuclear position. Moreover, RT is regulated during development and is altered in diseases. Here, we describe E/L Repli-seq, an extension of our Repli-chip protocol. E/L Repli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse-labeled with BrdU, and early and late S-phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S-phase fractions. The results are comparable to those of Repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single-nucleotide polymorphisms (SNPs) to analyze RT allelic asynchrony, and for direct comparison to Repli-chip data. This protocol can be performed in up to 3 d before sequencing, and requires basic cellular and molecular biology skills, as well as a basic understanding of Unix and R.

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